Download Regulation of Cytochrome bd Expression in Mycobacterium

Regulation of
Cytochrome bd Expression in
Mycobacterium smegmatis
Marion Ricarda Weimar (PGDip Sci)
A thesis submitted for the degree of Masters of Science at the
University of Otago, Dunedin, New Zealand
January 2010
Abstract
The metabolism and generation of energy by the majority of pathogenic bacteria
in the host remains enigmatic and emerging evidence suggests that the
identification of essential bacterial metabolic pathways that have no human
counterparts may represent a virtually untapped source of novel drug targets for
antibiotic development. The aim of this study was to determine how mycobacteria
metabolize and generate energy microaerobically. To identify potential regulatory
circuits that control the response to low oxygen tension, we targeted the enzyme
cytochrome bd oxidase, purportedly involved in the generation of energy under
these conditions, and isolated mutants that fail to activate expression of this
enzyme. Instead of finding regulators of cytochrome bd oxidase, our screen
revealed enzymes and regulators involved in redox homeostasis and defence
against electrophiles (oxidative stress). We discovered ProR, a novel regulator of
proline dehydrogenase and 1-pyrroline-5-carboxylate dehydrogenase, two
enzymes involved in proline degradation. Evidence is presented that ProR is
essential for growth on proline as a sole carbon source and that a proR mutant
experiences DNA damage due to methylglyoxal, an endogenously produced toxic
intermediate of glycolysis and glycerol metabolism. It is shown that proline
confers resistance to methylglyoxal. Furthermore, we characterized the
sodium/proline symporter PutP as a high-affinity transporter for proline and show
that it is the major uptake system for proline in Mycobacterium smegmatis.
A new model for methylglyoxal detoxification in Mycobacteria is presented.
ii
Acknowledgements
I would first like to thank my supervisor Professor Gregory Cook, for offering that
amazing project and his exceptional encouragement throughout my time in his
lab. The way he motivates his team is excellent and I was extremely lucky that I
was able to experience being part of his team in the last two years. His
exceedingly high interest in research was very inspiring to me and his energy and
support were outstanding.
I would also like to thank my second supervisor Dr. Michael Berney for all the
helpful advice and support he was giving me. His patience in answering endless
questions was phenomenal and I could not have asked for any more
encouragement. Thanks so much for making my time in this lab so successful.
Special thanks goes to all the current and former members of the Cook Lab team
for all the help and advice. Thanks to Susanne Gebhard for teaching me all the
molecular skills in an exceptional way. Special mention to Lisa, Nicola, Htin, Mae
and Shaaly for simply just being there when you need a friend to talk to and for all
the fun we had together. It was great!
I would also like to thank the University of Otago for their financial support which
enabled me to finish my degree in New Zealand.
The biggest thanks are for Joel and my family. Without the mental support they
were giving me I would have had a much harder time. Thanks mum and dad for
believing in me no matter what. I am very grateful to call myself your daughter.
Thanks so much Joel for going this journey with me and making it a wonderful
time.
iii
Table of Contents
Abstract
ii
Acknowledgements
iii
Table of Contents
iv
List of Tables
vi
List of Figures
vii
List of Abbreviations
x
Appendix
I
1. INTRODUCTION
1 1.1 MYCOBACTERIA
1 1.2 MYCOBACTERIUM TUBERCULOSIS
2 1.3 TREATMENT AND THE PROBLEM OF DRUG RESISTANCE OF M.
TUBERCULOSIS
5 1.4 VACCINES
6 1.5 PHYSIOLOGY OF MYCOBACTERIA
8 1.5.1 THE CELL ENVELOPE OF MYCOBACTERIA 8 1.5.2 NUTRIENTS FOR MYCOBACTERIAL METABOLISM 8 1.5.3 SENSING OF ENVIRONMENTAL FACTORS 9 1.5.3.1 Sensing oxygen availability and redox state of the cell 10 1.5.3.2 Sensing of oxidative stress 11 1.5.4 RESPIRATORY CHAIN IN MYCOBACTERIA 12 1.5.5 CYTOCHROME BD 15 1.6 AIMS OF THE STUDY
17
iv
2. MATERIALS AND METHODS
18 2.1 BACTERIAL STRAINS AND GROWTH CONDITIONS
18 2.2 TRANSPOSON MUTAGENESIS AND CLONING OF CONSTRUCTS
19 2.2.1 SCREENING FOR TRANSPOSON MUTANTS 21 2.2.2 GENOMIC DNA EXTRACTION 22 2.2.3 PCR AMPLIFICATION AND GEL‐EXTRACTION (GE) 22 2.2.4 DIGEST AND LIGATION 23 2.2.5 ELECTROPORATION OF PLASMID DNA 23 2.2.6 ALKALINE LYSIS MINIPLASMID PREPARATION 23 2.3 β-GALACTOSIDASE ASSAY
24 2.4 RNA ISOLATION AND REVERSE TRANSCRIPTION (RT)-PCR
25 2.5 PRIMER DESIGN AND REAL-TIME-QUANTITATIVE PCR (RT-QPCR)
26 2.6 MICROARRAY ANALYSIS
26 2.7 PROLINE TRANSPORT ASSAY
27 2.8 OXIDATIVE STRESS EXPERIMENTS
28 2.8.1 CULTURE EXPERIMENTS 28 2.8.2 DISK DIFFUSION ASSAYS 28 3. RESULTS
29 3.1 IDENTIFICATION OF TRANSPOSON MUTANTS
29 3.2 GROWTH CURVES AND CYDAB EXPRESSION
32 3.3 PROLINE TRANSPORT ASSAYS
39 3.4 REGULATION OF PUTP, PROR AND CYDAB
43 3.5 MICROARRAY OF PROR::TN611 MUTANT
45 3.6 SENSITIVITY TO METHYLGLYOXAL, ROS STRESS AND NACL
49
4. DISCUSSION
54
5. REFERENCES
65 v
List of Tables
Table 1
Bacterial strains and plasmids used in this study
20
Table 2
Primers used in this study
21
Table 3
Transposon mutants defective in cydA-lacZ expression
32
Table 4
Summary of specific growth rates and final optical densities of
M. smegmatis strain MB100 and mutants proR::Tn611 and
putP::Tn611 grown under various conditions
Table 5
Upregulated DNA metabolism genes of proR::Tn611 mutant
compared to M. smegmatis strain MB100 in Microarray
Table 6
33
46
Upregulated methylglyoxal synthesis and detoxification genes
of proR::Tn611 mutant compared to M. smegmatis strain
MB100 in Microarray
Table 7
Downregulated genes of proR::Tn611 mutant compared to M.
smegmatis strain MB100 in Microarray
Table 8
48
48
Genes with induction ratios (IR) significantly different from 1
(p ≤ 0.05) comparing M. smegmatis strain MB100 with the
proR::Tn611 mutant
I
vi
List of Figures
Figure 1
The Immune response to M. tuberculosis infection
Figure 2
Proposed respiratory chain of M. tuberculosis under A)
aerobic and B) hypoxic conditions
Figure 3
4
14
Region view of MSMEG_5120 taken from The Institute of
Genomic Research (TIGR)
30
Figure 4
Region view of MSMEG_5303 taken from TIGR
31
Figure 5
Growth (A) and β-galactosidase expression from a cydA-lacZ
fusion (B) of M. smegmatis strain MB100 (solid squares) and
proR::Tn611 (solid triangles) and putP::Tn611 (solid inverted
triangles) mutants in Luria-Bertani (LB) medium
supplemented with 0.05% (w/v) Tween-80 (LBT) in sealed
serum vials over the course of time
Figure 6
34
Growth (A) and β-galactosidase expression from a cydA-lacZ
fusion (B) of M. smegmatis strain MB100 (solid squares) and
proR::Tn611 (solid triangles) and putP::Tn611 (solid inverted
triangles) mutants in HdB minimal medium supplemented with
glycerol in sealed serum vials over the course of time
Figure 7
35
Growth (A) and β-galactosidase expression from a cydA-lacZ
fusion (B) of M. smegmatis strain MB100 (solid squares) and
proR::Tn611 (solid triangles) and putP::Tn611 (solid inverted
triangles) mutants in HdB minimal medium supplemented with
glycerol and L-proline (25 mM) in sealed serum vials over the
course of time
Figure 8
36
Growth (A) and β-galactosidase expression from a cydA-lacZ
fusion (B) of M. smegmatis strain MB100 (solid squares) and
proR::Tn611 (solid triangles) and putP::Tn611 (solid inverted
triangles) mutants in HdB minimal medium supplemented with
L-proline (25 mM) as a sole carbon source in sealed serum
vials over the course of time
37
vii
Figure 9
(A) Growth of M. smegmatis strain MB100 (solid squares) and
putP::Tn611 (solid triangles) and complemented putP::Tn611
(solid inverted triangles) mutants over the course of time. (B)
Growth of M. smegmatis strain MB100 (solid squares) and
proR::Tn611 (open squares) and complemented proR::Tn611
(open triangles) mutants over the course of time
Figure 10
Rate of proline transport of the M. smegmatis strain MB100
and mutants proR::Tn611 and putP::Tn611
Figure 11
38
40
Rate of proline transport of the M. smegmatis strain MB100
(solid squares), strain MB100 plus CCCP (solid diamonds)
and putP::Tn611 (solid triangles) mutant
Figure 12
Lineweaver-Burke plot of the M. smegmatis strain MB100
proline transport system
Figure 13
42
Gene expression ratios comparing proR::Tn611/strain MB100
(white bars) and putP::Tn611/strain MB100 (black bars)
Figure 14
41
44
Survival of the M. smegmatis strain MB100 (white bars) and
proR::Tn611 (lined bars) and putP::Tn611 (black bars)
mutants after cultures have been stressed with 5 mM, 10 mM
and 20 mM hydrogen peroxide
Figure 15
50
Hydrogen peroxide sensitivity of M. smegmatis strain MB100
and proR::Tn611 and putP::Tn611 mutants as determined by
disk diffusion assays
Figure 16
51
Methylglyoxal sensitivity of the M. smegmatis strain MB100
and proR::Tn611 and putP::Tn611 mutants as determined by
disk diffusion assays
Figure 17
52
Growth of M. smegmatis strain MB100 (A) and proR::Tn611
(B) and putP::Tn611 (C) mutants in Luria-Bertani (LB)
medium with 10g/l (solid squares), 1 g/l (solid triangles) or
0.1g/l (solid inverted triangles) of NaCl supplemented with
0.05% (w/v) Tween-80 (LBT) and in sealed serum vials over
the course of time
53
Figure 18
Proline catabolism
55
Figure 19
Synthesis and detoxification of methylglyoxal
62
viii
Figure 20
Model of proline and methylglyoxal metabolism in M.
smegmatis mutants defective in proR
64
ix
List of Abbreviations
°C
Degrees Celsius
µF
Microfarads
µg
Micrograms
µl
Microliters
µm
Micrometers
µM
Micromolar
Ω
Ohms
BCG
Bacille Calmette and Guerin
BLAST
Basic Local Alignment Search Tool
bp
Base pairs
CT
Cycle threshold value
CFU
Colony Forming Units
DEPC
Diethylpyrocarbonate
dH2O
Distilled water
DNA
Deoxyribonucleic Acid
DOS
Dormancy Survival System
DOTS
Directly Observed Treatment, Short course
EDTA
Disodium Ethylene Diamine Tetra Acetic Acid
FADH2
Flavin Adenine Dinucleotide, reduced form
g
Gram
g
Gravitational force
h
Hours
HdB
Hartmans de Bont
IR
Induction Ratios
l
Liters
LB
Luria-Bertani
M
Molar
MDR
Multi-Drug Resistant
mg
Milligrams
min
Minutes
ml
Milliliters
mM
Millimolar
x
MU
Miller Units
NADH
β-Nicotinamide Adenine Dinucleotide, reduced form
NCBI
National Center for Biotechnology Information
ng
Nanograms
nm
Nanometers
NRP
Non-Replicating Persistence
NTM
Nontuberculous Mycobacteria
OD600
Optical Density at 600 nm
PCR
Polymerase Chain Reaction
RNA
Ribonucleic Acid
RNase
Ribonuclease
ROS
Reactive Oxygen Species
rpm
Revolutions per minute
rRNA
Ribosomal RNA
RT
Room Temperature
RT-PCR
Reverse Transcriptase-PCR
RT-qPCR
Real-Time-quantitative PCR
s
Seconds
SDS
Sodium Dodecyl Sulphate
SSC
Saline Sodium Citrate
TIGR
The Institute of Genomic Research
Tris
Tris(hydroxymethyl)aminomethane
TB
Tuberculosis
V
Volts
w/v
Weight to volume ratio
U
Units
XDR
Extensively Drug-Resistant
xi
1. Introduction
1.1 Mycobacteria
Mycobacteria are Gram-positive and acid-fast bacteria, belonging to the phylum of
Actinobacteria. Mycobacteria possess G + C rich genomes with a content of about
62-70% (24). They are considered to be obligate aerobes and have a rod-shaped
appearance of about 0.3 - 0.5 µm in diameter and of variable length. The cell wall
in Mycobacteria is characteristic because it is thicker than in many other bacteria
and contains mycolic acid. Other Actinobacteria such as Corynebacterium,
Rhodococcus and Nocardia have related properties. There are over 100 species
that belong to the genus Mycobacterium and 16S rRNA sequence analyses
reveals that all mycobacterial species are closely related (143, 159). Mycobacteria
have been found to be widely distributed in the environment and most species
have been referred to as nontuberculous mycobacteria (NTM) (45). Unlike the
tubercle bacilli, NTM are not spread from person to person, however, NTM can still
cause disease in humans. Mycobacteria are opportunistic pathogens, especially in
a clinical environment, and therefore their detection is very important for treatment
of the infections (24). There are several reasons for why research on
mycobacterial species remains challenging. Some mycobacterial strains are not
easy to grow in the laboratory and Mycobacterium leprae (the cause of leprosy)
has never been grown in vitro (24). M. ulcerans and M. paratuberculosis are
examples of mycobacterial species that produce visible growth on solid medium
only after a month or greater. For most slow-growing mycobacterial strains,
colonies become visible on a solid medium after 10-28 days of growth. Other
difficulties that hinder and slow research on mycobacteria are the hazards of
handling pathogens, the tendency of mycobacterial cells to clump, and the
resistance of cells to lysis (24). These are reasons for why although M. leprae (50)
and Mycobacterium tuberculosis (64) were two of the first microbes that have been
identified our knowledge of their physiological properties still remain rudimentary.
1
1.2 Mycobacterium tuberculosis
M. tuberculosis was discovered to be the causative agent of tuberculosis (TB) by
Hermann Heinrich Koch in 1882 (64). M. tuberculosis spreads from person to
person through the air and in most cases affects the lungs (9). M. tuberculosis
causes around 8 million new infections per annum of which up to 3 million lead to
death (34). It is one of the most successful bacterial pathogens and approximately
2 billion people have been infected with M. tuberculosis (76). The World Health
Organisation (WHO) estimates a distribution of all global tuberculosis cases of
55% occur in Asia, 33% occur in Africa and only 12% occur in countries other than
African or Asian countries. Among that, about 15% of the annual cases of TB
infections are in people who are infected with the Human Immunodefiency Virus
(HIV).
In humans, tubercle bacilli enter the host through the respiratory system and
spread from person to person through the air. The bacilli are released by a patient
with active disease and inhaled by a healthy person. As soon as the bacilli reach
the pulmonary alveoli they are phagocytized by macrophages (Figure 1). As a
protective mechanism, the macrophages start producing lysosomal enzymes and
reactive oxygen species (ROS), which in most of the cases (50-80%) destroy the
pathogen (30, 110). The progress from tuberculosis (TB) infection to the
development of actual clinical TB disease occurs when the pathogen overcomes
the immune system defences of the host and starts replicating within the infected
macrophages (110). Infected macrophages then start to produce a series of
inflammatory chemokines which are recruiting cells of the immune system (natural
killer T cells, neutrophils, CD4+ and CD8+ T cells), which in turn start to produce
their own cytokines to amplify the immune response (9). To prevent the spread of
mycobacteria within the host, the infected macrophages aggregate together with T
and B cells to form a tuberculosis granuloma, which is then surrounded by
lymphocytes. Those granulomas are assumed to build a hypoxic (oxygen-limited)
environment for the pathogen (130). It is estimated that in 90-95% of the cases the
formation of a granuloma inhibits the replication of the tubercle bacilli, controls the
growth of the tuberculosis pathogen and leads to a latent infection (30). Most
tuberculosis infections are latent, showing no symptoms, but about one in every
ten individuals develops active disease (169). Environmental factors, such as poor
2
economic conditions, under nourishment and overcrowding and also human
immunodeficiency virus (HIV) infections or genetic variability in host defence are
the main factors that trigger the onset for developing an active disease state (86,
119).
M. tuberculosis has the ability to exist in various metabolic states. In acute phases
of disease the tubercle bacilli are fast replicating whereas they enter a nonreplicating persistence (NRP) state in a chronic infection in which they remain
metabolically active (primed) and ready to reinitiate growth (130, 153). Therefore,
these bacteria in the latter state should not be considered dormant (62). There are
numerous environmental factors that might cause the onset for M. tuberculosis to
enter a non-replicating state in the host organism. Low carbohydrate and
phosphate concentrations (nutrient depletion), shifts in pH to a mildly acidic pH,
production of specific growth-limiting products and the depletion of oxygen
(hypoxia) seem to be conditions that are likely to prevail in vivo and that initiate a
shifting towards a NRP state (153). Tubercle bacilli reside inside granulomas of
the human host and those appear to build a hypoxic environment for the bacteria
(130). This seems to be inconsistent with the classification of M. tuberculosis as an
obligate aerobe; however tubercle bacilli are able to survive oxygen deprivation if
they have time to adapt to its gradual depletion (151); sudden changes to
anaerobic conditions lead to cell death (152). The respiration chain of M.
tuberculosis contains both aerobic and anaerobic components (22) and is
proposed to play an important role in the adaptation to changes in oxygen
concentration and alternate electron acceptors (12, 26).
Current anti-tuberculosis drugs are not able to kill tubercle bacilli in their latent
state as they all target actively growing bacteria. The reason for this is that M.
tuberculosis in the NRP state shows increased resistance to many of the present
antibiotics leading to the failure of a successful treatment (63). The fact that no
new drug has been discovered in more than 40 years (101), shows how difficult it
is to identify drugs that can target the pathogen in its NRP state (129).
3
Figure 1: The Immune response to M. tuberculosis infection. Once the bacilli
reach the alveoli they are ingested by macrophages, that in most cases can
effectively kill the bacteria. However, in about 20-50% of people exposed to M.
tuberculosis, the bacilli overcomes the innate immune response, and is then able
to multiply while they are infecting nearby cells in the host organism. The cellmediated immune response of the host leads to a cellular infiltration in the site of
primary infection leading to the formation of a granuloma. In 90-95% of infections,
the host immune response will inhibit bacterial multiplication and controls the
growth of the bacilli, which then leads to a latent infection. People with latent TB
have a 5-10% chance of developing active TB during their lifetime. In 5-10% of
cases, the host immune response fails to control primary infection and the host
response itself is responsible for the general tissue damage and necrosis.
Modified from Delogu and Fadda, 2009 (30).
4
1.3 Treatment and the problem of drug resistance of M. tuberculosis
Tuberculosis became effectively curable for the first time ever between 1943 and
1965 when antitubercular drugs, such as streptomycin (30), para-aminosalicylic
acid (167), isoniazid (133), pyrazinamide (166), cycloserine (1), ethambutol (29)
and rifampicin (rifampin) (73) were developed. Unfortunately, even nowadays
tuberculosis is still the primary cause of death from a curable infectious disease (9,
33) and the reason for this is the problem of dispersion of multidrug-resistant
(MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) strains, which
make it very difficult to treat this disease (19, 43, 110). There are two
classifications of drugs that are used for the treatment of tuberculosis: first-line and
second-line drugs. The current treatment for tuberculosis recommended by the
WHO is the directly observed treatment, short course (DOTS), which consists of
four co-administered first-line drugs: rifampicin, isoniazid, pyrazinamide and
ethambutol that are given to the patient for six to nine months (9, 57). In the case
of MDR-TB strains that are resistant to isoniazid and rifampicin and/or resistant to
other drugs, the WHO recommends the DOTS-plus treatment that includes a
further treatment with first-line drugs plus the addition of second-line drugs, such
as quinolones, aminoglycosides, ethionamide and other peptides for an additional
2 years. However, the DOTS-plus treatment brings some major disadvantages
with it. First of all, the duration of the treatment and therefore the high costs of
treatment. Moreover, the problem of toxicity due to the second-line drugs plays a
major role (9). The biggest problem that arose recently however is the global
spread of XDR-TB strains, which are MDR-TB strains that are also resistant to two
major second-line drugs: aminoglycoside and fluoroquinolone (110). Another
problem is that DOTS is very personnel intensive and only one in five TB patients
worldwide receives DOTS treatment (83). The problem of MDR-TB and XDR-TB
strains plus the fact that current drugs only target active replicating tubercle bacilli
rather than bacilli in a NRP state makes it obvious that there is a major need for
new antitubercular drugs. As mentioned, no new drug has been discovered in the
last 40 years (101), which shows how difficult it is to design a new drug for the
treatment of this infectious disease. Another reason for why no new drug has been
discovered in the last four decades is that people believed that TB was cured and
therefore no new drugs were needed. However, currently there are seven potential
5
new anti-tuberculosis drugs in clinical trial (110). Of these, two substances,
gatifloxacin and moxifloxacin, both antibiotics belonging to the fluoroquinolone
family, target the DNA gyrase. Both agents are in Phase III clinical trials, however
their use will be to shorten the treatment to about 4 months and they cannot be
used to treat XDR-TB strains because of the problem of cross-resistance with
other fluoroquinolones (110). There are two nitroimidazole compounds, which are
currently in Phase II clinical trials and show in vitro activity against sensitive and
drug-resistant TB strains (110). The mode of action of these compounds has not
been completely defined; however, it has been shown that both compounds inhibit
mycolic acid synthesis in mycobacteria (80, 134). Another very promising
compound, which is currently in Phase II clinical trials, is TMC-207 (R207910), a
diarylquinoline. TMC-207 inhibits the synthesis of ATP in M. tuberculosis by
targeting the C subunit, encoded by atpE, of the respiratory ATP synthase (28).
1.4 Vaccines
Mycobacterium bovis [Bacille Calmette-Guérin (BCG)] is an avirulent strain and
the most widely used vaccine (30). BCG has been shown to be very efficient in
preventing tuberculosis disease in infants and children when administered at birth,
however clinical trials have demonstrated that the efficiency of BCG to protect
adults from active TB is rather low and is particularly poor in tropical and
subtropical regions (3, 30). Unfortunately, these are the regions with the highest
incidences of tuberculosis. The reasoning for this might be the pre-sensitisation of
adults with environmental mycobacteria that are fairly common in tropical areas or
areas with low hygienic standards and this might lead to less efficiency of the BCG
vaccine because it cannot provide an additive immune response (3, 30).
Therefore, the vaccine would be still highly effective in newborns as they have not
been exposed to environmental mycobacteria prior to their vaccination (30).
Consequently, there is a huge demand for new vaccines. Attenuated M.
tuberculosis mutant strains are new vaccine possibilities that are currently in
animal trails (77). Since BCG lacks several genes present in M. tuberculosis it has
been proposed that a live attenuated TB-causing strain containing a more
complete antigen selection would be more effective than BCG (30). Hinchey et al.
6
(52) found that vaccination with a M. tuberculosis ΔsecA2 mutant in guinea pigs
and mice lead to a higher resistance to M. tuberculosis challenge compared with
standard BCG vaccination. Protein-based vaccines are another promising new
vaccination method. Antigen candidates are expressed in Escherichia coli as
recombinant proteins and used to immunize animals with certain adjuvants. It has
been shown that this new vaccine delivered high protection in mice for M.
tuberculosis infection (154). However, recombinant proteins expressed in E. coli
might not always be useful as some mycobacterial proteins undergo posttranslational modifications that affect the immunological properties of the protein
itself (24, 30). DNA vaccines have also been tested in preclinical animal models
but none of the developed DNA vaccines have achieved protection greater than
the protection obtained with standard BCG vaccination (30). However, McShane et
al. (84) were able to show in Phase I clinical trials that the administration of a
recombinant modified vaccine virus Ankara (MVA) expressing antigen 85A
(MVA85A) amplified the immune response of vaccinees previously vaccinated with
BCG. Boosting vaccinations with MVA85A might offer a new strategy to achieve
enhanced and prolonged antimycobacterial immunity in humans (84).
Instead of trying to replace the already existing BCG vaccine, new strategies have
been approached called Prime-boost vaccination strategies that begin with a
vaccination of newborns using BCG. This first vaccination is then followed by a
booster vaccination in infancy, using recombinant proteins or viral vectors, which
will be repeated in the childhood and adolescence (126).
7
1.5 Physiology of Mycobacteria
1.5.1 The cell envelope of mycobacteria
Mycobacteria are classified as Gram-positive bacteria and like all actinobacteria,
they show a very complex cell wall that features a peptidoglycan-arabinogalactan
polymer with covalently bound mycolic acids, a large repertoire of extractable
lipids and pore forming proteins (5, 24, 94). The size of mycolic acids in the cell
wall of mycobacteria can reach up to 90 carbon atoms, compared to other species
of the actinobacteria family like Nocardia, Rhodoccocus and Corynebacterium
where the size of carbon atoms of mycolic acids does not exceed 66 carbon atoms
(24), which shows that the cell wall of mycobacteria is unusually thick compared to
most bacteria. The fact that most of the mycobacterial lipids are an essential part
of the cell wall of the organism makes mycobacteria highly resistant to a variety of
drugs, because those lipids form a very effective permeability barrier to many
diverse harmful compounds (17, 24). Barry et al. (5) were able to demonstrate that
there is a positive correlation between mycolic acid-containing glycolipids and the
virulence of mycobacteria. They showed that mycolic acid-containing glycolipids
influence the pathogenicity of M. tuberculosis and that a decrease in mycolic acid
biosynthesis leads to an increase of permeability and therefore causes a decrease
of virulence (24).
1.5.2 Nutrients for mycobacterial metabolism
It has been known for a long time that M. smegmatis is able to grow on an
enormous variety of carbon sources such as polyols, pentoses, and hexoses (35,
56). It has been shown that the genome of M. smegmatis harbours in total 28
permeases for carbohydrate uptake (142) compared to the genome of M.
tuberculosis, which only features five permeases for carbohydrate uptake (24, 95).
This information indicates that there are not many diverse sugars present for the
use of the pathogen in the phagosome. The results of several studies suggest that
a major source of carbon and energy for M. tuberculosis during infection might be
supplied by the use of fatty acids (117, 156). Phosphorous is needed as an energy
supply, but also for the biosynthesis of nucleic acids and phospholipids, and
8
various other cellular processes (24). For M. smegmatis, two high-affinity
phosphate uptake systems, the Pst system, encoded by the pstSCAB operon and
a second uptake system encoded by the phnDCE operon have been reported
(40). Furthermore, a third high-affinity phosphate transporter has been proposed
(40). M. tuberculosis features multiple copies of genes for components of the Pst
system in its genome (14, 41) and two of them have been shown to be virulence
factors (117). Amino acids are also taken up by mycobacteria and serve as a
carbon source and/or as a source of nitrogen (24). Recently, it has been
demonstrated that amino acids such as proline and glycine betaine are important
in the regulation of osmotic stress in M. tuberculosis, which has a glycine betaine
transporter, encoded by the proXVWZ operon (105).
In summary, it has been shown that mycobacteria can use many different carbon
sources, such as carbohydrates, fatty acids, and lipids or the carbon backbones of
amino acids.
1.5.3 Sensing of environmental factors
One of the most interesting features of mycobacteria is the ability to adapt to
various changes in their environment and to survive under the new conditions.
These changes vary from oxygen limitation (hypoxia), nutrient deprivation, several
exogenous stress conditions to shifts in pH. Therefore, mycobacteria possess
sensory elements that recognize the changes in environmental conditions (24).
The sequencing of the M. tuberculosis genome (22) revealed the presence of
several signal transduction systems, such as 11 complete two-component
regulatory systems, 14 eukaryotic type serine/threonine protein kinases (STPKs)
and phosphatases, and 10 extracytoplasmic function (ECF) sigma factors.
However, for most of these systems only little is known about what signal they are
actually responding to, highlighting a general lack of knowledge in this area (24).
Different mycobacterial species feature altered amounts of regulatory systems. For
examples the genome of M. tuberculosis contains 10 ECF sigma factors, M.
smegmatis possess 23 ECF sigma factors and the genome of M. leprae features
only 3 ECF sigma factors (23-24, 149). The reason for this might be in the specific
adaptations of different species to different environments they inhabit, i.e. M.
9
smegmatis is an environmental bacterium while M. tuberculosis is a pathogen and
resides within the host (granulomas).
1.5.3.1 Sensing oxygen availability and redox state of the cell
As mentioned above, mycobacteria are obligate aerobes and therefore need a
mechanism to detect variations in oxygen availability and adjust their metabolism
accordingly. Currently there are two known mechanisms by which mycobacteria
sense oxygen: the Dos system and WhiB3 (24).
The dormancy survival (Dos) system of M. tuberculosis consists of three proteins,
DosR (or DevR), DosS (or DevS) and DosT, which comprise a two-component
signalling system that is required to respond to hypoxia and nitric oxide stress
(112). Kumar et al. (69) demonstrated that DosS functions as a redox sensor while
DosT functions as a hypoxia sensor. Furthermore, it has been shown that DosR
acts as a transcriptional regulator and is regulated by the two sensor kinases
DosS and DosT (24, 69, 97). DosR and DosS homologues have also been found
in non-pathogenic mycobacteria such as M. smegmatis (82) and it has been
demonstrated that a M. smegmatis ΔdosR strain showed a decrease in survival
during adaptation to oxygen limitation in stationary phase (96).
A second oxygen and redox sensor in mycobacteria is thought to be WhiB3.
Recently, Singh et al. (125) demonstrated that the regulatory protein WhiB3
controls the differential production of lipids (e.g. triacylglycerol, sulfolipids) by
binding to the promoter regions of lipid biosynthetic genes in response to changes
in the intracellular redox environment. By this regulatory process, WhiB3 controls
fatty acid metabolism and maintains intracellular redox homeostasis by
channelling toxic reducing equivalents into lipid anabolism (125).
WhiB3 and the Dos system are the most studied systems regarding the sensing
abilities for oxygen tensions and redox state in mycobacteria, however, they are
probably not the only systems used by these bacteria. A recent study by Rustad et
al. (114) showed that about 50% of the DosR-dependent genes upregulated under
hypoxia return to baseline levels after only 24 h of oxygen limitation. More
10
interestingly, the authors were able to show that 230 genes were significantly
upregulated after 4 and 7 days of hypoxia, but not initially, and therefore named
this response the enduring hypoxic response (EHR). The regulation of these 230
genes was independent of DosR, indicating that there must be another sensory
mechanism in mycobacteria responsible for the detection of prolonged exposure to
hypoxia (114).
1.5.3.2 Sensing of oxidative stress
Oxidative stress caused by reactive oxygen species (ROS) such as peroxides and
free radicals can damage all components of the cell, including proteins, DNA and
lipids. Mycobacteria are obligate aerobes and therefore must be able to cope with
oxidative stress. For pathogenic mycobacteria it is even more important to
possess a mechanism to respond to oxidative stress, as macrophages produce
ROS, such as peroxides, hydroxyl radicals, superoxide and nitric oxide, as a selfdefence mechanism in response to bacterial infections (24, 31). In E. coli and
other enteric bacteria, the transcriptional regulators OxyR and SuxRS play central
roles in the defence against the endogenous oxidative damage associated with
active aerobic growth (44, 140). However, apart from M. avium and M. leprae, all
other mycobacterial genomes show several mutations in the oxyR homologues
resulting in the inactivation of this gene (24, 123). Also, no SuxRS system has
been
identified
in
mycobacteria.
Therefore,
mycobacteria
require
other
mechanisms to induce an oxidative stress response. Many studies have
investigated how mycobacteria respond to oxidative stress and a variety of
sensory and regulatory systems have been identified (78).
The expression of catalase-peroxidase (KatG) and iron-dependent superoxide
dismutase (SodA) in M. smegmatis have been shown to be related to the ironresponsive regulator IdeR (32). However, the mechanism of how katG and sodA
are regulated by IdeR in M. smegmatis remains unknown and in M. tuberculosis it
has been shown that IdeR does not play a role in the regulation of these two
genes (113). FurA, a ferric uptake regulator (22) encoded by a gene immediately
upstream of katG in all mycobacteria (88), has been shown to be involved in the
repression of katG of M. smegmatis and M. tuberculosis (106, 168). However, the
sensory mechanism for oxidative stress of FurA and how it represses the
11
expression of katG remain unidentified (24). Various alternative and ECF sigma
factors have been shown to play a role in the oxidative stress response in
mycobacteria, but not much is known about these defence mechanisms.
1.5.4 Respiratory chain in mycobacteria
Respiration in bacteria involves a series of energetically coupled electron and
proton transfers and translocations, which result in the formation of a protonmotive force (ΔµH+) across the inner bacterial membrane (127). During
mycobacterial growth, electrons enter the electron transport chain when
cytoplasmic electron carriers (substrates) are oxidized by membrane-bound
dehydrogenases leading to the formation of reduced cytoplasmic electron carriers,
e.g. NADH, FADH2, or ferredoxin (iron-sulfur proteins) (24, 85). Under aerobic
conditions, NADH oxidation in M. tuberculosis is carried out mainly by membranebound NADH dehydrogenases (Figure 2). NADH dehydrogenase transfers
electrons from NADH to quinones (e.g. uniquinone or menaquinone), which are
then reduced to quinols. M. tuberculosis features two types of NADH
dehydrogenases,
NDH-1
and
NDH-2
(155).
NDH-1,
encoded
by
the
nuoABCDEFGHIJKLMN operon, pumps protons across the membrane to
generate a ΔµH+ and therefore conserves energy. NDH-2, encoded by ndh and
ndhA, is a non-proton pumping dehydrogenase and hence does not conserve
energy (155). Electrons can also enter the electron transport chain via succinate
dehydrogenase, encoded by the sdhABCD operon. Succinate dehydrogenase is a
non-proton pumping dehydrogenase and oxidizes succinate to fumarate while
electrons are transferred to quinones. Under aerobic conditions, electrons that
have entered the menaquinone (oxidized state)- menaquinol (reduced state) pool
are transferred to one of the terminal oxidases, either to the cytochrome c pathway
or directly to the cytochrome bd oxidase (12, 61, 79). Terminal oxidases provide
the final step in aerobic respiration by reducing oxygen (85). Cytochrome bd,
encoded by the cydABDC operon, is bioenergetically less efficient and induced
under low oxygen levels (61, 79). The cytochrome c pathway consists of a bc1
cytochrome reductase, encoded by the qcrCAB operon, and a proton-translocating
aa3 cytochrome c oxidase, encoded by the ctaBCDE operon, that are believed to
form a supercomplex (79). This has been shown for other Actinomycetales such
12
as C. glutamicum (13). For M. smegmatis, an additional terminating oxidase for
the respiratory chain, a YthAB (bd-type) menaquinol oxidase, has been proposed
(61). In mycobacteria, ATP is synthesized via substrate level phosphorylation and
oxidative phosphorylation using the membrane-bound F1Fo-ATP synthases,
encoded by the atpBEFHAGDC operon. The F1Fo-ATP synthase in bacteria is
powered by a ΔµH+ generated by the proton-pumping components of the
respiratory chain (NADH dehydrogenase and aa3 cytochrome c oxidase). It has
been shown that the process of ATP generation by the membrane-bound F1FoATP synthase is essential for the survival of mycobacteria (117, 145). Moreover,
this enzyme has been the target of the new anti-tuberculosis drug (diarylquinoline
R207910, TMC-207). Recent research has highlighted the importance of metabolic
enzymes, especially those involved in the generation of energy under low oxygen
conditions. These enzymes could provide a new source of drug targets for
mycobacterial infections (91-92, 141, 145, 155). To date, it is unclear how a proton
gradient is generated in a low oxygen or hypoxic environment. Therefore, it is of
interest how mycobacteria power their F1Fo-ATP synthase under these conditions.
Two enzymes purportedly involved in the energy generation and adaptation to
hypoxic conditions are a respiratory nitrate reductase, encoded by the narGHIJ
operon (124, 131), and cytochrome bd oxidase, encoded by the cydABDC operon
(61). However, this proposal has not been proven yet at a biochemical level.
13
A
B
Figure 2: Proposed respiratory chain of M. tuberculosis under A) aerobic and B)
hypoxic conditions. Dashed line for nitrate reductase indicates the proposed
proton-pumping function of the enzyme to generate a membrane potential under
hypoxia. Taken from Cook et al., 2009.
14
1.5.5 Cytochrome bd
Cytochrome bd oxidase belongs to the widely distributed family of quinol oxidases
found in prokaryotes. Through the complete genome sequencing of M.
tuberculosis (22), a cydABDC cluster was found. The cydAB genes, encode for
subunit I (cydA) and subunit II (cydB) (70) and show high similarity to the
corresponding genes in E. coli. Cytochrome bd does not contain copper and the
two membrane-integrated subunits harbour three redox cofactors: two protoheam
IX groups (low spin heam b558 and high spin heam b595) and a chlorine (heme d)
(11). The cydD and cydC genes are located immediately downstream of cydB and
have been shown to be required for the formation of active cytochrome bd in E.
coli (104) and B. subtilis (158). These two genes encode an ABC transporter
which was reported to catalyze the export of L-cysteine and glutathione (102).
Cytochrome bd exhibits typical properties of bd oxidases, in that it is induced
under oxygen limitation (hypoxia) and mutants are more sensitive to cyanide (27,
85). It has been shown for E. coli and for several Bacillus species that cytochrome
bd is involved in energy-transducing respiration (58, 115). E. coli mutants that did
not possess a functional cytochrome bd oxidase showed lowered ability of the
organism to cope with oxidative stress which lead to increased sensitivity to higher
temperatures or peroxide (42). Furthermore, it has been demonstrated that
cytochrome bd is related to aerotolerant nitrogen fixation and protection against
metal toxicity and oxidative stress in Azotobacter vinelandii (36), which represents
an organism with the most active respiratory chain known where cytochrome bd is
thought to be the major pathway from ubiquinol to oxygen (58). Moreover, a
positive correlation between the virulence of the bacterial pathogen, Shigella
flexneri, and the cytochrome bd expression level has been shown (150). These
results suggest that cytochrome bd might be particularly important for the growth
and survival of pathogens that enter hypoxic environments.
15
On the basis of the acknowledged importance of the cytochrome bd system for
microaerobic growth (61, 131), strong regulatory circuits are expected to control
the expression of these genes in the absence of appropriate stimuli. E. coli
precisely controls cydAB expression using two global regulators that respond to
oxygen (FNR, ArcBA), but no homologues of these genes have been identified in
mycobacterial genomes (26, 103). For Streptomyces coelicolor A3(2), a redoxsensing repressor (Rex) that binds to operator sites located upstream of several
respiratory genes, including cydABCD, has been identified (16). It has been shown
that the DNA-binding activity of Rex is controlled by the redox poise of the
NADH/NAD+ pool (16). Rex homologues have been found in most Gram-positive
bacteria
including
pathogens
like
Staphylococcus
aureus
and
Listeria
monocytogenes, however this does not include mycobacterial genomes. It also
has been shown that cydABDC expression is independent of DosR, a
transcriptional regulator regulated by a redox sensor DosS and hypoxia sensor
DosT (114). In this study, these authors were able to show that the upregulation of
cydA is part of the enduring hypoxic response (EHR), however this response is
independent of DosR.
16
1.6 Aims of the study
Kana et al. (61) reported that the M. smegmatis cyd gene cluster coding for
cytochrome bd is transcriptionally regulated in response to oxygen concentration
of the growth medium. Induction of cytochrome bd in M. smegmatis occurs at
about 1% air saturation and this value is significantly lower than in E. coli where
cytochrome bd is already induced at an air saturation of 10% (61). Recently,
Berney and Cook (8) demonstrated that cytochrome bd in M. smegmatis is
upregulated 160-fold at 0.6% oxygen saturation compared to 50% oxygen
saturation, whereas the cytochrome bc1-aa3 supercomplex is only upregulated 6fold under the same conditions.
Cytochrome bd is not part of the Dos regulon and the signal transduction
pathways and molecular signals that activate cytochrome bd expression have not
been identified. Escherichia coli precisely controls cytochrome bd expression
using two global regulators that respond to oxygen (FNR, ArcBA), but no
homologues of these genes have been identified in mycobacterial genomes (26,
103-104). For Streptomyces coelicolor, a close relative of M. tuberculosis, a
redox-sensing repressor (Rex), that binds to operator sites located upstream of
several respiratory genes, including cydABCD, has been identified. Rex
homologues have been found in most Gram-positive bacteria, however this does
not include mycobacterial genomes.
The aims of this thesis are to identify the regulator(s) of cytochrome bd
expression in M. smegmatis and characterize the regulator(s) at a biochemical
and molecular level.
To address these aims, we will carry out the following objectives:
Objective 1: Identify genes controlling the expression of the cydABDC operon by
transposon mutagenesis in M. smegmatis harbouring a cydA-lacZ reporter gene.
Isolate mutants that either fail to activate expression of cydA-lacZ or show
constitutive expression (repressors).
Objective 2: Characterization of the genes at a biochemical and molecular level.
17
2. Materials and Methods
2.1 Bacterial strains and growth conditions
Escherichia coli strains were maintained on Luria-Bertani (LB) agar plates
containing (per liter): 10 g BactoTM tryptone (Becton Dickinson), 5 g BactoTM yeast
extract (Becton Dickinson), 10 g sodium chloride (Scharlau), 15 g bacteriological
agar (Coast Biologicals limited). Mycobacterium smegmatis strain mc2155 (128)
and derived strains (Table 1) were maintained on LB agar plates supplemented
with 0.05% (w/v) Tween-80 (Sigma-Aldrich) (LBT) and appropriate antibiotics. For
broth culture, E. coli was grown in LB-medium at 37°C. M. smegmatis was grown
at 37°C in either LBT-medium or in Hartmans de Bont minimum medium
supplemented with or without 0.2% (w/v) glycerol and/or L-proline (25 mM) as
carbon sources. The composition of Hartmans de Bont medium per liter was as
follows: 0.01 g EDTA, 0.1 g MgCl2 hexahydrate, 1 mg CaCl2 dihydrate, 0.22 mg
Na2MoO4 dihydrate, 0.4 mg CoCl2 hexahydrate, 1.22 mg MnCl2 tetrahydrate. 2 mg
ZnSO4 heptahydrate, 5 mg FeSO4 heptahydrate, 0.2 mg CuSO4 pentahydrate, 2 g
(NH4)2SO4, 0.05% (w/v) Tween-80, 3.75 g Na2HPO4 dihydrate and 1.5 g KH2PO4
trihydrate. Aerobic cultures (25 ml medium in a 250 ml flask) were inoculated to an
initial optical density (OD600) of 0.001 and grown with agitation (200 rpm) (New
Brunswick Scientific, TM1250-9700). Two ml samples to measure β-galactosidase
expression (see section 2.3) and the optical density were taken and stored at 20°C. Growth of M. smegmatis under hypoxic conditions was performed as
follows: cultures were grown in 120 ml sealed serum vials at 37°C with agitation
(200 rpm). Serum vials were inoculated to an initial optical density of 0.005. Two
ml samples to measure β-galactosidase expression (see section 2.3) were taken
and stored at -20°C. Medium was autoclaved at 121°C for 15 min before
inoculation. Solid selective media contained streptomycin (100 μg ml-1), kanamycin
(50 μg ml-1), spectomycin (50 μg ml-1) or hygromycin B (200 μg ml-1 for E. coli; 50
μg ml-1 for M. smegmatis).
The optical density at 600 nm (OD600) was measured in a Jenway 6300
spectrophometer. Cultures were diluted in 0.85% saline to bring the OD600 to
below 0.5 when measured in cuvettes of 1 cm light path length.
18
2.2 Transposon mutagenesis and cloning of constructs
All molecular biology techniques were carried out according to standard
procedures (116). Restriction enzymes and other molecular biology reagents were
obtained from Roche Diagnostics or New England Biolabs. All bacterial strains,
plasmids and primer sequences are listed in Tables 1 and 2. A mutant library was
generated by transposon mutagenesis performed by Dr. Michael Berney.
Therefore, strain M. smegmatis mc2155 with a transcriptional cydA-lacZ fusion
(pSM128) integrated into the chromosome (strain MB100) and Tn611 (pCG79)
were used as described previously (47, 146). To complement the transposon
mutants with the corresponding genes, MSMEG_5120 and MSMEG_5303 were
amplified from genomic DNA using the primers (comp_MSMEG_5120 and
comp_MSMEG_5303) listed in Table 2. The products were cloned into the multiple
cloning site of the pMind vector using SpeI and BamHI restriction and transformed
into the transposon mutants of M. smegmatis mc2155 (proR::Tn611 and
putP::Tn611).
19
Table 1: Bacterial strains and plasmids used in this study
Strain or plasmid
Description*
E. coli
DH10B
F¯mcrA Δ (mrr-hsdRMS-mcrBC) Ф80dlacZ ΔM15
ΔlacX74 deoR recA1 araD139 Δ(ara leu)7697 galU
galK rpsL endA1 nupG (49)
M. smegmatis
mc2155
electrocompetent wild type strain of M. smegmatis
(128)
MB100
mc2155 with a transcriptional cydA-lacZ fusion
integrated into the chromosome
proR::Tn611
MB100 with Tn611 insertion in MSMEG_5120
putP::Tn611
MB100 withTn611 insertion in MSMEG_5303
Plasmids
pCG79
Temperature sensitive plasmid containing Tn611
(47); Kmr, Strr, Spr
pSM128
Promotor probe vector, lacZ reporter gene; Strr, Spr
pMind
E. coli-mycobacteria shuttle vector with tetracycline
inducible promoter (10); Hygr, Kmr
* Kmr, kanamycin resistance; Strr, streptomycin resistance; Spr, spectomycin resistance;
Hygr, hygromycin resistance.
20
Table 2: Primers used in this study
Primers
Description*
Oligo_G
GAGCGACAGCCTACCTCTGACT
Oligo_F_trunc
GTTCCGTCCGTCCAATCTCC
MSMEG_5117fw
GTTCTGCAGGCCTACCTCAA
MSMEG_5117rev
AGGCACGTCAGGTACGAGTC
MSMEG_5119fw
CGCAGGCAACTTCTACATCA
MSMEG_5119rev
CTCCATGTGGGGATAGGTGT
MSMEG_5120fw
CGGATCTGTTCTTCCTCCTG
MSMEG_5120rev
TGTTCGAAGACGTGGTTGAC
MSMEG_5303fw
CCGAGCTTCTTCGAGAACAG
MSMEG_5303rev
AGCGTGTAGCACAACGTGAC
MSMEG_3232fw
GAACGACAGGTGGATCTGCT
MSMEG_3232rev
ATGTTCTCGGGCCTGTACCT
sigAfw
GACTCTTCCTCGTCCCACAC
sigArev
GAAGACACCGACCTGGAACT
comp_MSMEG_5120fw
AAATTTGGATCCGGAGGGAATATTGGCCCTCGA
CGCGACGCT
comp_MSMEG_5120rev
AAATTTACTAGTACGCCTCGCCGGTTAGTTCA
comp_MSMEG_5303fw
AAATTTGGATCCGGAGGGAATAATGTCCGACCT
CACGTTTCA
comp_MSMEG_5303rev
AAATTTACTAGTGATCCGGCGTGCCGGATTCA
* Primer sequences are given in 5’-3’ direction; restriction sites (underlined) and
ribosomal binding sites (dashed lines) are included in the primer sequences.
2.2.1 Screening for transposon mutants
The transposon library was plated out on LBT agar plates, containing bromochloro-indolyl-galactopyranoside (X-Gal) (40 μg ml-1) and appropriate antibiotics,
and incubated at 39°C for about 3 to 4 days. The plates were then put in an
anaerobic jar (AnaeroGen) and incubated overnight at 39°C. Colonies that were
unable to express cydA-lacZ (i.e. β-galactosidase) were white and picked on a
fresh LBT agar plate containing X-Gal and appropriate antibiotics and incubated
under aerobic conditions at 39°C for about 3 days. The plates were again put in an
21
anaerobic jar and incubated overnight at 39°C. For genomic DNA extraction, white
mutant colonies were then streaked out on a LBT agar plate and incubated at
39°C for 48 h.
2.2.2 Genomic DNA extraction
Cells were taken off an initial streak of plate, suspended (20 μg ml-1 cells wet
weight) in lysis buffer (4 M guanidinum thiocynate, 1 mM 2-mercaptoethanol, 10
mM EDTA, 0.1% Tween-80) and snap frozen in a dry ice/ethanol bath.
Suspension was then incubated at 65°C for 10 min, snap frozen and incubated
again at 65°C for 10 min. After incubation for 5 min on ice, the volume of the
suspension was increased to 500 μl with milliQ H2O and the aqueous phase
extracted twice with chloroform (300 μl). DNA was then precipitated using 500 μl
isopropanol. After centrifugation (16100 x g, 15 min, room temperature [RT]) the
pellet was washed with 70% ethanol and dried at 37°C for about 10 min. DNA was
resuspended in 20 μl milliQ H2O.
2.2.3 PCR amplification and Gel-extraction (GE)
To complement the transposon mutants with the corresponding genes,
MSMEG_5120 and MSMEG_5303 were amplified from genomic DNA using the
primers
(comp_MSMEG_5120fw,
comp_MSMEG_5120rev,
comp_MSMEG_5303fw and comp_MSMEG_5303rev) listed in Table 2 and the
Phusion High-Fidelity PCR Kit (New England Biolabs). PCR reactions (50 μl)
contained 10 μl 5x HF buffer, 1 μl 10 mM dNTP mix, 20 nM primer A and primer B,
100 ng μl-1 template DNA, 1.5 μl DMSO and 0.5 μl Phusion DNA polymerase. The
following PCR program steps were used: Step 1) Initial duration at 98°C for 30
sec, Step 2) Denaturation at 98°C for 10 sec, Annealing at 63°C (for
MEMEG_5303) and 63.1°C (for MSMEG_5120) for 30 sec (35 cycles), Extension
at 72°C for 1 min, Step 3) Final extension at 72°C for 10 min. The PCR products
were loaded on a 1% agarose gel; gel extracted overnight at 21 V and cleaned up
using a QIAEX II Gel Extraction Kit (QIAGEN) following the manufacturers
protocol.
22
2.2.4 Digest and Ligation
Purified PCR product (39 μl) was digested with 1 μl BamHI and 1 μl SpeI
restriction enzymes (Roche, 10 U μl-1) in a 50 μl reaction with 5 μl 10x buffer B and
4 μl H2O. For extracted DNA, a 20 μl reaction was set up with 1 μl restriction
enzyme EcoRI (Roche, 10 U μl-1) and 2 μl 10x buffer H. After incubation at 37°C
for 3 hours, a High Pure PCR product purification Kit (Roche) was used to clean
up the digested product. To ligate the PCR product with the pMind vector, a 20 μl
reaction with 50 ng vector, 100-150 ng insert, 2 μl 10x T4 Ligation buffer and 0.5 μl
T4 Ligase (400 U μl-1, New England Biolabs) were mixed and incubated overnight
at 12°C. For a self-ligation of the extracted and digested DNA the same amounts
of ligation reagents were used in a volume of 20 μl (containing approx. 400 ng
genomic DNA).
2.2.5 Electroporation of plasmid DNA
Electrocompetent cells were made using the protocol of Sheng Y et al. (122). M.
smegmatis strains (5 ml cultures in Hartmans de Bont supplemented with 0.2%
glycerol) or E. coli DH10B (500 ml culture in LB) were grown at 37°C with agitation
(200 rpm) to mid-log phase (OD600 0.5 - 0.7) and washed twice with cold 10%
glycerol equal to the original culture volume. Harvested cell pellets were
resuspended in 10% glycerol at a volume of 2 ml l-1 of original culture. Cold cell
suspension (50 μl) was added to 2 μl of ligation product and electroporated using
the following parameters: for E. coli 1.8 V, 25 μF and 200 Ω, for M. smegmatis 2.5
V, 25 μF and 1000 Ω. Immediately after transformation, 1ml medium was added
and the cultures incubated for 30 min at 37°C with 200 rpm agitation and then
plated on agar plates containing selective antibiotics to maintain the plasmids.
2.2.6 Alkaline lysis miniplasmid preparation
Overnight cultures (3 ml) were harvested and resuspended in 300 μl P1 buffer (50
mM Tris [pH 8.0], 50 mM EDTA and 100 μg ml-1 RNase). Lysis solution (300 μl,
0.2 M NaOH, 1% SDS) was added and mixed by inverting 6 times. Three M
potassium acetate/ 5% formic acid solution (pH ≈ 5.8) (300 μl) was added and
23
again mixed by inverting 6 times followed by centrifugation (16100 x g, 10 min,
RT). The plasmid was precipitated by adding 560 μl of isopropanol to 800 μl
supernatant following a centrifugation step (16100 x g, 15 min, RT). The pellet was
then washed in 70% ethanol, dried at 37°C for about 10 min and resuspended in
50 μl milliQ H2O. Plasmid preparations (2 μl) were then digested either with
restriction enzymes BamHI and SpeI (see section 2.2.4), for complementation, or
with restriction enzymes EcoRI and HindIII to check for a transposon insertion.
Plasmids that seemed to have a transposon insertion were sent for sequencing
(Allan Wilson Centre, Palmerston North) using the primers Oligo_G or
Oligo_F_trunc (Table 2).
2.3 β-Galactosidase assay
To measure β-galactosidase activities, cell pellets were resuspended in 5 μl 10%
Tween-80 and 1 ml Z-buffer (61 mM Na2HPO4, 39 mM NaH2PO4, 10 mM KCl, 1
mM MgSO4). Cells were permeabilized by adding 40 μl chloroform and 20 μl 0.1 %
(w/v) SDS and vortexing for 5 sec. Tubes were incubated at RT for 10 min and of
this suspension, 300 and 600 μl were used in each assay and the volume adjusted
to 1 ml with Z-buffer. The reaction was started by adding 200 μl 4 mg ml-1 ONPG
stock solution. Reactions were incubated at 37°C for 30 min and then stopped by
adding 500 μl 1 M sodium carbonate. Cell debris was removed by centrifugation at
16100 x g for 3 min and the absorption at a wavelength of 420 nm (A420)
determined using cuvettes of 1 cm light path length and a Jenway 6300
spectrophotometer. Z-buffer (1 ml) was used in the assay as a blank control.
Activity of β-galactosidase was expressed as Miller Units (MU) and was calculated
as the increase in A420 per minute, per 1 ml cell suspension (normalized to an
OD600 of 1.0) used, multiplied by a factor of 1000.
24
2.4 RNA isolation and reverse transcription (RT)-PCR
RNA was extracted from 6 ml of hypoxic broth culture grown for 48 hours in HdB
medium supplemented with 0.2% glycerol and with or without L-proline (25 mM).
Cells were harvested by centrifugation (16100 x g, 2 min, RT) and resuspended in
1 ml TRIzol reagent (Invitrogen). Cells were ruptured by bead-beating with 0.5 ml
zirconia beads (0.1 mm diameter) in a Mini-Beadbeater (Biospec) at 5000 rpm
three times for 30 sec. In between each bead-beating cycle, cells were cooled
down on ice for 30 sec. After centrifugation (16100 x g, 30sec, RT), the
supernatant was shaken with 200 μl chloroform for 15 sec and incubated at RT for
2 min while inverted occasionally. After centrifugation (16100 x g, 15 min, 4°C)
RNA was precipitated by incubation of the aqueous phase (clear) with 500 μl of
isopropanol for 10 min at RT and centrifuged at 12000 x g for 10 min at 4°C. The
pellet was washed in 1 ml of 75% ethanol (made up with diethylpyrocarbonate
[DEPC] treated water). After centrifugation (7500 x g, 5 min, 4°C), RNA was dried
at RT and dissolved in 50 μl DEPC water by incubation at 55°C for 10 min.
Contaminating DNA in RNA samples was removed with application of Turbo DNAfree (Ambion) as described in the manufacturer’s protocol. 10x Turbo DNase
buffer (5.5 μl) and 1 μl Turbo DNase were added to the dissolved RNA followed by
incubation at 37°C for 30 min. Then DNase inactivation reagent (6 μl) was added
and incubated at RT for 2 min. After centrifugation (10000 x g, 90 sec, RT) RNA
was quantified in the supernatant using a NanoDrop ND-1000 spectrophotometer.
The integrity of the DNA-free RNA extract was confirmed by using agarose gel
electrophoresis (1% agarose gels made up using 1 x TAE (Tris-acetate-EDTA)
buffer. RT-PCR reactions were performed using the SuperScriptTM III Reverse
Transcriptase Kit (Invitrogen) following the First-Strand cDNA Synthesis protocol.
Therefore, 1 μg total RNA, 250 ng random primers and 1 μl 10 mM dNTP mix were
used in a total of 13 μl reaction. The mixture was first incubated at 65°C for 5 min
followed by a incubation on ice for 2 min. 5 x First-Strand buffer (4 μl), 1 μl 0.1 M
DTT, 1 μl RNaseOUTTM Recombinant RNase Inhibitor and 1 μl SuperScriptTM III
Reverse Transcriptase were added and incubated at 25°C for 5 min. The RT step
was carried out at 50°C for 60 min followed by an inactivation step at 70°C for 15
min. cDNA was stored at -80°C.
25
2.5 Primer design and Real-Time-quantitative PCR (RT-qPCR)
RT-qPCR analysis was performed using Platinum® SYBR® Green qPCR
SuperMix-UDG Kit (Invitrogen) (containing internal ROX dye to normalise any
resulting fluorescence in each reaction) in a 7500 Fast Real-Time PCR System
(Applied Biosystems). Primers (Invitrogen) listed in Table 2 were designed using
the Primer3-web 0.3.0 software and primer concentrations were optimized using
combinations of 50 nM, 300 nM and 900 nM to ensure optimal amplification and
that no non-specific amplification occurred. The cycle threshold value (CT)
obtained for each gene of interest was normalized with that of sigA
(MSMEG_2758), a housekeeping gene with constant expression under different
experimental conditions as previously shown, in order to obtain the normalized CT
value. RT-qPCR analysis was performed using a reaction volume of 25 μl and the
following assay conditions: 1) 50°C for 2 min, 2) 95°C for 2 min, 3) Quantification
at 95°C for 15 sec and 60°C for 1 min for 40 cycles, 4) Dissociation at 95°C for 15
sec, 60°C for 1 min, 95°C for 15 sec and 60°C for 15 sec. Amplifications were
performed in at least triplicates with technical replicates that enabled the
calculation of the standard error of the mean (SEM). Contamination was
determined by using no template controls for each gene. Amplification of the
housekeeping control gene was performed on each plate for extra accuracy and
reliability in the quantization. To confirm the specificity of the primers a melting
curve analysis was performed after each run. 7500 Fast Real-Time PCR System
SDS software version 1.4 was used for the acquisition of data.
2.6 Microarray analysis
Microarray analysis was performed by Dr. Michael Berney using arrays provided
by the Pathogen Functional Genomics Research Center (PFGRC) funded by the
National Institute of Allergy and Infectious Diseases using protocols SOP# M007
and M008 from The Institute of Genomic Research (TIGR) (51). Total RNA (10 µg)
was used to create cDNA labelled with aminoallyl dUTP (Sigma). Fluorescent Cy3
and Cy5 dyes (GE Healthcare) were then covalently attached to the aminoallyl
tags. Each pair of differentially labelled probes was resuspended in 60 μl of
hybridization buffer (500 μl formamide, 250 μl 20 x SSC, 5 μl 10% SDS, 245 μl
26
ultrapure water) and hybridized to the microarray slide overnight in a 42°C water
bath. Slides were then washed in increasingly stringent wash conditions (3 times 5
min 1 x SSC 0.1% SDS, 3 times 5 min 0.1 x SSC 0.1% SDS, 3 x 5 min 0.1 x SSC
and a final dip in 0.22 μm-filtered MQ water). Arrays were scanned in a Genepix
4000A scanner and spots quantified with TIGR Spotfinder. The data was then
processed in TIGR MIDAS with total and Lowess normalization. Data from in-slide
replicate spots were averaged before expression ratios were calculated. The
results from 5 biological replicates including two dye swaps were then subjected to
a t-test without false discovery correction in TIGR MeV software. The analysis was
used as a ranking method. For a general overview, genes with expression ratios of
> 2 and < 0.5 and a p-value < 0.05 were used for data interpretation.
2.7 Proline transport assay
To measure proline transport in M. smegmatis strain MB100 and proR::Tn611 and
putP::Tn611 mutants, cells were grown in HdB medium (with 0.2% glycerol) with
and without L-proline (25 mM) in sealed serum vials to late exponential phase
(approximately 22 hours). Cultures were harvested by centrifugation (9820 x g, 15
min, 4°C) and washed twice in HdB medium (no carbon source added)
supplemented with 0.05% Tween-80 (7310 x g, 10 min, 4°C). Cells were
resuspended to a final OD600 of about 1.5 in a volume of about 35 ml. The cell
suspension was then incubated for 2 hours in a 25 ml flask at 37°C with agitation
(200 rpm) to de-energize the cells. After de-energizing, 20 mM glycerol was added
to the cell suspension to energize the cells. The transport of radioactive proline
into the mycobacterial cell was measured by adding a final concentration of 6.65
nM of L-[2,3,4,53H]proline (specific activity, 75 Ci mmol-1) (NENTM) and 1 µM of Lproline to 200 µl of cell suspension. The reaction was stopped after the samples
were incubated for 0, 15, 30, 60 and 120 sec at 37°C by the addition of 2 ml cold
0.1 M LiCl and rapid filtration through a nitrate cellulose filter (pore size 0.45 µm;
Millipore), using a vacuum manifold (Millipore) with an applied vacuum of
approximately 80 p.s.i. Filters were washed again with 2 ml 0.1 M LiCl, air-dried in
4 ml scintillation vials and covered in 2 ml scintillation fluid (Amersham).The
amount of radioactivity taken up by the cells was determined with a 1214
27
Rackbeta liquid scintillation counter (LKB Wallac) using the [3H]TPP- window and
counting each vial for 1 min. Internal concentrations of solutes taken up by the cell
membrane were calculated using a value of 3.45 µl of intravesicular volume per
mg of membrane protein (108).
2.8 Oxidative stress experiments
2.8.1 Culture experiments
Stress experiments were performed with mid-exponential phase (OD600 0.6 - 0.7)
cultures grown in serum vials with HdB medium supplemented with 0.2% glycerol
and with 25 mM L-proline. Five ml of culture were transferred into 125 ml flasks
and hydrogen peroxide was added at concentrations between 5 and 20 mM.
Cultures were incubated at 37°C for 2 h. Stress experiments were carried out in
triplicates. Survival was determined by counts of colony forming units (CFUs) on
LBT agar plates. Therefore, the CFUs of a positive control, cultures that were
incubated for 2 h, but not stressed (no hydrogen peroxide), represented the
number of 100% survival. CFUs of stressed cultures were compared to the
positive control to be able to calculate the survival (%) of the stressed cultures.
2.8.2 Disk diffusion assays
Assays were performed with mid-exponential phase (OD600 0.6 - 0.7) cultures
grown in serum vials with HdB medium supplemented with 0.2% glycerol and with
and without 25 mM L-proline. Cultures (150 µl) were plated onto HdB agar plates
supplemented with and without L-proline (25 mM). Filter disks (7mm diameter,
0.45 μm pore size) were placed at the centre of the agar plates, and aliquots (5 μl)
of 10 M hydrogen peroxide or 5.55 M methylglyoxal were deposited on the filter
disk. The diameters of growth inhibition areas were measured after incubation at
37°C for 2 days.
28
3. Results
3.1 Identification of transposon mutants
To elucidate the genetic determinants that regulate the expression of cydABDC
encoding cytochrome bd oxidase, Tn611-interrupted mutants of M. smegmatis
strain MB100 (cydA-lacZ) were screened to identify mutants that exhibit increased
or decreased cydA-lacZ activity compared to strain MB100. A cydA-lacZ promoter
fusion integrated at the attB site was used to differentiate between low promoter
activity (white colonies) and high promoter activity (dark blue colonies). Out of
45,000 mutants we screened, we found 25 transposon mutants with zero betagalactosidase activity (white colonies) under inducing conditions (microaerobic
jars) (Table 3), while no mutants with an increased beta-galactosidase expression
were found in our screen. Out of the 22 mutans, 14 mutans were carrying a
transposon insertion in the lacZ and only 8 mutans showed a transposon insertion
in other genes than the lacZ gene. Among these 8 mutants, we found two of
particular interest. One insertion was found in the gene MSMEG_5120 encoding a
conserved hypothetical protein (Figure 3) and another insertion was found in
MSMEG_5303, annotated as a sodium/proline symporter also called PutP (Figure
4). The gene MSMEG_5120 is upstream of the genes MSMEG_5119 and
MSMEG_5117 encoding for 1-pyrroline-5-carboxylate dehydrogenase (referred to
as PruA) and proline dehydrogenase (referred to as PruB) respectively (Figure 3).
The organization of these three genes is unique to mycobacteria and is highly
conserved also in the tuberculosis complex. A bacterial regulatory helix-turn-helix
domain of the LysR family type (Pfam) indicates that this gene encodes a
regulatory protein. We hypothesized that MSMEG_5120 (referred to as proR)
encodes a regulatory protein that regulates the expression of pruA and pruB,
which are predicted to be in an operon. Hence, our transposon mutagenesis
screen highlighted the involvement of proline metabolism in the regulation of
cytochrome bd activity. Other mutants we found had a transposon insertion in
gene MSMEG_0096 encoding for a peroxisomal hydrates-dehydrogenaseepimerase,
MSMEG_0817
encoding
for
a
LysR-transcriptional
regulator,
MSMEG_1902 encoding for a transcriptional regulator, MSMEG_3461 encoding
for catalase/peroxidise (katG), MSMEG_6053 encoding for a cob(III)yrinic acid a,cdiamide reductase and MSMEG_6923 encoding for a hypothetical protein.
29
Figure 3: Region view of MSMEG_5120 taken from The Institute of Genomic
Research (TIGR).
30
Figure 4: Region view of MSMEG_5303 taken from TIGR.
31
Table 3: Transposon mutants defective in cydA-lacZ expression
Gene locus of
transposon insertion
Gene
symbol
Gene description
Number
of Hits
MSMEG_0096
-
Peroxisomal hydratesdehydrogenase-epimerase
1
MSMEG_0817
-
LysR-transcriptional regulator
2
MSMEG_1902
-
Transcriptional regulator
1
MSMEG_3461
katG
Catalase/peroxidase HPI
2
MSMEG_5120
proR
Hypothetical protein (now
ProR)
1
MSMEG_5303
putP
Sodium/Proline symporter
(PutP)
2
MSMEG_6053
bluB
Cob(II)yrinic acid a,c-diamide
reductase
1
MSMEG_6923
-
hypothetical protein
1
3.2 Growth curves and cydAB expression
Mycobacterium smegmatis strain MB100 and both mutants proR::Tn611 and
putP::Tn611 were grown in LBT in flasks (data not shown) or serum vials (Figure
5). Under these conditions the mutant cultures started to clump considerably so
that OD measurements after 24 h cannot be considered as correct because of the
clumps in the medium. At the same time none of the mutants showed cydA-lacZ
activity (data not shown) compared to M. smegmatis strain MB100 which showed
cydA-lacZ activity of about 3 MU after 48 h. In order to see if this growth defect is
dependent on the medium, we grew all strains in Hartmans de Bont (HdB) minimal
medium with glycerol as the sole carbon source (Figure 6). All strains grew to the
same final OD without clumping but strain MB100 had a significantly faster growth
rate (Table 4). Both mutants had no cydA-lacZ activity. This indicates that proR
and putP are essential genes for optimal growth in the absence of proline. In
cultures with 25 mM proline added, strain MB100 grew to an OD600 of 2.5
compared to an OD600 of 2.0 for the two mutants, indicating that only strain MB100
32
is able to use proline as a growth substrate (Figure 7). When strain MB100 and the
mutants proR::Tn611 and putP::Tn611 were grown in HdB medium with proline as
the sole carbon source two phenotypes emerged (Figure 8). The proR::Tn611
mutant could not grow at all and the putP::Tn611 mutant exhibited a reduced
growth rate (0.025 ± 0.002 h-1) compared to strain MB100 (0.13 ± 0.012 h-1). This
indicates that ProR is essential for growth on proline and that PutP is important for
optimal growth.
Table 4: Summary of specific growth rates and final optical densities of the M.
smegmatis strain MB100 and mutants proR::Tn611 and putP::Tn611 grown under
various conditions
a*
Medium
HdB&
glycerol
HdB&
glycerol
&
proline
HdB&
proline
b*
specific growth
Final
rate (h-1)
OD600
specific growth
rate (h-1)
Final
OD600
MB100
0.20 ± 0.006
2.1 ± 0.08
0.22 ± 0.011
3.92 ± 0.16
proR::Tn611
0.17 ± 0.015
1.9 ± 0.07
0.18 ± 0.008
3.70 ± 0.14
putP::Tn611
0.17 ± 0.001
1.9 ± 0.16
0.19 ± 0.01
3.86 ± 0.19
MB100
0.22 ± 0.022
2.4 ± 0.03
n.m.*
n.m.*
proR::Tn611
0.17 ± 0.007
1.8 ± 0.16
n.m.*
n.m.*
putP::Tn611
0.18 ± 0.012
1.8 ± 0.09
n.m.*
n.m.*
MB100
0.13 ± 0.012
2.02 ± 0.1
n.m.*
n.m.*
proR::Tn611
< 0.05
0.08 ± 0.01
n.m.*
n.m.*
putP::Tn611
0.03 ± 0.002
0.76 ± 0.16
n.m.*
n.m.*
Strain
* a = serum vials, b = flasks, n.m. = not measured
To validate the transposon insertions being responsible for the observed
phenotypes, we complemented both mutations proR::Tn611 and putP::Tn611 with
the gene reinserted on the tetracycline inducible vector pMind. The complemented
strains both grew on HdB medium supplemented with proline as a sole carbon
source to strain MB100 levels (Figure 9).
33
A
B
Figure 5: Growth (A) and β-galactosidase expression from cydA-lacZ fusion (B) of
M. smegmatis strain MB100 (solid squares) and proR::Tn611 (solid triangles) and
putP::Tn611 (solid inverted triangles) mutants in Luria-Bertani (LB) medium
supplemented with 0.05% (w/v) Tween-80 (LBT) in sealed serum vials over the
course of time. The error bars in (A) indicate the standard deviation (SD) and the
error bars in (B) indicate the error of the mean (SEM) of two technical replicates
from three independent experiments.
34
A
B
Figure 6: Growth (A) and β-galactosidase expression from a cydA-lacZ fusion (B)
of M. smegmatis strain MB100 (solid squares) and proR::Tn611 (solid triangles)
and putP::Tn611 (solid inverted triangles) mutants in HdB minimal medium
supplemented with glycerol in sealed serum vials over the course of time. The
error bars in (A) indicate the standard deviation (SD) and the error bars in (B)
indicate the error of the mean (SEM) of two technical replicates from three
independent experiments.
35
A
B
Figure 7: Growth (A) and β-galactosidase expression from cydA-lacZ fusion (B) of
M. smegmatis MB100 (solid squares) and proR::Tn611 (solid triangles) and
putP::Tn611 (solid inverted triangles) mutants in HdB minimal medium
supplemented with glycerol and L-proline (25 mM) in sealed serum vials over the
course of time. The error bars in (A) indicate the standard deviation (SD) and the
error bars in (B) indicate the error of the mean (SEM) of two technical replicates
from three independent experiments.
36
A
B
Figure 8: Growth (A) and β-galactosidase expression from cydA-lacZ fusion (B) of
M. smegmatis strain MB100 (solid squares) and proR::Tn611 (solid triangles) and
putP::Tn611 (solid inverted triangles) mutants in HdB minimal medium
supplemented with L-proline (25 mM) as a sole carbon source in sealed serum
vials over the course of time. The error bars in (A) indicate the standard deviation
(SD) and the error bars in (B) indicate the error of the mean (SEM) of two technical
replicates from three independent experiments.
37
putP
A
proR
B
Figure 9: (A) Growth of M. smegmatis strain MB100 (solid squares) and
putP::Tn611 (solid triangles), both carrying an empty pMind vector as a control,
and complemented putP::Tn611 (solid inverted triangles) mutants over the course
of time. (B) Growth of M. smegmatis strain MB100 (solid squares) and
proR::Tn611 (open squares), both carrying an empty pMind vector as a control,
and complemented proR::Tn611 (open triangles) mutants over the course of time.
When the complemented proR::Tn611 mutant culture (open triangles) reached an
OD600 of about 0.75, we used that culture to re-inoculated fresh medium and
measured again the growth over the course of time (open diamonds) to see if this
will shorten the long lag-phase observed in the growth of the complemented
proR::Tn611 (open triangles). Cultures were grown in HdB minimal medium
supplemented with L-proline (25 mM) as a sole carbon source, hygromycin B and
tetracycline in sealed serum vials. The error bars in (A) indicate the standard
deviation (SD) and the error bars in (B) indicate the error of the mean (SEM) of
two technical replicates from three independent experiments.
38
3.3 Proline transport assays
To validate the proposed transport function of PutP, transport studies were
conducted with radioactively labelled L-proline. Cultures were grown on HdB
minimal medium supplemented with glycerol with or without proline. Figure 10
shows the rate of proline transport for M. smegmatis strain MB100 and mutants
proR::Tn611 and putP::Tn611. When strain MB100 was grown in medium without
proline,
the
proline
transport
rate
reached
a
value
of
about
3 nmol.min-1(mg protein) compared to the proline transport rate of about 23
nmol.min-1(mg protein) when strain MB100 was grown in medium supplemented
with proline (Figure 10). While the proR::Tn611 mutant showed a diminished
proline uptake rate in the presence of proline, almost no uptake was detected in
the putP::Tn611 mutant even when grown in medium supplemented with proline.
The putP gene was annotated as a sodium/proline symporter and therefore we
studied the effect of monensin, a sodium ionophore that would dissipate the
sodium gradient across the cell membrane. When strain MB100 cells were
incubated with monensin, the rate of proline uptake was reduced to 1 and 11%
depending on the presence of proline.
Figure 11 shows the rate of proline transport as a function of proline concentration
for strain MB100 and putP::Tn611 mutant when grown HdB in medium
supplemented with proline. As a negative control, we also measured the proline
transport rate of strain MB100 pre-incubated with CCCP (2.5 mM), a protonophore
that dissipates the membrane potential. The apparent Km of the proline uptake
system in strain MB100 is 2.5 µM with a Vmax of 100 nmol.min-1(mg protein) as
shown in Figure 12. This indicates that PutP is a high affinity proline uptake
system in M. smegmatis as previously described for other organisms, e.g. E. coli
and S. aureus.
39
Figure 10: Rate of proline transport of the M. smegmatis strain MB100 and
mutants proR::Tn611 and putP::Tn611. Cell suspensions were prepared from
cultures grown in HdB minimal medium supplemented with glycerol and plus or
minus proline in sealed serum vials to late exponential phase (approx. 22 hours).
The uptake of radioactive proline into the mycobacterial cells was measured after
the samples were incubated with proline for 0, 15, 30, 60 and 120 sec at 37°C.
The rate of proline transport per minute was calculated out of the values of the
linear range for the proline uptake. The error bars indicate the standard deviation
(SD) of three independent experiments.
40
Figure 11: Rate of proline transport of the M. smegmatis strain MB100 (solid
squares), strain MB100 plus CCCP (solid diamonds) and putP::Tn611 (solid
triangles) mutant. Cell suspensions were prepared from cells grown as described
in Figure 10. The uptake of radioactive proline into the mycobacterial cells was
measured after the samples were incubated with different concentrations of proline
for 0, 15 and 60 sec at 37°C to determine the maximal rate of proline transport.
The rate of proline transport per minute was calculated out of the values from the
linear range for the proline uptake. M. smegmatis strain MB100 cell suspensions
were incubated with CCCP (2.5 mM), as a control, for 10 minutes prior the
performance of the transport assay. Representative data is shown.
41
Figure 12: Lineweaver-Burke plot of M. smegmatis strain MB100 proline transport
system. The Km and Vmax of the proline transport system were calculated using the
values from Figure 11.
42
3.4 Regulation of putP, proR and cydAB
We compared the transcriptional response of M. smegmatis strain MB100 and the
mutants putP::Tn611 and proR::Tn611 growing on HdB minimal medium
supplemented with 0.2% glycerol and with 25 mM proline using RT-qPCR (Figure
13A). Compared to strain MB100, expression of pruA, pruB, proR and putP was
low in the proR::Tn611 mutant. Cytochrome bd expression was downregulated 2fold, which corresponded well with the approx. 2-fold increased promoter activity in
strain MB100 determined with β-galactosidase assays (Figure 7). In the
putP::Tn611 mutant, only putP was significantly downregulated compared to strain
MB100. In the absence of proline as an inducer, we found pruA and pruB were
significantly downregulated in the proR::Tn611 mutant compared to strain MB100,
but no other gene showed a significant induction or repression (Figure 13B). No
significant difference in gene expression was noted between the putP::Tn611
mutant compared to strain MB100 when the cultures were grown in the absence of
proline (Figure 13B).
43
A
B
Figure 13: Gene expression ratios comparing proR::Tn611/MB100 (white bars)
and putP::Tn611/MB100 (black bars). RNA for RT-qPCR was extracted from
hypoxic broth cultures grown for 48 hours in the HdB minimal medium
supplemented with glycerol plus (A) and minus (B) proline. Primers were designed
to determine gene expression of proline dehydrogenase (pruB), 1-pyrroline-5carboxylate dehydrogenase (pruA), proR, Sodium/proline symporter (putP) and
subunit II of cytochrome bd (cydB). Amplifications were performed in triplicates
and the standard error of the mean (SEM) is shown calculated using the ΔΔCt
method. ** p-value ≤ 0.01, * p-value ≤ 0.05
44
3.5 Microarray of proR::Tn611 mutant
In order to gain more information about the regulon controlled by ProR and provide
insight into the observed phenotypes, we conducted a whole genome microarray
analysis comparing M. smegmatis strain MB100 with the proR::Tn611 mutant.
Both strains were grown in serum vials (4 replicates) on HdB medium
supplemented with 0.2% glycerol and 25 mM proline, and cells were harvested in
late exponential phase when oxygen started to become limiting. The microarray
data confirmed the RT-qPCR results for pruA, pruB, proR and putP and cydAB
(Table 7). In total, we found 877 genes with induction ratios (IR) significantly
different from 1 (p ≤ 0.05). Of these, 52 genes had and IR < 0.5 and 181 genes an
IR > 2. All
877 genes with induction ratios (IR) significantly different from 1
(p ≤ 0.05) are listed in Table 8 (see appendix).
The most striking result was the upregulation of almost the entire DNA
repair/metabolism machinery (53 genes) in the proR::Tn611 mutant (Table 5),
including the alternative sigma factor SigG (MSMEG_0219) and the transcription
factor NrdR (MSMEG_2743), two regulators purportedly involved in the regulation
of DNA repair and de novo DNA synthesis. Additionally, we found five glyoxalase
family proteins strongly induced (Table 6). This pointed to DNA damage and the
involvement of methylglyoxal, because glyoxalases are known to be part of the
methylglyoxal degradation pathway. Apart from pruA, pruB, proR and putP and
cydAB, we found several genes in phosphate transport significantly downregulated
(Table 7). Additionally, two gene couples encoding for MmpS5 protein/MmpL4
protein and MmpS5 protein/MmpL5 were significantly downregulated (Table7).
45
Table 5: Upregulated DNA metabolism genes of proR::Tn611 mutant compared
to strain MB100 in microarray analysis
M.
smegmatis
locus
M. tb
H37Rv
locus
>40%
similarity
MSMEG_0003
Rv0003
MSMEG_0005
Rv0005
MSMEG_0006
Rv0006
MSMEG_0219
Rv0182c
MSMEG_0457
n.h.*
MSMEG_1254
Gene
symbol
IR
recF
3.45
0.04
DNA gyrase, B subunit
gyrB
2.63
0.01
DNA gyrase, A subunit
gyrA
1.68
0.05
sigG
11.29
0.02
DNA topoisomerase IV subunit B
-
4.72
0.03
n.h.*
DEAD/DEAH box helicase
-
5.28
0.02
MSMEG_1275
n.h.*
HNH nuclease, putative
-
2.49
< 0.01
MSMEG_1325
Rv0629c
recD
3.16
0.02
MSMEG_1328
Rv0631c
recC
4.10
< 0.01
MSMEG_1383
Rv0670
endonuclease IV
-
3.72
0.01
MSMEG_1656
n.h.*
exodeoxyribonuclease III
xth
4.03
< 0.01
MSMEG_1756
Rv3297
-
3.00
0.05
MSMEG_1757
Rv3296
DEAD/DEAH box helicase
-
4.62
0.02
MSMEG_1941
Rv3202c
helicase, UvrD/Rep family protein
-
8.34
< 0.01
MSMEG_1943
Rv3201c
ATP-dependent DNA helicase
-
13.74
< 0.01
MSMEG_2277
Rv3062
DNA ligase I, ATP-dependent
dnl1
7.31
0.02
MSMEG_2362
Rv3014c
DNA ligase, NAD-dependent
ligA
8.18
0.05
MSMEG_2403
Rv2973c
ATP-dependent DNA helicase RecG
recG
2.20
0.04
MSMEG_2723
Rv2737c
protein RecA
recA
6.18
< 0.01
MSMEG_2724
Rv2736c
regulatory protein RecX
recX
8.86
< 0.01
MSMEG_2740
Rv2720
LexA repressor
lexA
3.74
0.01
MSMEG_2742
Rv2719c
DNA-damage-inducible protein
-
7.82
0.01
MSMEG_2743
Rv2718c
nrdR
3.52
0.01
MSMEG_2944
Rv2593c
holliday junction DNA helicase RuvA
ruvA
4.33
< 0.01
MSMEG_3078
Rv1420
excinuclease ABC, C subunit
uvrC
2.05
0.01
MSMEG_3172
Rv1537
DNA polymerase IV 1
-
2.11
0.04
MSMEG_3178
Rv1547
DNA polymerase III alpha subunit
-
3.20
< 0.01
MSMEG_3404
Rv2015c
HNH endonuclease domain protein
-
18.00
0.01
Gene description
DNA replication and repair protein
RecF
RNA polymerase sigma-70 factor,
family protein
exodeoxyribonuclease V, alpha
subunit
exodeoxyribonuclease V, gamma
subunit
endonuclease VIII and dna nglycosylase with an ap lyase activity
transcriptional regulator, NrdR family
protein
pvalue
46
M.
smegmatis
locus
M. tb
H37Rv
locus
>40%
similarity
Gene description
Gene
symbol
IR
MSMEG_3808
Rv1638
excinuclease ABC, A subunit
uvrA
4.11
0.01
MSMEG_3816
Rv1633
excinuclease ABC, B subunit
uvrB
6.85
< 0.01
MSMEG_3839
Rv1629
DNA polymerase I
-
3.54
0.01
MSMEG_3907
Rv2119
RecB family protein exonuclease
-
16.20
0.05
MSMEG_4259
Rv2191
DNA polymerase III, epsilon subunit
-
6.75
0.02
MSMEG_4491
Rv2362c
DNA repair protein RecO
recO
2.82
< 0.01
MSMEG_4691
n.h.*
HNH nuclease
-
2.30
0.04
rdgB
5.81
< 0.01
pvalue
non-canonical purine NTP
MSMEG_4899
Rv1341
pyrophosphatase, RdgB/HAM1 family
protein
MSMEG_5004
Rv1277
DNA repair exonuclease
-
18.00
0.01
MSMEG_5044
Rv1251c
ATPase (RecB family-like nuclease)
-
10.09
0.01
MSMEG_5082
Rv1210
DNA-3-methyladenine glycosylase I
tag
7.32
< 0.01
MSMEG_5534
Rv0949
ATP-dependent DNA helicase PcrA
pcrA
3.75
0.02
MSMEG_5583
Rv2100
HNH endonuclease
-
4.95
0.02
MSMEG_5706
Rv0861c
-
3.18
0.01
MSMEG_5876
n.h.*
H-N-H endonuclease F-TflIV
-
6.88
0.02
MSMEG_5935
Rv3585
ATP-dependent DNA helicase
-
5.97
< 0.01
MSMEG_6079
Rv3585
DNA repair protein RadA
radA
7.47
0.02
MSMEG_6080
Rv3586
-
5.70
< 0.01
MSMEG_6187
Rv3674c
nth
2.91
0.02
MSMEG_6285
Rv3721c
-
2.51
< 0.01
MSMEG_6443
Rv1537
dinP
4.05
0.04
DNA or RNA helicase of superfamily
protein II
conserved hypothetical protein (DNA
integrity scanning protein DisA)
endonuclease III
DNA polymerase III gamma/tau
subunit
DNA polymerase IV
* n.h. = no homologue
47
Table 6: Upregulated methylglyoxal synthesis and detoxification genes of
proR::Tn611 mutant compared to strain MB100 in microarray analysis
M.
smegmatis
locus
M. tb
H37Rv
locus
>40%
similarity
Gene description
MSMEG_0884
n.h.*
glyoxalase family protein
-
2.05
0.04
MSMEG_1417
Rv0689c
glyoxalase family protein
-
2.35
0.03
MSMEG_5216
n.h.*
glyoxalase family protein
-
15.15
0.03
MSMEG_5680
Rv0887c
glyoxalase family protein
-
4.73
0.01
MSMEG_5827
Rv0801
glyoxalase family protein
-
6.43
< 0.01
MSMEG_2402
Rv2974c
dihydroxyacetone kinase
-
2.72
0.05
Gene
symbol
IR
pvalue
*n.h. = no homologue
Table 7: Downregulated genes of proR::Tn611 mutant compared to strain MB100
in microarray analysis
M.
smegmatis
locus
M. tb
H37Rv
locus
>40%
similarity
MSMEG_5119
Rv1187
MSMEG_5117
Rv1188
MSMEG_5303
n.h.*
MSMEG_3232
Rv1622c
MSMEG_6919
Rv0040c
MSMEG_0649
Gene
symbol
IR
pvalue
pruA
0.02
< 0.01
proline dehydrogenase
pruB
0.03
< 0.01
sodium/proline symporter
putP
0.12
< 0.01
cydB
0.62
0.01
proline-rich 28 kDa antigen
-
0.38
< 0.01
Rv1680
phosphonate-binding periplasmic protein
phnD
0.42
0.02
MSMEG_0936
Rv0490
sensor histidine kinase SenX3
senX3
0.54
0.02
MSMEG_0937
Rv0491
DNA-binding response regulator RegX3
regX3
0.47
< 0.01
MSMEG_1064
Rv0545c
phosphate/sulphate permease
pit
0.58
0.02
MSMEG_5780
Rv0930
pstA
0.48
< 0.01
MSMEG_5781
Rv0929
pstC
0.29
< 0.01
MSMEG_5782
Rv0928
pstS
0.27
< 0.01
MSMEG_0225
Rv0676c
MmpL4 protein
-
0.50
< 0.01
MSMEG_0226
Rv0677c
MmpS5 protein
-
0.50
0.01
MSMEG_1381
Rv0677c
MmpS5 protein
-
0.22
0.01
MSMEG_1382
Rv0676c
MmpL5 protein
-
0.14
< 0.01
Gene description
1-pyrroline-5-carboxylate
dehydrogenase
cytochrome D ubiquinol oxidase, subunit
II
phosphate ABC transporter, permease
protein PstA
phosphate ABC transporter, permease
protein PstC
phosphate ABC transporter, phosphatebinding protein PstS
48
3.6 Sensitivity to methylglyoxal, ROS stress and NaCl
The microarray data demonstrated the upregulation of DNA repair genes in the
proR::Tn611 mutant and this suggested that the cells were experiencing DNA
damage. Therefore, we investigated what causes the DNA damage in the mutants
and conducted stress experiments with H2O2 and methylglyoxal. Cultures of M.
smegmatis strain MB100 and
proR::Tn611 and putP::Tn611 mutants were
exposed to different concentrations of H2O2 in a growing culture stress experiment
(Figure 14) and were exposed to 10 M H2O2 in filter disk assays (Figure 15). None
of the mutants were more sensitive to H2O2 than strain MB100 and therefore we
repeated the filter disk assays using methylglyoxal and found a significant
difference in sensitivity to methylglyoxal between the mutants and strain MB100.
On plates supplemented with proline, the difference in inhibition zone between
strain MB100 and proR::Tn611 or putP::Tn611 mutants was more than 1.5 cm
indicating that proline plays an important role in the protection against
methylglyoxal toxicity (Figure 16). In the absence of proline, sensitivity of strain
MB100 increased significantly, although not to the same level as the two mutants
(proR::Tn611 and putP::Tn611) (Figure 16).
Proline is a known osmoprotectant in bacteria and plants. Therefore, we
investigated if the growth defect of mutant cultures (proR::Tn611 and putP::Tn611)
grown in LBT medium could be rescued by lowering the salt concentration in the
medium. We demonstrated that in LBT medium with lower salt concentrations the
mutants reached a higher final OD600 (about 1.25), compared to the final OD600
(about 0.7) reached when grown in LBT medium with higher salt concentrations.
However, both mutants could not restore to MB100 levels (OD600 = 2.0) when
grown in LBT medium with lower salt concentrations (Figure 17). Increased proline
concentrations in LBT medium did also not lead to optimal growth of the mutants
compared to MB100.
49
Figure 14: Survival of M. smegmatis strain MB100 (white bars) and proR::Tn611
(lined bars) and putP::Tn611 (black bars) mutants after cultures have been
stressed with 5 mM, 10 mM and 20 mM hydrogen peroxide. Mid-exponential
phase cells growing in HdB minimal medium supplemented with glycerol and
proline were exposed to different concentrations of hydrogen peroxide. After 2 h
incubation, cultures were plated out onto agar plates to determine the CFUs.
CFUs of a positive control (cultures that were incubated for 2 h, but not stressed
with hydrogen peroxide), represented the number of 100% survival. CFUs of
stressed cultures were compared to the positive control to be able to calculate the
survival (%) of the stressed cultures. The error bars indicate the standard error of
the mean (SEM) of two plates for each strain from three independent experiments.
50
Figure 15: Hydrogen peroxide sensitivity of M. smegmatis strain MB100 and
proR::Tn611 and putP::Tn611 mutants as determined by disk diffusion assays.
Mid-exponential phase cells growing in HdB minimal medium supplemented with
glycerol and proline were plated on HdB agar plates (supplemented with glycerol
and proline). Filter disks (7mm diameter, 0.45 μm pore size) were placed on each
agar plates and 5 µl of 10 M hydrogen peroxide was added to each disk. The
diameters of growth inhibition areas were measured after incubation at 37°C for 2
days. The error bars indicate the standard error of the mean (SEM) of three plates
for each strain from three independent experiments.
51
Figure 16: Methylglyoxal sensitivity of the M. smegmatis strain MB100 and
proR::Tn611 and putP::Tn611 mutants as determined by disk diffusion assays.
Mid-exponential phase cells growing in HdB medium supplemented with glycerol
and plus and minus proline were plated on HdB agar plates (supplemented with
glycerol and plus and minus proline). Filter disks (7mm diameter, 0.45 μm pore
size) were placed on each agar plates and 5 µl of 5.55 M methylglyoxal was added
to each disk. The zone of inhibition was measured after incubation at 37°C for 2
days. The error bars indicate the standard error of the mean (SEM) of three plates
for each strain from three independent experiments. ** p-value ≤ 0.01,
* p-value ≤ 0.05
52
A
B
C
Figure 17: Growth of M. smegmatis strain MB100 (A) and proR::Tn611 (B) and
putP::Tn611 (C) mutants in Luria-Bertani (LB) medium with 10g/l (solid squares), 1
g/l (solid triangles) or 0.1g/l (solid inverted triangles) of NaCl supplemented with
0.05% (w/v) Tween-80 (LBT) and in sealed serum vials over the course of time.
The error bars indicate the standard deviation (SD) from three independent
experiments.
53
4. Discussion
This study aimed to identify possible regulators of the high affinity terminal oxidase
cytochrome bd in Mycobacterium smegmatis. Two of the mutants we identified
showed the transposon insertion in katG (MSMEG_3461). The katG gene encodes
a catalase-peroxidase and catalyzes the decomposition of hydrogen peroxide to
water and oxygen (20). Therefore, katG plays an important role for the protection
against oxidative stress in the cell (88). That is why the katG::Tn611 mutant
experiences more oxidative stress than strain MB100 and therefore expresses
less cytochrome bd (i.e. cydA-lacZ). Another mutant we found in the transposon
screen showed an insertion in the Cob(II)yrinic acid a,c-diamide reductase, bluB
(MSMEG_6053). Recent studies discovered that bluB is necessary for the
biosynthesis of 5,6-dimethylbenzimidazole (DMB), the lower ligand of vitamin B12
in Sinorhizobium meliloti (136). These authors were able to show that BluB
resembles an NAD(P)H-flavin oxidoreductase and triggers the fragmentation and
contraction of the bound flavin mononucleotide cofactor to form DMB and Derythrose 4-phosphate (136), while oxygen gets reduced to water. A BluB
homologue of S. meliloti also exists in mycobacteria (136). Like a mutation in katG,
a mutation in bluB leads to a more oxidised state of the cytoplasm of the cell and
therefore results in the failure to activate cydA-lacZ. We found four transposon
mutants
(1
x
MSMEG_0096::Tn611,
2
x
MSMEG_0817::Tn611,
1
x
MSMEG_1902::Tn611, 1 x MSMEG_6923::Tn611) which we cannot explain why
these mutants are not able to express cydA-lacZ as there is no literature available.
More interestingly, we found three transposon mutants (2 x putP::Tn611 and 1 x
proR::Tn611) that failed to activate the expression of cytochrome bd and showed a
mutation in the proline metabolism pathway. We therefore isolated these mutants
to be able to do further investigations.
Proline is an amino acid and is known to accumulate in many bacterial and plant
cells in response to osmotic stress and acts as an osmoprotectant (137). The first
step of proline metabolism requires the uptake of this amino acid into the cells.
Most bacteria feature several proline transport systems that show differences in
their affinity for proline. PutP is a high affinity proline symporter, which has been
shown to be solely responsible for the symport of L-proline and sodium or lithium
54
ions (21, 48, 59). PutP of M. smegmatis shows homology to the well characterized
PutP protein from Staphylococcus aureus, which has been shown to be a highaffinity proline transporter (144) and important for virulence (6, 121). PutP has
been shown to be a high-affinity proline transporter in various bacterial species.
These include the Gram-positive Corynebacterium glutamicum (98-99), which is a
close relative to mycobacteria, and also Gram-negative bacteria like Escherichia
coli (160, 165) and Pseudomonas putida (148). In this study, we characterized the
sodium/proline symporter PutP in M. smegmatis as a high-affinity transporter for
proline with a Km of 2.5 µM. This is comparable to a Km of 1.74 to 7 µM for PutP in
S. aureus (4, 144) and a Km of 2 µM for PutP in E. coli (164). M. smegmatis
appears to be the only mycobacterial species to harbour a high-affinity Na+/proline
symporter. Mycobacterium tuberculosis lacks homologues of putP in its genome.
Recently, proXVWZ, annotated as encoding a putative glycine betaine or proline
transporter, has been shown to protect M. tuberculosis against osmotic stress and
thereby provides a growth advantage in vitro and in macrophages (105). However,
the results from this study indicate that PutP is not the only proline transporter in
M. smegmatis. A putP::Tn611 mutant showed slow growth compared to strain
MB100 in a minimal medium supplemented with proline as a sole carbon source
and was also able to transport low amounts of proline into cells, as was
demonstrated in proline transport assays. This suggests that M. smegmatis must
feature at least one low-affinity proline transporter. E. coli has two more proline
transporter proteins, ProP and ProU, however, both proteins transport glycinebetaine and other betaines, in response to osmotic stress (59, 162). ProU, an
ATP-binding cassette (ABC)-type carrier, is a high-affinity glycine betaine
transporter and shows only low affinity for proline (15). Both genes, proP and
proU, are regulated by external osmolarity (74, 99). S. aureus also features ProP
as a low-affinity proline uptake system other than the high-affinity symporter PutP
(4, 120). Peter et al. (99) demonstrated that C. glutamicum has a proP homologue
in its genome that shows high similarity to that of E. coli transporting
proline/ectoine, but has a preference for proline with a Km of 48 µM. In M.
smegmatis, there are three genes that show similarity to proP of C. glutamicum;
MSMEG_2486,
annotated
as
a
major
facilitator
superfamily
(58.8%);
MSMEG_1447, annotated as a major facilitator superfamily (58.7%) which shows
similarity to a proline/betaine transporter in Rhodococcus species, and
55
MSMEG_4889 annotated as a integral membrane transport protein (53%) that
shows similarity to a proline/betaine transporter in Rhodococcus species (TIGR),
which are closely related to mycobacteria. This suggests that there might be
several low-affinity systems for proline in M. smegmatis as there are in many other
bacterial species. However, at this stage it is unknown how many proline transport
systems exist in M. smegmatis and how these are regulated.
In various Gram-negative bacteria, the high-affinity proline symporter PutP is
regulated by the proline utilization A (PutA) flavoenzyme (7, 87, 139). In M.
smegmatis, putP is located in an operon together with putA. PutA is the key
metabolic enzyme in the proline catabolism of various Gram-negative bacteria and
combines the enzyme activities of proline dehydrogenase (PruB) and Δ1-pyrroline5-carboxylate dehydrogenase (PruA) (65-66, 72, 87, 118, 148, 161). The enzymes
proline dehydrogenase (PruB) and Δ1-pyrroline-5-carboxylate dehydrogenase
(PruA) catalyze the oxidation of proline to glutamate while 4-electrons are
transferred to the respiratory chain (139) (Figure 18). The first step in proline
oxidation is performed by the flavin adenine dinucleotide (FAD)–dependent proline
dehydrogenase. The product of this first reaction is Δ1-pyrroline-5-carboxylate,
which is hydrolyzed to glutamate-γ-semialdehyde (GSA). This intermediate product
is then oxidized to glutamate via the NAD+-dependent Δ1-pyrroline-5-carboxylate
dehydrogenase. The PutA flavoenzyme can act as a transcriptional repressor and
as a membrane-bound proline dehydrogenase and this is dependent on proline
availability and the redox state of the FAD cofactor (7, 135). However, in all
eukaryotes and several bacterial species PruB and PruA are separated enzymes
(139, 157). Our data suggests that the novel transcriptional regulator ProR,
located upstream of PruB and PruA in the mycobacterial genome, is responsible
for the regulation of PruB and PruA. ProR is highly conserved amongst all
mycobacterial species and might also be regulating PutP in the presence of
proline. The RT-qPCR results show that in the presence and absence of proline
the expression of pruB (0.02) and pruA (0.03) are highly reduced in the
proR::Tn611 mutant compared with strain MB100. This explains why the
proR::Tn611 mutant is not able to grow on proline as a sole carbon and energy
source. A mutation in proR results in a defect in the metabolism of proline. The
expression of putP in both mutant (proR::Tn611 and putP::Tn611) is 10-fold
56
reduced compared with strain MB100 in the presence of proline. However, in the
absence of proline there is no difference in putP expression between any of the
strains. This supports the proline transport assay data that demonstrated that
strain MB100 only induces proline permeases when grown in the presence of
proline. Therefore, no differences in putP expression between strain MB100 and
the mutants were expected in the absence of proline. Interestingly, the
putP::Tn611 mutant does not show a downregulation of pruB or pruA. This
confirms the idea that M. smegmatis must have at least one low-affinity proline
transporter and that the putP::Tn611 mutant would be still able to metabolise
proline. RT-qPCR results confirmed the results we gained from the βGalactosidase assays that showed, there is less cytochrome bd expression in both
mutants compared to strain MB100. In the presence of proline, both mutants show
a reduction of cytochrome bd expression of approximately 40%.
57
Figure 18: Proline catabolism. PRODH, Proline dehydrogenase; P5CR, Δ1pyrroline-5-carboxylate
reductase;
P5C,
Δ1-pyrroline-5-carboxylate;
ORN,
ornithine; OAT, acetylornithine aminotransferase; GSA, glutamate-γ-semialdehyde;
P5CDH, Δ1-pyrroline-5-carboxylate dehydrogenase; Glu, glutamate. Taken from
Tanner, 2008 (139).
58
To validate the result that ProR is the regulator of pruB and pruA and to study the
global genome-wide transcriptional response to proR inactivation, we performed a
whole genome microarray analysis comparing the gene expression profile of the
proR::Tn611 mutant and strain MB100. The results of the microarray confirmed
the qPCR results for pruA, pruB, proR and putP and cydAB. Furthermore, we
found several genes involved in phosphate transport significantly downregulated.
M. smegmatis features two high-affinity phosphate ABC-transport systems, the Pst
system (pstSCAB) and the Phn system (phnDCE) (40). Here, we show that the
whole pst operon in the proR::Tn611 mutant is significantly downregulated (IR
0.27 - 0.52) as well as phnD (IR 0.42) of the Phn system. Moreover, the
expression of the two-component regulatory system senX3-regX3, which controls
the transcription of pstSCAB and phnDCE (39, 41) in M. smegmatis was
downregulated (IR 0.53 and 0.47 respectively). Additionally, the proline-rich 28
kDa antigen (MSMEG_6919) was significantly downregulated (IR 0.38). The most
remarkable result of the microarray was the upregulation of almost the entire DNA
repair/metabolism machinery (53 genes up to an IR of 18) in the proR::Tn611
mutant. Significantly upregulated genes comprised the alternative sigma factor
SigG (IR 11.3), which recently has been shown to be involved in the SOS
response in M. tuberculosis (71) as well as NrdR (IR 3.5), a transcriptional
regulator in all mycobacteria (90). Moreover, we show that five glyoxalase family
proteins were strongly induced in the proR::Tn611 mutant (IR 2.1 - 15.2). DNA
damage could be an indicator for that the cells are experiencing oxidative stress
(18, 75).
ROS, especially singlet oxygen and free radicals cause oxidative stress and are
known to damage DNA (81). ROS, include various oxygen-derived molecules,
including oxygen radicals, such as superoxide anion (O2·-) and hydroxyl radical
(·OH), as well as nonradicals that are oxidizing agents, such as hydrogen peroxide
(H2O2) and singlet oxygen (1O2) (75, 81). ROS are produced by most enzymes that
are metabolising oxygen and under normal conditions; concentrations of ROS
remain low because of the cell protective enzymes, such as catalase and
superoxide dismutase, responsible for "detoxification". Proline accumulation has
been shown to protect mammalian cells (67), yeast cells (137-138), plant cells
(107) and bacterial cells (Helicobacter hepaticus) (68) against oxidative stress by
59
quenching ROS, especially singlet oxygen and free radicals (2, 81), and therefore
stabilizes proteins and DNA. In our study proR::Tn611 and putP::Tn611 mutants
were not significantly more sensitive to H2O2 compared to strain MB100. This
result is supported by the microarray analysis, which showed that the gene
expression rate of the catalases in the proR::Tn611 mutant were not significant
different than compared to strain MB100.
Regarding the upregulation of five glyoxalase family proteins and the fact that
hydrogen peroxide did not cause DNA damage in the mutants, we further
proposed that the DNA damage might be caused by methylglyoxal, a toxic
electrophile, produced as a side-product of several metabolic pathways
(degradation of glucose, glycerol, fatty acids and proteins) (37, 55, 60). E. coli and
various other prokaryotic and mammalian systems can synthesize methylglyoxal
via the enzyme methylglyoxal synthase (25, 109). Mycobacteria do not comprise a
methylglyoxal synthase homologue; however, this toxic compound is also
generated through the non-enzymatic formation from glyceraldehyde-3-phosphate
and
dihydroxyacetone
phosphate
(132)
(Figure
19).
Methylglyoxal
is
predominantly derived from the triose phosphate intermediates (dihydroxyacetone
phosphate and glyceraldehyde-3-phosphate) in the glycolytic pathway through
fragmentation or elimination of the phosphate group from enediol intermediate at
the active site of triose phosphate isomerise (TPI) (100, 111). Both TPI and
cytosolic phosphorylating glyceraldehyde 3-phosphate dehydrogenase (GapC) are
involved in the glycolytic pathway. TPI catalyzes the reversible interconversion of
dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, whereas GapC
catalyzes the catabolism of glyceraldehyde 3-phosphate. The toxicity of
methylglyoxal is believed to be due to its ability to interact with the nucleophilic
centres of macromolecules such as DNA (37). Bacteria show an array of
detoxification mechanisms for methylglyoxal. The main degradation pathway is via
the glyoxalase system. E. coli uses the glutathione-dependent glyoxalase I-II
pathway as the major detoxification pathway to convert methylglyoxal to D-lactate
and methylglyoxal derivatives to the corresponding α-hydroxyacids. Methylglyoxal
first reacts spontaneously with glutathione (GSH) to form hemithiolacetal, which is
then converted into S-lactoylglutathione via the glyoxalase I enzyme. Glyoxalase II
degrades S-lactoylglutathione to D-lactate while glutathione is released and
60
available to react with methylglyoxal. However, GSH is absent in mycobacteria
(93). As a second detoxification pathway for methylglyoxal, E. coli contains
glyoxalase III, a glutathione-independent enzyme that converts methylglyoxal
directly into D-lactate (89). Mycobacteria features several genes encoding for
glyoxalase family proteins, however their specific roles remain unknown. Due to
the fact that five glyoxalase family proteins were significantly upregulated in the
proR::Tn611 mutant in the microarray analysis, we tested the sensitivity of both
mutants and strain MB100 for methylglyoxal. We demonstrated that a mutation in
the proline metabolism pathway of the mutants (proR::Tn611 and putP::Tn611)
results in increased sensitivity to methylglyoxal compared to M. smegmatis strain
MB100.
61
Figure 19: Synthesis and detoxification of methylglyoxal (dashed lines indicating
non-enzymatic reactions).
62
Recent literature proposes a detoxification mechanism for methylglyoxal through
L-proline. It is thought for Bacillus subtilis subsp. natto (53), Aromatic Rice (Oryza
sativa L.) (54) and Vegetable Soybean (Glycine max L.) (163) that the product Δ1pyrroline-5-carboxylic acid, derived from the conversion of L-proline, can react with
methylglyoxal to form the non-toxic compound 2-acetyl-1-pyrroline. This has
already been shown for Lactobacillus bulgaricus by Griffith and Hammond (1989)
(46) during an investigation into the generation of swiss cheese flavour
components by the reaction of amino acids with carbonyl compounds like
methylglyoxal. Griffith and Hammond (46) were able to demonstrate that
methylglyoxal can react with proline to form the non-toxic compounds 2-acetyl-1pyrroline and pyrrolidine. Because methylglyoxal is the major electrophile
produced intracellularly by bacterial cells (147), it can also cause oxidative stress
(38) and therefore changes the redox state of the cell. This mechanism is
comparable to oxidative stress caused by hydrogen peroxide. A mutation in
catalases results in more oxidative stress for the cell which then changes the
redox state of the cell and this leads to failure to activate expression of cytochrome
bd. We therefore propose that the interruption of the proline metabolism in M.
smegmatis changes the redox state of the cell via the electrophile methylglyoxal
resulting in a downregulation of cytochrome bd (Figure 20).
63
Figure 20: Model of proline and methylglyoxal metabolism in an M. smegmatis
mutant defective in proR. Numbers represent gene expression ratios of 4
independent biological samples (p ≤ 0.05) determined with microarray analysis.
64
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Appendix
Table 8: Genes with induction ratios (IR) significantly different from 1 (p ≤ 0.05)
comparing M. smegmatis strain MB100 with the proR::Tn611 mutant
M. smegmatis
locus
Gene description
IR
MSMEG_0003
DNA replication and repair protein RecF
3.45
0.04
MSMEG_0004
hypothetical protein
1.91
< 0.01
MSMEG_0005
DNA gyrase, B subunit
2.63
0.01
MSMEG_0006
DNA gyrase, A subunit
1.68
0.05
MSMEG_0032
cell cycle protein, FtsW/RodA/SpoVE family protein
1.34
0.02
MSMEG_0056
hypothetical protein
1.25
0.02
MSMEG_0063
PE family protein
1.79
0.04
MSMEG_0067
conserved hypothetical protein
1.92
0.05
MSMEG_0071
conserved hypothetical protein
1.09
0.01
MSMEG_0072
conserved hypothetical protein
1.46
0.02
MSMEG_0082
conserved hypothetical protein
1.52
0.04
MSMEG_0083
membrane-anchored mycosin mycp1
1.54
0.02
MSMEG_0095
methyltransferase
0.80
0.04
MSMEG_0096
peroxisomal hydratase-dehydrogenase-epimerase
0.55
0.01
MSMEG_0108
acyl-CoA dehydrogenase
1.70
0.02
MSMEG_0118
conserved hypothetical protein
0.29
0.01
MSMEG_0156
transcriptional regulator, LysR family protein
1.44
0.05
0.86
0.02
MSMEG_0177
ABC polar amino acid family protein transporter, inner
membrane subunit
pvalue
MSMEG_0194
serine esterase, cutinase family protein
0.55
0.01
MSMEG_0209
ribonuclease inhibitor
0.81
0.05
MSMEG_0211
ABC transporter, ATP-binding protein
0.78
0.01
MSMEG_0215
YhhW family protein
1.22
0.02
MSMEG_0219
RNA polymerase sigma-70 factor, family protein
11.29
0.02
MSMEG_0222
conserved hypothetical protein
12.88
0.02
MSMEG_0223
conserved hypothetical protein
8.47
0.01
MSMEG_0224
O-methyltransferase MdmC
3.10
0.03
MSMEG_0225
MmpL4 protein
0.50
< 0.01
MSMEG_0226
MmpS5 protein
0.50
0.01
MSMEG_0234
metallopeptidase
0.64
0.02
MSMEG_0240
conserved hypothetical protein
0.71
0.02
MSMEG_0256
conserved hypothetical protein
2.74
0.03
I
pvalue
M. smegmatis
locus
Gene description
IR
MSMEG_0257
acyl-CoA synthase
4.61
0.05
MSMEG_0268
transcriptional regulator, GntR family protein
1.79
< 0.01
MSMEG_0292
conserved hypothetical protein
0.70
0.03
MSMEG_0294
3-oxoacyl-(acyl-carrier-protein) reductase
0.48
< 0.01
MSMEG_0299
Rieske [2Fe-2S] domain protein
0.76
< 0.01
1.35
0.04
MSMEG_0312
2-dehydro-3-deoxyphosphogluconate aldolase/4hydroxy-2-oxoglutarate aldolase
MSMEG_0318
AMP-dependent synthetase and ligase
0.67
0.01
MSMEG_0353
conserved hypothetical protein
1.26
0.01
MSMEG_0364
hypothetical protein
1.43
0.05
MSMEG_0369
hypothetical protein
1.97
0.01
MSMEG_0370
conserved hypothetical protein
2.09
0.01
1.72
< 0.01
MSMEG_0372
oxidoreductase, short chain dehydrogenase/reductase
family protein
MSMEG_0373
3-ketoacyl-CoA thiolase
2.26
0.01
MSMEG_0375
phospholipase D family protein
0.48
0.01
MSMEG_0380
MmpS4 protein
0.69
0.01
MSMEG_0381
Mmp14a protein
0.76
< 0.01
MSMEG_0383
conserved hypothetical protein
0.64
0.02
MSMEG_0395
hypothetical protein
0.74
0.01
MSMEG_0408
type I modular polyketide synthase
2.26
0.05
MSMEG_0415
NADH-fmn oxidoreductase
1.13
0.03
MSMEG_0416
hypothetical protein
0.89
0.04
MSMEG_0422
transferase
1.46
0.05
MSMEG_0427
nitrite reductase [NAD(P)H], large subunit
0.76
0.02
MSMEG_0436
allophanate hydrolase subunit 1
1.40
0.03
MSMEG_0440
hypothetical protein
0.80
< 0.01
MSMEG_0456
DNA gyrase subunit A
7.58
0.05
MSMEG_0457
DNA topoisomerase IV subunit B
4.72
0.03
MSMEG_0458
two-component system sensor kinase
1.30
0.02
MSMEG_0490
enoyl-CoA hydratase
0.79
0.05
MSMEG_0502
glucosidase
1.81
0.04
MSMEG_0504
carbohydrate kinase
2.14
0.04
MSMEG_0514
alpha-galactosidase
1.27
0.04
MSMEG_0519
conserved hypothetical protein
0.68
0.03
MSMEG_0539
transcriptional regulator, Crp/Fnr family protein
1.50
< 0.01
MSMEG_0547
ISMsm5, transposase
0.64
0.01
II
pvalue
M. smegmatis
locus
Gene description
IR
MSMEG_0557
hypothetical protein
1.42
0.05
MSMEG_0558
conserved hypothetical protein
1.35
0.01
MSMEG_0559
conserved domain protein
0.64
< 0.01
MSMEG_0579
helix-turn-helix, Fis-type
1.52
0.05
MSMEG_0586
stas domain, putative
1.68
0.05
MSMEG_0593
conserved hypothetical protein
0.68
0.02
MSMEG_0594
iron-sulfur cluster binding protein
0.59
0.01
MSMEG_0596
bacterial regulatory protein, GntR family protein
1.43
0.01
MSMEG_0604
glyoxylate reductase
0.57
0.01
MSMEG_0619
ppe family protein
0.80
< 0.01
MSMEG_0624
subtilase family protein
1.18
0.04
MSMEG_0625
conserved hypothetical protein
0.80
0.04
MSMEG_0635
putative conserved exported protein
0.49
0.05
MSMEG_0636
conserved hypothetical protein
0.78
0.02
MSMEG_0649
phosphonate-binding periplasmic protein
0.42
0.02
MSMEG_0671
S-(hydroxymethyl)glutathione dehydrogenase
0.71
0.02
MSMEG_0673
hypothetical protein
0.59
< 0.01
MSMEG_0679
conserved hypothetical protein
0.63
0.04
MSMEG_0694
conserved hypothetical protein
0.38
0.01
MSMEG_0695
isoniazid inductible protein IniA
0.34
0.01
MSMEG_0702
monooxygenase
0.68
0.01
MSMEG_0708
transcriptional regulator, LysR family protein
3.36
< 0.01
MSMEG_0726
hypothetical protein
0.88
0.02
MSMEG_0760
thioesterase family protein
0.81
0.02
MSMEG_0762
cytochrome P450 FAS1
0.73
0.01
MSMEG_0769
O-succinylhomoserine sulfhydrylase
0.74
0.01
0.81
0.04
0.69
< 0.01
0.55
0.01
MSMEG_0777
MSMEG_0786
MSMEG_0787
F420-dependent glucose-6-phosphate
dehydrogenase
serine/threonine protein kinase
Bacterial extracellular solute-binding protein, family
protein 3
MSMEG_0788
putative conserved membrane protein
0.76
< 0.01
MSMEG_0791
glycine oxidase ThiO
0.89
0.01
MSMEG_0797
conserved hypothetical protein
0.72
0.04
MSMEG_0803
hypothetical protein
1.55
< 0.01
MSMEG_0804
hypothetical protein
0.73
0.01
MSMEG_0818
transporter, major facilitator family protein
1.59
0.05
III
pvalue
M. smegmatis
locus
Gene description
IR
MSMEG_0826
thiamine biosynthesis protein ThiC
0.65
0.02
MSMEG_0833
conserved hypothetical protein
1.91
< 0.01
MSMEG_0835
copper/zinc superoxide dismutase
0.71
0.02
0.68
0.01
MSMEG_0847
NADH-ubiquinone/plastoquinone oxidoreductase
chain 4l family protein
MSMEG_0855
conserved hypothetical protein
0.15
< 0.01
MSMEG_0857
conserved hypothetical protein
0.11
< 0.01
MSMEG_0882
conserved hypothetical protein
1.30
0.05
MSMEG_0884
glyoxalase family protein
2.05
0.04
MSMEG_0886
serine/threonine-protein kinase PknD
0.72
0.04
MSMEG_0893
hypothetical protein
0.70
0.02
MSMEG_0904
probable conserved membrane protein
0.65
< 0.01
MSMEG_0913
methoxy mycolic acid synthase 1
0.72
0.01
MSMEG_0919
heparin-binding hemagglutinin
0.57
0.03
MSMEG_0936
sensor histidine kinase SenX3
0.54
0.02
MSMEG_0937
DNA-binding response regulator RegX3
0.47
< 0.01
MSMEG_0940
conserved hypothetical protein
1.30
0.05
MSMEG_0944
DNA binding domain, excisionase family protein
0.76
0.02
MSMEG_0965
porin
0.68
< 0.01
MSMEG_0970
phosphoglycerate mutase family protein
0.62
0.01
1.43
0.03
MSMEG_0972
cytochrome C biogenesis protein transmembrane
region
MSMEG_0985
sugar transporter family protein
0.67
0.01
MSMEG_0989
conserved hypothetical protein
1.47
0.04
MSMEG_0998
oxidoreductase, molybdopterin binding
1.55
0.02
MSMEG_1062
O-succinylbenzoic acid--CoA ligase
0.87
0.04
MSMEG_1064
phosphate/sulphate permease
0.58
0.02
MSMEG_1065
conserved hypothetical protein
0.45
< 0.01
MSMEG_1066
conserved hypothetical protein
0.44
< 0.01
MSMEG_1086
ABC transporter permease protein
0.71
< 0.01
MSMEG_1089
hypothetical protein
0.77
0.04
MSMEG_1115
menaquinone biosynthesis methyltransferase UbiE
0.71
< 0.01
MSMEG_1129
D-amino-acid dehydrogenase
1.54
0.02
MSMEG_1151
DNA-binding protein
1.23
0.02
MSMEG_1162
nitrilotriacetate monooxygenase component A
0.89
0.03
MSMEG_1165
conserved hypothetical protein
0.83
0.02
MSMEG_1169
integral membrane transport protein
0.68
0.01
IV
pvalue
M. smegmatis
locus
Gene description
IR
MSMEG_1170
conserved domain protein
0.72
0.04
MSMEG_1222
ISMsm6, transposase
0.70
0.02
MSMEG_1225
conserved hypothetical protein
7.76
< 0.01
MSMEG_1236
Mpr protein
13.54
0.03
MSMEG_1242
cysteine desulfurase IscS
4.81
< 0.01
MSMEG_1243
conserved hypothetical protein
4.27
< 0.01
MSMEG_1244
conserved hypothetical protein
5.14
< 0.01
MSMEG_1245
phosphoadenosine phosphosulfate reductase
6.49
0.01
MSMEG_1246
conserved hypothetical protein
7.08
< 0.01
MSMEG_1251
conserved hypothetical protein
8.68
0.01
MSMEG_1252
conserved hypothetical protein
3.97
0.01
MSMEG_1253
conserved hypothetical protein
6.02
0.04
MSMEG_1254
DEAD/DEAH box helicase
5.28
0.02
MSMEG_1264
prophage Lp1 protein 5
9.59
0.01
MSMEG_1266
ankyrin-repeat containing protein
2.11
0.02
MSMEG_1269
Ser/Thr protein phosphatase family protein
6.45
< 0.01
MSMEG_1270
hypothetical protein
6.59
< 0.01
MSMEG_1271
hypothetical protein
3.72
0.03
MSMEG_1272
putative ribosylglycohydrolase
13.34
0.01
MSMEG_1273
conserved hypothetical protein
2.18
< 0.01
MSMEG_1275
HNH nuclease, putative
2.49
< 0.01
MSMEG_1279
conserved hypothetical protein
7.56
0.03
MSMEG_1281
conserved hypothetical protein
3.03
0.03
MSMEG_1288
conserved hypothetical protein
4.37
0.01
MSMEG_1302
alkylphosphonate uptake protein
3.50
0.03
MSMEG_1309
conserved hypothetical protein
0.70
0.04
MSMEG_1312
hypothetical protein
7.29
0.02
MSMEG_1325
exodeoxyribonuclease V, alpha subunit
3.16
0.02
MSMEG_1328
exodeoxyribonuclease V, gamma subunit
4.10
< 0.01
MSMEG_1353
ABC1 family protein
1.20
0.02
MSMEG_1356
conserved hypothetical protein
3.68
0.02
MSMEG_1357
conserved hypothetical protein
2.80
0.04
MSMEG_1358
conserved hypothetical protein
1.48
0.02
MSMEG_1376
putative xylulose kinase
0.73
0.03
MSMEG_1381
MmpS5 protein
0.22
0.01
MSMEG_1382
MmpL5 protein
0.14
< 0.01
MSMEG_1383
endonuclease IV
3.72
0.01
V
pvalue
M. smegmatis
locus
Gene description
IR
MSMEG_1388
enoyl-CoA hydratase
0.79
0.01
MSMEG_1389
conserved hypothetical protein
0.82
0.03
MSMEG_1393
conserved hypothetical protein
0.77
0.01
MSMEG_1408
arginine/ornithine antiporter
0.71
0.04
MSMEG_1414
Amidinotransferase
2.79
0.03
MSMEG_1417
glyoxalase family protein
2.35
0.03
MSMEG_1418
RNA polymerase ECF-type sigma factor
2.55
0.04
MSMEG_1419
conserved hypothetical protein
2.21
0.02
MSMEG_1422
conserved hypothetical protein
0.74
0.04
MSMEG_1423
radical SAM domain protein
0.67
0.02
MSMEG_1439
ribosomal protein L2
0.53
< 0.01
MSMEG_1440
ribosomal protein S19
0.81
0.04
MSMEG_1465
ribosomal protein L14
1.32
0.03
MSMEG_1469
ribosomal protein S8
0.81
0.03
MSMEG_1476
signal peptide peptidase SppA, 67K type
0.60
0.02
MSMEG_1500
TetR family protein transcriptional regulatory protein
0.68
0.02
MSMEG_1502
ABC transporter, ATP-binding protein
0.57
0.02
MSMEG_1510
dTDP-4-dehydrorhamnose 3,5-epimerase
0.79
0.03
MSMEG_1511
putative oxidoreductase
0.58
0.02
MSMEG_1523
ribosomal protein S4
0.72
0.01
MSMEG_1524
DNA-directed RNA polymerase, alpha subunit
1.47
0.05
MSMEG_1530
integral membrane protein
0.86
< 0.01
MSMEG_1542
transcriptional regulator
1.28
0.04
0.72
0.03
MSMEG_1568
glucosamine--fructose-6-phosphate aminotransferase,
isomerizing
MSMEG_1576
alpha/beta hydrolase fold
1.41
0.04
MSMEG_1584
hypothetical protein
1.18
0.05
MSMEG_1600
conserved hypothetical protein
0.58
0.01
MSMEG_1606
benzoylformate decarboxylase
1.17
0.02
MSMEG_1610
glutamine-hydrolyzing GMP synthase
0.82
0.03
MSMEG_1624
universal stress protein family protein
0.86
0.01
MSMEG_1635
nitroreductase family protein
0.77
0.04
MSMEG_1637
periplasmic sensor signal transduction histidine kinase
0.58
0.01
MSMEG_1656
exodeoxyribonuclease III
4.03
< 0.01
MSMEG_1659
multidrug resistance protein, SMR family protein
0.69
0.01
MSMEG_1676
adenosine deaminase
0.81
0.01
MSMEG_1688
cupin domain protein
0.72
0.04
VI
pvalue
M. smegmatis
locus
Gene description
IR
MSMEG_1692
ECF-family protein RNA polymerase sigma factor
1.57
0.02
MSMEG_1744
hypothetical protein
0.92
0.04
3.00
0.05
MSMEG_1756
endonuclease VIII and dna n-glycosylase with an ap
lyase activity
MSMEG_1757
DEAD/DEAH box helicase
4.62
0.02
MSMEG_1764
L-lysine-epsilon aminotransferase
0.56
0.01
MSMEG_1786
stas domain, putative
1.87
< 0.01
MSMEG_1808
Fe-S metabolism associated SufE
0.85
0.01
MSMEG_1811
septum formation protein Maf
0.65
< 0.01
MSMEG_1813
propionyl-CoA carboxylase beta chain
0.70
0.03
0.79
0.04
MSMEG_1819
phosphoribosylaminoimidazole carboxylase, ATPase
subunit
MSMEG_1822
biotin-[acetyl-CoA-carboxylase] ligase
1.48
0.03
MSMEG_1828
Nucleotidyl transferase
1.28
0.04
MSMEG_1831
Transcription factor WhiB
0.64
0.04
MSMEG_1837
secreted protein
0.78
0.04
MSMEG_1843
adenosylhomocysteinase
0.55
< 0.01
MSMEG_1843
adenosylhomocysteinase
0.63
< 0.01
MSMEG_1862
transposase
0.84
0.02
MSMEG_1874
DNA-binding response regulator MtrA
1.47
0.01
MSMEG_1905
acyl-CoA dehydrogenase protein
1.12
0.03
MSMEG_1906
toluate 1,2-dioxygenase electron transfer component
0.87
< 0.01
MSMEG_1911
catechol 1,2-dioxygenase
0.59
0.05
MSMEG_1912
muconolactone delta-isomerase 1
0.75
0.02
MSMEG_1914
RNA polymerase sigma-70 factor, family protein
2.01
0.01
MSMEG_1915
anti-sigma factor, family protein
1.24
0.03
MSMEG_1917
conserved domain protein
1.52
0.01
MSMEG_1927
cobyrinic Acid a,c-diamide synthase
0.81
< 0.01
MSMEG_1941
helicase, UvrD/Rep family protein
8.34
< 0.01
MSMEG_1943
ATP-dependent DNA helicase
13.74
< 0.01
MSMEG_1947
conserved hypothetical protein
1.40
0.05
MSMEG_1974
propane monooxygenase coupling protein
0.75
0.03
MSMEG_1980
monooxygenase
0.76
0.05
MSMEG_1982
acyl-CoA synthase
0.76
0.03
MSMEG_1985
transcriptional regulator, LysR family protein
0.66
0.01
MSMEG_1998
hydrolase, alpha/beta fold family protein
0.60
0.01
MSMEG_2007
putative HpcE protein
0.82
0.01
VII
M. smegmatis
locus
MSMEG_2010
Gene description
stress responsive A/B Barrel Domain superfamily
protein
IR
pvalue
0.51
0.01
MSMEG_2026
short chain dehydrogenase
0.65
< 0.01
MSMEG_2027
conserved hypothetical protein TIGR00026
1.63
0.02
MSMEG_2028
chloramphenicol resistance protein
0.85
< 0.01
0.84
0.05
MSMEG_2033
oxidoreductase, zinc-binding dehydrogenase family
protein
MSMEG_2041
conserved hypothetical protein
0.53
0.01
MSMEG_2067
methyltransferase type 12
0.39
0.01
MSMEG_2078
antigen 85-C
0.61
< 0.01
MSMEG_2081
putative acyl-CoA dehydrogenase
2.70
0.03
MSMEG_2090
putative cell division protein FtsX
0.64
0.02
MSMEG_2094
56kDa selenium binding protein
1.27
0.01
MSMEG_2101
taurine ABC transporter, permease protein
0.83
0.03
MSMEG_2107
conserved hypothetical protein
0.49
0.01
MSMEG_2128
malonyl CoA decarboxylase
0.72
0.04
MSMEG_2143
hypothetical protein
0.67
0.04
MSMEG_2151
transposase IstA protein, putative
0.75
0.03
MSMEG_2161
FadD9 protein
0.73
0.01
MSMEG_2163
aldehyde dehydrogenase
0.73
0.02
MSMEG_2190
acyl-CoA dehydrogenase domain protein
0.75
0.01
MSMEG_2201
ZbpA protein
0.81
0.03
MSMEG_2225
transcriptional regulator, TetR family protein
1.32
< 0.01
MSMEG_2277
DNA ligase I, ATP-dependent
7.31
0.02
MSMEG_2278
membrane-bound oxidoreductase
0.69
0.01
MSMEG_2280
pyruvate dehydrogenase
0.73
< 0.01
MSMEG_2286
aminoglycoside phosphotransferase
0.64
0.02
MSMEG_2293
conserved hypothetical protein
2.66
0.01
MSMEG_2293
conserved hypothetical protein
4.10
< 0.01
MSMEG_2294
DNA polymerase IV
0.70
0.01
MSMEG_2299
ribonucleoside-diphosphate reductase, alpha subunit
0.52
< 0.01
MSMEG_2301
glyoxalase/bleomycin resistance protein/dioxygenase
0.76
0.05
MSMEG_2309
probable transcriptional regulatory protein
0.86
0.04
MSMEG_2315
conserved hypothetical protein
0.62
0.01
MSMEG_2315
conserved hypothetical protein
0.78
0.04
MSMEG_2317
alcohol dehydrogenase, zinc-containing
0.66
0.05
MSMEG_2322
hypothetical protein
1.82
0.02
VIII
M. smegmatis
locus
MSMEG_2326
Gene description
ABC-type molybdenum transport system, ATPase
component
IR
pvalue
1.31
0.02
MSMEG_2329
methyltransferase, UbiE/COQ5 family protein
1.19
0.03
MSMEG_2346
phytoene synthase
0.51
0.01
MSMEG_2356
acyltransferase
0.66
0.02
MSMEG_2359
methionine synthase, vitamin-B12 independent
0.71
0.01
MSMEG_2361
phosphoribosylglycinamide formyltransferase
1.29
0.03
MSMEG_2362
DNA ligase, NAD-dependent
8.18
0.05
MSMEG_2369
LppZ protein
0.83
0.03
MSMEG_2378
D-3-phosphoglycerate dehydrogenase
0.69
< 0.01
MSMEG_2381
conserved hypothetical protein
0.58
0.01
MSMEG_2383
glutamyl-tRNA synthetase
0.79
< 0.01
MSMEG_2387
3-isopropylmalate dehydratase, large subunit
1.49
0.05
MSMEG_2391
polyphosphate kinase
0.55
< 0.01
MSMEG_2397
transcriptional regulatory protein, AsnC family protein
0.84
0.01
MSMEG_2403
ATP-dependent DNA helicase RecG
2.20
0.04
MSMEG_2406
transcriptional regulator, TetR family protein, putative
0.84
0.03
MSMEG_2408
2,5-diketo-D-gluconic acid reductase A
0.68
< 0.01
MSMEG_2410
putative serine-threonine protein kinase
0.47
< 0.01
MSMEG_2411
conserved integral membrane protein
0.55
< 0.01
MSMEG_2412
pyruvate carboxylase
0.62
0.01
MSMEG_2413
putative methyltransferase
0.60
< 0.01
MSMEG_2417
conserved hypothetical protein
1.68
0.02
MSMEG_2423
chromosome segregation protein SMC
0.66
0.03
MSMEG_2424
signal recognition particle-docking protein FtsY
1.71
0.04
MSMEG_2432
D-alanyl-D-alanine carboxypeptidase
0.54
< 0.01
MSMEG_2449
methylmalonate-semialdehyde dehydrogenase
3.85
0.04
MSMEG_2486
major facilitator superfamily protein
0.73
< 0.01
MSMEG_2489
transcriptional regulator, GntR family protein, putative
0.50
0.01
MSMEG_2492
D-lactate dehydrogenase
0.55
0.01
MSMEG_2497
amidase family protein
0.46
< 0.01
MSMEG_2498
conserved hypothetical protein
0.52
0.01
MSMEG_2500
conserved hypothetical protein
0.66
0.01
MSMEG_2518
peptidase M23B
0.72
0.02
MSMEG_2521
amidase
0.62
0.02
0.59
0.02
MSMEG_2530
4-carboxymuconolactone decarboxylase domain
protein
IX
pvalue
M. smegmatis
locus
Gene description
IR
MSMEG_2534
putative carboxylesterase protein
0.65
0.04
MSMEG_2536
3-oxoacyl-[acyl-carrier-protein] reductase
0.70
0.04
MSMEG_2545
radical SAM enzyme, Cfr family protein
0.85
0.04
MSMEG_2547
transcriptional regulator
0.95
0.05
MSMEG_2578
1-deoxy-D-xylulose 5-phosphate reductoisomerase
0.80
0.02
0.72
0.01
MSMEG_2580
4-hydroxy-3-methylbut-2-en-1-yl diphosphate
synthase
MSMEG_2582
conserved hypothetical protein
0.55
0.03
MSMEG_2584
penicillin-binding protein, putative
2.89
0.01
MSMEG_2613
malate:quinone-oxidoreductase
0.80
< 0.01
MSMEG_2620
hypothetical protein
0.68
0.05
MSMEG_2622
conserved hypothetical alanine rich protein
0.77
0.01
MSMEG_2625
transcription termination factor NusA
0.55
0.01
MSMEG_2642
conserved hypothetical protein
1.24
0.04
MSMEG_2647
metallophosphoesterase
0.82
0.04
MSMEG_2700
conserved hypothetical protein
0.77
0.01
MSMEG_2723
protein RecA
6.18
< 0.01
MSMEG_2724
regulatory protein RecX
8.86
< 0.01
MSMEG_2727
glutamate binding protein
0.59
0.01
MSMEG_2728
glutamate transport ATP-binding protein GluA
1.59
< 0.01
MSMEG_2730
conserved hypothetical protein
0.74
0.01
MSMEG_2740
LexA repressor
3.74
0.01
MSMEG_2741
hypothetical protein
19.38
< 0.01
MSMEG_2742
DNA-damage-inducible protein
7.82
0.01
MSMEG_2743
transcriptional regulator, NrdR family protein
3.52
0.01
MSMEG_2747
conserved hypothetical protein
0.73
0.01
MSMEG_2758
sigma factor MysA
1.13
0.04
MSMEG_2761
putative secreted alanine rich protein
1.44
0.04
MSMEG_2767
conserved hypothetical protein
0.77
0.05
MSMEG_2768
OB-fold nucleic acid binding domain protein
1.42
0.01
MSMEG_2776
1-deoxy-D-xylulose-5-phosphate synthase
0.81
0.02
MSMEG_2779
conserved hypothetical protein
1.89
0.03
MSMEG_2782
conserved hypothetical protein
0.70
0.02
MSMEG_2783
hypothetical protein
1.21
0.03
MSMEG_2784
methionine-R-sulfoxide reductase
0.64
0.02
MSMEG_2798
hypothetical protein
1.18
0.04
MSMEG_2805
ISMsm5, transposase
0.62
0.02
X
pvalue
M. smegmatis
locus
Gene description
IR
MSMEG_2815
putative tape-measure protein;
1.44
0.02
MSMEG_2818
ISMsm1, transposase orfB
0.72
0.04
MSMEG_2820
hypothetical protein
0.78
0.04
MSMEG_2824
IS1549, transposase
1.17
0.03
MSMEG_2832
hypothetical protein
1.25
0.04
MSMEG_2837
nitrate reductase NarB
0.91
0.05
MSMEG_2839
transcriptional accessory protein
8.25
0.02
MSMEG_2859
virulence factor Mce family protein
1.30
0.02
MSMEG_2879
acyl-CoA dehydrogenase
0.87
0.01
MSMEG_2900
2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase
0.81
0.03
MSMEG_2903
transposase
0.68
0.01
MSMEG_2924
permease binding-protein component
0.59
0.02
MSMEG_2925
permease membrane component
0.65
0.03
MSMEG_2943
crossover junction endodeoxyribonuclease RuvC
3.57
0.01
MSMEG_2944
holliday junction DNA helicase RuvA
4.33
< 0.01
MSMEG_2951
[2Fe-2S] binding domain protein
0.77
0.03
1.61
0.02
MSMEG_2963
bacterial extracellular solute-binding protein, family
protein 5
MSMEG_2970
polysaccharide deacetylase
0.74
0.01
MSMEG_3001
conserved hypothetical protein
1.60
0.02
MSMEG_3011
steroid monooxygenase
0.71
0.04
MSMEG_3012
acetyl-CoA acetyltransferases
1.72
< 0.01
MSMEG_3024
conserved hypothetical protein
3.12
< 0.01
MSMEG_3027
conserved hypothetical protein
1.51
0.05
MSMEG_3033
3-dehydroquinate synthase
1.70
0.02
MSMEG_3051
guanylate kinase
1.20
0.05
MSMEG_3053
DNA-directed RNA polymerase, omega subunit
1.31
0.04
MSMEG_3056
ABC transporter ATP-binding protein
0.65
0.01
MSMEG_3066
ribulose-phosphate 3-epimerase
1.12
0.05
MSMEG_3070
LprG protein
1.20
0.05
MSMEG_3071
riboflavin synthase, alpha subunit
0.67
0.04
MSMEG_3078
excinuclease ABC, C subunit
2.05
0.01
MSMEG_3080
conserved hypothetical protein
1.51
0.01
MSMEG_3081
conserved hypothetical protein
1.68
0.04
MSMEG_3096
hypothetical protein
0.77
0.01
MSMEG_3103
transketolase
0.55
< 0.01
MSMEG_3121
DNA-binding protein
0.52
0.01
XI
pvalue
M. smegmatis
locus
Gene description
IR
MSMEG_3122
FeS assembly protein SufB
0.68
0.04
MSMEG_3124
FeS assembly ATPase SufC
0.64
0.01
MSMEG_3125
cysteine desulfurase
0.69
0.04
MSMEG_3127
conserved protein, DUF59
0.50
< 0.01
MSMEG_3129
putative HTH-type transcriptional regulator
0.56
< 0.01
MSMEG_3136
heme peroxidase superfamily protein
1.89
0.04
MSMEG_3146
invasin 1
0.59
0.03
MSMEG_3147
ATPase, MoxR family protein
0.81
0.03
MSMEG_3155
band 7 protein
0.70
< 0.01
MSMEG_3157
conserved hypothetical protein
1.60
< 0.01
MSMEG_3172
DNA polymerase IV 1
2.11
0.04
MSMEG_3174
lipoprotein signal peptidase
2.50
0.01
MSMEG_3178
DNA polymerase III alpha subunit
3.20
< 0.01
MSMEG_3194
biotin synthase
0.51
0.02
MSMEG_3196
conserved hypothetical protein
0.67
0.02
MSMEG_3197
lipase
0.61
0.04
MSMEG_3198
hydrolase, NUDIX family protein
1.58
0.02
MSMEG_3205
histidinol dehydrogenase
1.37
0.03
MSMEG_3213
conserved hypothetical protein
8.53
0.02
MSMEG_3214
hypothetical protein
7.88
0.04
MSMEG_3224
conserved hypothetical protein
0.87
0.05
MSMEG_3232
cytochrome D ubiquinol oxidase, subunit II
0.62
0.01
1.31
0.03
MSMEG_3242
starvation-inducible DNA-binding protein or fine
tangled pili major subunit
MSMEG_3265
arabitol-phosphate dehydrogenase
2.57
0.05
MSMEG_3268
ABC transporter, permease protein
2.07
0.03
MSMEG_3274
MerR-family protein transcriptional regulator
0.69
0.05
MSMEG_3290
regulatory protein
0.82
0.03
MSMEG_3315
hypothetical protein
0.69
0.01
0.53
0.03
MSMEG_3317
dihydrodipicolinate reductase, N-terminus domain
protein
MSMEG_3331
hypothetical protein
1.44
0.02
MSMEG_3336
hydrolase
0.49
0.04
MSMEG_3350
TRAP transporter, DctM-like membrane protein
0.88
0.04
MSMEG_3353
hypothetical protein
0.85
0.05
MSMEG_3357
metal-dependent phosphohydrolase, HD subdomain
0.55
< 0.01
XII
M. smegmatis
locus
MSMEG_3380
Gene description
pyridoxamine 5'-phosphate oxidase-related, FMNbinding
IR
pvalue
0.78
0.02
MSMEG_3392
acyl-CoA dehydrogenase domain protein
0.80
0.03
MSMEG_3404
HNH endonuclease domain protein
18.00
0.01
MSMEG_3454
hypothetical protein
0.87
0.01
MSMEG_3455
heat shock protein 15
0.73
< 0.01
MSMEG_3470
NAD-dependent epimerase/dehydratase
1.79
0.03
MSMEG_3477
possible inv protein
0.51
< 0.01
MSMEG_3489
conserved hypothetical protein, putative
0.70
0.01
MSMEG_3493
putative secreted protein
0.70
0.04
MSMEG_3495
MmpS5 protein
0.74
0.02
MSMEG_3497
transcriptional regulator, ArsR family protein
0.52
0.01
MSMEG_3513
enhanced intracellular survival protein
1.77
0.05
MSMEG_3522
dopamine receptor D4
0.52
< 0.01
MSMEG_3528
ErfK/YbiS/YcfS/YnhG family protein
0.57
0.01
MSMEG_3545
hypothetical protein
0.51
0.01
0.53
0.01
MSMEG_3550
ectoine/hydroxyectoine ABC transporter, ATP-binding
protein
MSMEG_3563
drug transporter
0.58
0.03
MSMEG_3572
transcriptional regulator, TetR family protein
0.47
0.05
MSMEG_3573
integral membrane protein, putative
0.17
< 0.01
MSMEG_3574
TPR domain protein
0.31
< 0.01
MSMEG_3580
antigen 85-C
0.66
0.02
MSMEG_3581
FabG protein
0.71
< 0.01
MSMEG_3596
ATPase
1.44
0.02
2.69
0.04
MSMEG_3601
ribose/xylose/arabinose/galactoside ABC-type
transport systems, permease components
MSMEG_3602
ribose transport ATP-binding protein RbsA
2.48
0.02
MSMEG_3606
transcriptional regulator
1.83
0.03
MSMEG_3608
acetyl-CoA acetyltransferase
1.56
0.01
MSMEG_3615
zinc-binding alcohol dehydrogenase family protein
0.46
0.01
MSMEG_3616
integral membrane protein
0.48
0.01
MSMEG_3620
conserved hypothetical protein
0.66
0.02
MSMEG_3621
NADH dehydrogenase
0.65
0.01
MSMEG_3625
urease, alpha subunit
0.69
0.04
MSMEG_3627
urease, gamma subunit
1.40
0.05
MSMEG_3631
integral membrane protein
1.20
0.04
XIII
M. smegmatis
locus
MSMEG_3636
Gene description
ferric iron-binding periplasmic protein of ABC
transporter
IR
pvalue
0.73
< 0.01
MSMEG_3647
forkhead-associated protein
0.79
0.04
MSMEG_3649
conserved hypothetical protein
1.66
0.04
MSMEG_3662
mannose-binding lectin
0.46
< 0.01
MSMEG_3667
para-nitrobenzyl esterase
0.64
0.03
MSMEG_3675
metal-dependent hydrolase
0.67
0.02
MSMEG_3676
phenoxybenzoate dioxygenase beta subunit
0.62
0.04
MSMEG_3685
conserved hypothetical protein
0.83
0.01
MSMEG_3740
ribosomal large subunit pseudouridine synthase B
1.42
0.03
MSMEG_3741
transcriptional regulator
1.34
0.03
MSMEG_3746
CTP synthase
1.43
0.02
MSMEG_3747
conserved hypothetical protein
2.24
0.01
MSMEG_3748
thiamin pyrophosphokinase, catalytic domain protein
2.05
0.03
MSMEG_3749
DNA repair protein RecN
1.94
0.03
1.13
0.01
MSMEG_3750
inorganic polyphosphate/ATP-NAD kinase
(Poly(P)/ATP NADkinase)
MSMEG_3764
conserved hypothetical protein
0.65
0.02
MSMEG_3767
acyl-CoA synthase
0.68
0.01
MSMEG_3786
D-amino acid deaminase
1.36
0.01
MSMEG_3791
ribosomal protein L20
0.80
0.01
MSMEG_3808
excinuclease ABC, A subunit
4.11
0.01
MSMEG_3815
possible drug efflux membrane protein
4.08
0.04
MSMEG_3816
excinuclease ABC, B subunit
6.85
< 0.01
MSMEG_3824
conserved hypothetical protein
1.40
0.03
MSMEG_3839
DNA polymerase I
3.54
0.01
MSMEG_3850
alkanesulfonate monooxygenase family protein
0.69
< 0.01
MSMEG_3862
FxsA cytoplasmic membrane protein
0.84
0.05
MSMEG_3863
pyridoxamine 5'-phosphate oxidase family protein
0.84
0.03
1.58
0.04
MSMEG_3880
nitrilase/cyanide hydratase and apolipoprotein Nacyltransferase
MSMEG_3886
twin arginine-targeting protein translocase TatC
0.90
0.03
MSMEG_3896
conserved hypothetical protein
0.81
0.03
MSMEG_3897
proteasome component
1.34
0.03
MSMEG_3898
ectoine hydroxylase
0.73
< 0.01
MSMEG_3899
ectoine synthase
0.52
0.01
MSMEG_3903
Low molecular weight antigen MTB12
0.64
0.02
XIV
pvalue
M. smegmatis
locus
Gene description
IR
MSMEG_3905
conserved hypothetical protein
0.64
0.04
MSMEG_3907
RecB family protein exonuclease
16.20
0.05
MSMEG_3908
conserved hypothetical protein
11.13
0.02
MSMEG_3910
conserved hypothetical protein
2.65
0.03
MSMEG_3915
NAD-dependent alcohol dehydrogenase
0.75
< 0.01
MSMEG_3916
hypothetical protein
0.77
0.05
MSMEG_3935
conserved hypothetical protein
1.83
0.02
MSMEG_3939
universal stress protein family protein
2.67
0.02
MSMEG_3940
universal stress protein family protein
2.94
0.03
MSMEG_3941
GAF family protein
1.85
0.01
MSMEG_3942
conserved hypothetical protein
2.42
0.04
MSMEG_3943
conserved hypothetical protein
1.82
0.02
MSMEG_3946
probable conserved transmembrane protein
2.64
< 0.01
MSMEG_3948
acyltransferase, ws/dgat/mgat subfamily protein
2.43
0.05
MSMEG_3949
hypothetical protein
2.58
0.02
MSMEG_3950
universal stress protein family protein
1.49
0.03
MSMEG_3960
transcriptional regulator
2.08
0.03
MSMEG_3982
acyl-CoA dehydrogenase
0.89
< 0.01
MSMEG_3992
integral membrane transport protein
0.77
0.03
MSMEG_3998
MmsAB operon regulatory protein
0.71
0.05
MSMEG_4013
oxidoreductase
1.60
0.03
MSMEG_4023
oxidoreductase
0.40
< 0.01
MSMEG_4025
transcriptional regulator, LysR family protein
0.35
< 0.01
MSMEG_4026
hypothetical protein
0.59
0.01
0.56
0.02
MSMEG_4027
zinc-containing alcohol dehydrogenase superfamily
protein
MSMEG_4030
conserved hypothetical protein
0.62
0.01
MSMEG_4031
hypothetical protein
0.67
< 0.01
MSMEG_4054
hypothetical protein
0.74
0.03
MSMEG_4066
conserved hypothetical protein
0.83
0.03
MSMEG_4070
transcriptional regulator, TetR family protein, putative
0.79
0.02
MSMEG_4072
ISMsm5, transposase
0.57
0.01
MSMEG_4074
peroxisomal trans-2-enoyl-CoA reductase
0.82
0.04
MSMEG_4083
putative monooxygenase
2.59
0.02
MSMEG_4084
putative acyl-CoA dehydrogenase
1.18
0.02
MSMEG_4121
GntR-family protein transcriptional regulator
1.54
0.02
MSMEG_4152
phenoxybenzoate dioxygenase beta subunit
0.90
0.03
XV
pvalue
M. smegmatis
locus
Gene description
IR
MSMEG_4180
ATP phosphoribosyltransferase
0.74
0.02
MSMEG_4181
phosphoribosyl-ATP pyrophosphohydrolase
1.28
0.01
MSMEG_4187
conserved hypothetical protein
0.61
0.05
MSMEG_4191
conserved hypothetical protein
0.86
0.02
MSMEG_4197
conserved hypothetical protein
0.82
0.03
MSMEG_4199
conserved hypothetical protein
0.74
0.01
MSMEG_4206
Molybdopterin oxidoreductase
1.10
0.02
MSMEG_4217
DivIVA protein
1.41
0.02
MSMEG_4218
possible conserved transmembrane protein
0.72
0.03
MSMEG_4219
conserved hypothetical protein
2.81
< 0.01
MSMEG_4221
conserved hypothetical protein
1.67
< 0.01
MSMEG_4222
cell division protein FtsZ
2.30
0.01
MSMEG_4226
UDP-N-acetylmuramate--alanine ligase
1.76
0.02
MSMEG_4229
UDP-N-acetylmuramoylalanine--D-glutamate ligase
1.54
0.04
MSMEG_4236
MraZ protein
1.66
< 0.01
MSMEG_4244
3-deoxy-7-phosphoheptulonate synthase
0.75
0.01
MSMEG_4247
probable conserved integral membrane protein
0.74
0.05
MSMEG_4249
conserved hypothetical protein
0.75
0.04
MSMEG_4256
NLP/P60 family protein
5.73
0.01
MSMEG_4257
conserved hypothetical protein
9.82
< 0.01
MSMEG_4259
DNA polymerase III, epsilon subunit
6.75
0.02
MSMEG_4271
conserved hypothetical protein
1.22
0.01
MSMEG_4276
branched-chain amino acid aminotransferase
1.39
0.01
MSMEG_4285
lipoyltransferase
0.82
0.02
MSMEG_4294
glutamine synthetase, type I
1.25
0.02
MSMEG_4307
conserved hypothetical protein
0.75
0.02
MSMEG_4313
glyoxalase/bleomycin resistance protein/dioxygenase
1.47
0.03
0.81
0.02
MSMEG_4320
alkyl hydroperoxide reductase/ Thiol specific
antioxidant/ Mal allergen
MSMEG_4332
glycerol-3-phosphate dehydrogenase 1
0.46
0.04
MSMEG_4334
flavoprotein
0.24
0.01
MSMEG_4336
conserved hypothetical protein
1.72
0.04
MSMEG_4338
possible transcriptional regulatory protein
0.75
0.01
0.80
0.02
MSMEG_4340
NAD/mycothiol-dependent formaldehyde
dehydrogenase
MSMEG_4342
metallo-beta-lactamase family protein
0.79
0.04
MSMEG_4362
universal stress protein family protein
1.28
0.02
XVI
pvalue
M. smegmatis
locus
Gene description
IR
MSMEG_4379
isochorismatase hydrolase
0.57
< 0.01
MSMEG_4399
conserved hypothetical protein
7.09
0.03
MSMEG_4449
putative transcriptional regulator
0.68
0.02
MSMEG_4450
alpha/beta hydrolase
0.17
< 0.01
MSMEG_4451
probable monooxygenase
0.16
< 0.01
MSMEG_4459
agmatinase
0.66
0.05
MSMEG_4461
conserved hypothetical integral membrane protein
1.36
0.02
MSMEG_4474
acyl-CoA oxidase
0.79
< 0.01
MSMEG_4490
undecaprenyl diphosphate synthase
3.55
< 0.01
MSMEG_4491
DNA repair protein RecO
2.82
< 0.01
MSMEG_4505
heat-inducible transcription repressor HrcA
1.29
0.03
MSMEG_4512
polyketide synthetase mbtd
0.52
0.02
0.73
0.02
MSMEG_4517
TetR-type transcriptional regulator of sulfur
metabolism
MSMEG_4522
ISMsm2, transposase
0.52
0.02
MSMEG_4522
ISMsm2, transposase
0.58
0.02
MSMEG_4535
glycoside hydrolase
0.76
0.01
MSMEG_4555
LppR protein
1.19
0.04
MSMEG_4556
GTP-binding protein LepA
0.66
< 0.01
MSMEG_4558
conserved hypothetical protein
1.46
0.04
MSMEG_4571
ribosomal protein S20
1.15
0.05
MSMEG_4582
conserved hypothetical protein
0.95
0.03
MSMEG_4613
hypothetical protein
0.82
0.04
MSMEG_4615
replicative DNA helicase
0.76
0.04
MSMEG_4617
glutamine-dependent NAD+ synthetase
0.65
0.04
MSMEG_4626
ribonuclease, Rne/Rng family protein
2.07
0.02
0.72
0.02
MSMEG_4645
alpha oxoglutarate ferredoxin oxidoreductase, beta
subunit
MSMEG_4646
pyruvate synthase
0.71
0.01
MSMEG_4650
hypothetical oxidoreductase YisS
2.46
0.04
MSMEG_4652
D-oliose 4-ketoreductase
1.87
0.03
MSMEG_4658
sugar ABC transporter substrate-binding protein
0.61
0.01
MSMEG_4674
trigger factor
3.25
0.03
MSMEG_4690
aminopeptidase N
4.02
< 0.01
MSMEG_4691
HNH nuclease
2.30
0.04
MSMEG_4693
conserved hypothetical protein
1.73
0.05
MSMEG_4695
protozoan/cyanobacterial globin family protein
1.14
0.02
XVII
M. smegmatis
locus
MSMEG_4712
MSMEG_4713
MSMEG_4716
Gene description
pyruvate dehydrogenase E1 component, alpha
subunit
HpcH/HpaI aldolase/citrate lyase family protein
acetyl-/propionyl-coenzyme A carboxylase alpha
chain
IR
pvalue
0.66
0.04
0.56
0.05
0.54
0.02
MSMEG_4717
Carboxyl transferase domain protein
0.42
0.01
MSMEG_4735
conserved hypothetical protein
1.80
0.02
MSMEG_4749
hypothetical protein
0.80
0.04
MSMEG_4759
hypothetical protein
0.45
< 0.01
MSMEG_4767
major facilitator superfamily protein permease
0.80
0.02
MSMEG_4769
conserved hypothetical protein
0.79
0.03
MSMEG_4788
virulence factor mce family protein
0.65
0.02
MSMEG_4789
hypothetical protein
0.68
< 0.01
MSMEG_4804
conserved hypothetical protein
1.55
0.04
0.73
0.04
MSMEG_4812
respiratory-chain NADH dehydrogenase domain, 51
kda subunit
MSMEG_4825
transcriptional repressor, TetR family protein, putative
0.79
0.04
MSMEG_4826
putative acyl-CoA dehydrogenase
0.52
< 0.01
MSMEG_4827
aminoglycoside phosphotransferase
0.51
< 0.01
MSMEG_4835
3-oxoacyl-[acyl-carrier-protein] reductase
1.28
0.04
MSMEG_4883
AMP-dependent synthetase and ligase
1.07
0.04
0.68
0.01
0.59
0.01
5.81
< 0.01
MSMEG_4888
MSMEG_4891
MSMEG_4899
periplasmic binding proteins and sugar binding
domain of the LacI family protein, putative
alkylhydroperoxide reductase
non-canonical purine NTP pyrophosphatase,
RdgB/HAM1 family protein
MSMEG_4901
ribonuclease PH
2.36
< 0.01
MSMEG_4918
1,4-alpha-glucan branching enzyme
0.70
< 0.01
MSMEG_4920
acetyl-CoA acetyltransferase
0.76
0.02
MSMEG_4923
conserved hypothetical protein
1.34
0.03
MSMEG_4927
IS1096, tnpR protein
1.15
0.04
MSMEG_4928
methylated-DNA--protein-cysteine methyltransferase
0.80
0.03
MSMEG_4935
ATP synthase F1, epsilon subunit
1.48
0.03
MSMEG_4944
hypothetical protein
1.56
< 0.01
MSMEG_4949
modification methylase, HemK family protein
1.44
0.04
MSMEG_4950
peptide chain release factor 1
1.69
0.04
MSMEG_4951
ribosomal protein L31
1.60
< 0.01
XVIII
pvalue
M. smegmatis
locus
Gene description
IR
MSMEG_4964
transcriptional regulator, TetR family protein
1.55
0.04
MSMEG_4965
hypothetical protein
1.48
0.01
MSMEG_4972
acetyltransferase
1.53
< 0.01
MSMEG_4974
rrf2 family protein (putative transcriptional regulator)
1.54
0.05
MSMEG_4977
3'(2'),5'-bisphosphate nucleotidase, putative
0.46
0.04
0.36
0.05
3.18
0.02
11.08
0.05
MSMEG_4978
MSMEG_5000
MSMEG_5002
sulfate adenylytransferase, large
subunit/adenylylsulfate kinase
transcriptional regulator, LysR family protein
conserved hypothetical protein (BLASTx: ATPase
involved in DNA repair)
MSMEG_5004
DNA repair exonuclease
18.00
0.01
MSMEG_5006
phosphohistidine phosphatase
2.17
< 0.01
MSMEG_5033
Tap protein
1.33
0.02
MSMEG_5041
probable acyltransferase
1.23
0.03
MSMEG_5044
ATPase
10.09
0.01
MSMEG_5058
ABC transporter, ATP-binding protein SugC
1.82
0.01
MSMEG_5065
Mg/Co/Ni transporter MgtE
1.61
0.01
MSMEG_5068
Mrp protein
1.45
0.05
MSMEG_5081
conserved hypothetical protein
2.04
< 0.01
MSMEG_5082
DNA-3-methyladenine glycosylase I
7.32
< 0.01
MSMEG_5083
conserved hypothetical protein
9.04
< 0.01
MSMEG_5084
glycosyl transferase, group 2 family protein
2.00
0.01
MSMEG_5101
hypothetical protein
2.15
0.02
MSMEG_5102
ABC transporter ATP-binding protein
1.86
0.04
MSMEG_5103
succinyl-diaminopimelate desuccinylase
0.80
0.04
MSMEG_5117
proline dehydrogenase
0.03
< 0.01
MSMEG_5119
1-pyrroline-5-carboxylate dehydrogenase
0.02
< 0.01
MSMEG_5123
acyl-CoA dehydrogenase, C- domain protein
1.71
< 0.01
MSMEG_5131
hypothetical protein
1.59
0.05
MSMEG_5136
helix-turn-helix motif
2.14
0.01
MSMEG_5142
beta-glucosidase A
1.48
0.02
MSMEG_5149
conserved transmembrane protein
1.91
0.03
MSMEG_5152
hypothetical protein
1.39
0.02
MSMEG_5152
hypothetical protein
1.85
0.02
1.81
0.02
18.53
0.02
MSMEG_5156
MSMEG_5157
base excision DNA repair protein, HhH-GPD family
protein
hypothetical protein
XIX
pvalue
M. smegmatis
locus
Gene description
IR
MSMEG_5165
integral membrane protein
1.87
0.02
MSMEG_5167
major facilitator superfamily protein MFS_1
1.96
0.02
MSMEG_5170
pyridoxamine 5'-phosphate oxidase family protein
1.38
0.03
MSMEG_5175
NAD-dependent deacetylase
1.54
0.02
MSMEG_5176
methyltransferase type 11
0.75
0.04
MSMEG_5177
regulatory protein, TetR
0.55
0.02
MSMEG_5180
conserved hypothetical protein
2.44
0.01
MSMEG_5182
2-Nitropropane dioxygenase
1.37
< 0.01
MSMEG_5183
3-Hydroxyacyl-CoA dehydrogenase
1.34
0.05
MSMEG_5186
HicB family protein
5.21
0.02
MSMEG_5187
tetracycline-resistance determinant TetV
3.06
< 0.01
MSMEG_5200
hypothetical protein
0.78
0.04
MSMEG_5201
regulatory protein GntR, HTH
1.45
0.01
MSMEG_5203
DoxX subfamily protein, putative
0.60
< 0.01
MSMEG_5205
conserved hypothetical protein
0.77
0.01
MSMEG_5210
hydrolase
1.49
0.03
MSMEG_5215
conserved hypothetical protein
1.92
0.02
MSMEG_5216
glyoxalase family protein
15.15
0.03
MSMEG_5220
esterase/lipase/thioesterase
1.27
0.02
MSMEG_5223
conserved hypothetical protein
1.49
0.01
MSMEG_5230
conserved hypothetical protein
1.26
0.04
MSMEG_5233
RmlD substrate binding domain superfamily protein
2.04
0.01
MSMEG_5235
short chain dehydrogenase family protein
1.47
0.04
MSMEG_5238
conserved hypothetical protein
1.36
0.01
MSMEG_5242
acyltransferase, ws/dgat/mgat subfamily protein
2.26
0.01
MSMEG_5243
helix-turn-helix motif
2.27
0.01
MSMEG_5244
two component transcriptional regulatory protein
1.56
0.04
MSMEG_5245
universal stress protein family protein
1.49
0.02
MSMEG_5248
acyl-[ACP] desaturase
2.20
0.01
MSMEG_5249
serine hydroxymethyltransferase
1.58
0.01
MSMEG_5263
transcription elongation factor GreA
1.44
0.02
MSMEG_5264
conserved hypothetical protein
1.59
< 0.01
MSMEG_5268
conserved hypothetical protein
1.36
< 0.01
MSMEG_5269
hypothetical protein
0.86
0.02
MSMEG_5303
sodium/proline symporter
0.12
< 0.01
MSMEG_5304
two-component sensor kinase
0.61
0.03
MSMEG_5308
conserved hypothetical protein
1.80
0.01
XX
pvalue
M. smegmatis
locus
Gene description
IR
MSMEG_5335
formamidase
0.46
< 0.01
MSMEG_5336
amidate substrates transporter protein
0.37
< 0.01
MSMEG_5337
putative regulatory protein, FmdB family
0.44
0.02
MSMEG_5341
dipeptidyl aminopeptidase/acylaminoacyl peptidase
1.88
0.02
MSMEG_5362
hypothetical protein
2.50
0.02
MSMEG_5363
hypothetical protein
3.07
0.04
MSMEG_5405
transcriptional regulator, ArsR family protein
1.22
0.02
MSMEG_5427
ribose-phosphate pyrophosphokinase
0.58
0.01
MSMEG_5429
LpqT protein
0.69
0.02
MSMEG_5432
peptidyl-tRNA hydrolase
1.50
0.03
MSMEG_5435
acyl-CoA synthase
0.58
0.01
MSMEG_5438
dimethyladenosine transferase
1.44
0.02
MSMEG_5449
conserved hypothetical protein
4.03
0.03
MSMEG_5468
conserved hypothetical protein
3.08
0.01
MSMEG_5472
5,10-methenyltetrahydrofolate synthetase
0.78
0.03
MSMEG_5477
2-hydroxy-3-oxopropionate reductase
1.75
0.04
MSMEG_5481
conserved hypothetical protein
0.78
0.01
MSMEG_5484
conserved hypothetical protein
2.35
0.02
MSMEG_5485
molybdopterin biosynthesis protein
1.84
0.02
MSMEG_5489
ribosomal protein L32
1.76
0.02
MSMEG_5511
von Willebrand factor, type A
1.14
0.03
MSMEG_5512
magnesium chelatase
1.57
0.02
MSMEG_5518
antigen 34 kDa
1.28
0.05
MSMEG_5534
ATP-dependent DNA helicase PcrA
3.75
0.02
MSMEG_5538
[NADP+] succinate-semialdehyde dehydrogenase
0.71
0.02
MSMEG_5546
membrane spanning protein
1.73
0.01
MSMEG_5551
stas domain protein
0.76
0.01
MSMEG_5552
conserved hypothetical protein
0.76
0.03
MSMEG_5556
MFS permease
1.47
0.05
MSMEG_5576
D-mannonate oxidoreductase
1.42
0.04
MSMEG_5582
hypothetical protein
2.49
0.02
MSMEG_5583
HNH endonuclease
4.95
0.02
MSMEG_5587
hypothetical protein
1.24
0.02
MSMEG_5594
ferredoxin-dependent glutamate synthase
1.39
0.04
MSMEG_5595
MarR-family protein transcriptional regulator
2.22
0.01
MSMEG_5625
cyclododecanone monooxygenase
1.76
0.03
MSMEG_5633
feruloyl-CoA synthetase
0.78
0.05
XXI
pvalue
M. smegmatis
locus
Gene description
IR
MSMEG_5634
conserved hypothetical protein
1.45
0.01
MSMEG_5643
conserved hypothetical protein
4.12
0.04
MSMEG_5658
epoxide hydrolase
0.77
0.02
MSMEG_5659
ABC transporter, ATP-binding protein
2.36
0.02
MSMEG_5660
ABC transporter ATP-binding protein
5.41
0.04
MSMEG_5672
citrate synthase I
0.60
< 0.01
MSMEG_5680
glyoxalase family protein
4.73
0.01
MSMEG_5685
DNA-binding protein
3.71
0.01
MSMEG_5704
conserved hypothetical protein
1.36
0.03
MSMEG_5706
DNA or RNA helicase of superfamily protein II
3.18
0.01
MSMEG_5717
pyridoxamine 5'-phosphate oxidase family protein
4.63
< 0.01
MSMEG_5732
monooxygenase
1.37
0.01
MSMEG_5733
universal stress protein family protein
2.15
0.01
MSMEG_5749
hypothetical protein
0.69
0.02
MSMEG_5751
putative adhesin/hemolysin
1.35
0.03
MSMEG_5753
serine/threonine specific protein phosphatase
9.54
0.03
MSMEG_5779
phosphate ABC transporter, ATP-binding protein
0.52
< 0.01
MSMEG_5780
phosphate ABC transporter, permease protein PstA
0.48
< 0.01
MSMEG_5781
phosphate ABC transporter, permease protein PstC
0.29
< 0.01
0.27
< 0.01
MSMEG_5782
phosphate ABC transporter, phosphate-binding
protein PstS
MSMEG_5784
transcriptional regulatory protein
1.25
0.04
MSMEG_5791
conserved hypothetical protein
1.33
0.02
MSMEG_5792
conserved hypothetical protein
1.24
0.04
MSMEG_5796
Glycine cleavage T-protein (aminomethyl transferase)
1.56
0.03
MSMEG_5797
conserved hypothetical protein
1.46
0.01
MSMEG_5805
hypothetical protein
0.80
0.03
MSMEG_5824
phosphoribosylformylglycinamidine synthase II
1.13
0.01
MSMEG_5827
glyoxalase family protein
6.43
< 0.01
MSMEG_5831
phosphoribosylformylglycinamidine synthase I
0.77
0.02
MSMEG_5842
conserved hypothetical protein
0.69
0.03
MSMEG_5861
cytochrome P450 109
0.89
0.04
MSMEG_5863
cytochrome P450 51
0.75
0.01
MSMEG_5876
H-N-H endonuclease F-TflIV
6.88
0.02
MSMEG_5894
conserved hypothetical protein
1.33
0.02
MSMEG_5900
virulence factor Mce family protein
1.35
0.01
MSMEG_5920
FMN-dependent monooxygenase
0.69
0.01
XXII
pvalue
M. smegmatis
locus
Gene description
IR
MSMEG_5931
short chain dehydrogenase
0.57
0.02
MSMEG_5934
conserved hypothetical protein
3.74
< 0.01
MSMEG_5935
ATP-dependent DNA helicase
5.97
< 0.01
MSMEG_6005
conserved hypothetical protein
4.39
< 0.01
MSMEG_6006
conserved hypothetical protein
5.85
0.02
MSMEG_6007
cation diffusion facilitator family transporter
0.63
0.02
MSMEG_6032
transcriptional regulator, TetR family protein
1.52
0.04
MSMEG_6053
cob(II)yrinic acid a,c-diamide reductase
1.20
0.02
MSMEG_6074
cysteinyl-tRNA synthetase
1.49
0.01
1.51
0.05
MSMEG_6076
2-C-methyl-D-erythritol 4-phosphate
cytidylyltransferase
MSMEG_6079
DNA repair protein RadA
7.47
0.02
MSMEG_6080
conserved hypothetical protein
5.70
< 0.01
MSMEG_6081
conserved hypothetical protein
2.08
0.02
MSMEG_6087
beta-lactamase
1.50
< 0.01
MSMEG_6090
conserved hypothetical protein
1.21
0.02
MSMEG_6092
Lsr2 protein
1.62
< 0.01
1.79
0.04
MSMEG_6099
probable conserved transmembrane protein rich in
alanine
MSMEG_6104
GTP cyclohydrolase I
0.71
0.01
MSMEG_6110
hypoxanthine phosphoribosyltransferase
1.51
0.01
MSMEG_6163
conserved hypothetical protein
1.43
0.04
MSMEG_6168
bacterial type II secretion system protein F domain
0.53
0.01
MSMEG_6187
endonuclease III
2.91
0.02
MSMEG_6225
proton antiporter efflux pump
0.58
0.01
0.79
0.03
MSMEG_6235
thiopurine S-methyltransferase (tpmt) superfamily
protein
MSMEG_6246
pyridoxal-phosphate-dependent transferase
3.99
0.02
MSMEG_6248
conserved hypothetical protein
3.83
0.01
MSMEG_6249
conserved hypothetical protein
4.63
0.01
MSMEG_6250
glutamate--cysteine ligase
6.14
< 0.01
MSMEG_6272
NAD-glutamate dehydrogenase
2.57
0.05
MSMEG_6281
N-acetylmuramoyl-L-alanine amidase
0.68
0.03
MSMEG_6284
cyclopropane-fatty-acyl-phospholipid synthase
0.76
< 0.01
MSMEG_6285
DNA polymerase III gamma/tau subunit
2.51
< 0.01
MSMEG_6286
aspartate transaminase
1.59
0.03
MSMEG_6302
DNA ligase
0.74
0.05
XXIII
pvalue
M. smegmatis
locus
Gene description
IR
MSMEG_6327
cytidine and deoxycytidylate deaminase family protein
0.89
0.03
MSMEG_6329
conserved hypothetical protein
1.44
0.02
MSMEG_6342
oxidoreductase
0.71
0.03
MSMEG_6345
conserved hypothetical protein
1.26
0.01
MSMEG_6354
serine esterase, cutinase family protein
1.60
0.02
MSMEG_6368
DNA-binding protein
0.70
0.04
MSMEG_6370
4-carboxymuconolactone decarboxylase
0.65
< 0.01
MSMEG_6379
transporter, major facilitator family protein
0.63
0.01
MSMEG_6385
hypothetical oxidoreductase in MprA 5'region
0.70
0.01
MSMEG_6397
hypothetical protein
0.68
0.03
MSMEG_6398
antigen 85-A
0.56
0.01
MSMEG_6403
bifunctional udp-galactofuranosyl transferase
0.86
0.01
MSMEG_6406
N-acetylmuramoyl-L-alanine amidase
1.94
0.03
MSMEG_6409
acyltransferase family protein
1.24
0.01
MSMEG_6410
Rieske 2Fe-2S family protein
0.41
< 0.01
MSMEG_6412
conserved hypothetical protein
0.81
0.02
MSMEG_6414
conserved hypothetical protein
0.76
0.03
MSMEG_6416
phosphoglycerate mutase family protein
1.39
< 0.01
MSMEG_6417
conserved hypothetical protein
1.79
< 0.01
MSMEG_6418
prephenate dehydratase
1.34
< 0.01
1.28
< 0.01
MSMEG_6423
Glycerophosphoryl diester phosphodiesterase family
protein
MSMEG_6425
rhodanese-like domain protein
1.33
< 0.01
MSMEG_6426
conserved hypothetical protein
1.62
0.03
MSMEG_6443
DNA polymerase IV
4.05
0.04
MSMEG_6453
sulfate permease
0.47
< 0.01
MSMEG_6467
starvation-induced DNA protecting protein
1.84
0.04
MSMEG_6468
conserved hypothetical protein
3.08
0.02
MSMEG_6478
putative cytochrome P450 135B1
0.48
0.01
MSMEG_6490
major facilitator superfamily protein
1.76
0.03
MSMEG_6491
aldehyde dehydrogenase
1.70
0.01
0.81
0.05
MSMEG_6495
ABC nitrate/sulfonate/bicarbonate family protein
transporter, inner membrane subunit, putative
MSMEG_6498
hypothetical protein
1.37
0.05
MSMEG_6507
glycogen debranching enzyme GlgX
0.55
< 0.01
MSMEG_6515
trehalose synthase
0.72
0.04
MSMEG_6543
GAF domain protein
2.00
0.04
XXIV
pvalue
M. smegmatis
locus
Gene description
IR
MSMEG_6558
putative enoyl-CoA hydratase
0.78
< 0.01
0.69
0.04
1.30
0.04
MSMEG_6581
MSMEG_6594
oxidoreductase, short chain dehydrogenase/reductase
family protein
amino acid ABC transporter, permease protein, 3-TM
region, His/Glu/Gln/Arg/opine
MSMEG_6619
conserved hypothetical protein
3.06
0.04
MSMEG_6620
conserved hypothetical protein
2.98
< 0.01
MSMEG_6635
alpha subunit of malonate decarboxylase
1.34
0.02
MSMEG_6641
nitrilotriacetate monooxygenase component A
1.29
0.01
MSMEG_6645
2-methylcitrate dehydratase 2
0.53
0.03
MSMEG_6646
methylisocitrate lyase
0.30
< 0.01
MSMEG_6647
citrate synthase 2
0.58
0.01
MSMEG_6654
TnpC protein
1.65
0.04
0.26
< 0.01
MSMEG_6668
ABC transporter, periplasmic substrate-binding
protein, putative
MSMEG_6688
regulatory protein
0.83
0.05
MSMEG_6694
conserved domain protein
1.14
0.05
MSMEG_6726
ABC transporter, permease protein
0.81
0.05
MSMEG_6739
hypothetical protein
5.19
0.01
MSMEG_6743
lactoylglutathione lyase
0.34
0.05
MSMEG_6757
glycerol operon regulatory protein
2.76
0.04
MSMEG_6779
conserved hypothetical protein, putative
1.83
0.02
MSMEG_6783
integral membrane protein
4.41
< 0.01
MSMEG_6808
hypothetical protein
0.79
0.03
MSMEG_6829
transcriptional regulatory protein
1.46
0.01
MSMEG_6880
hydrophobic amino acid ABC transporter, putative
0.61
< 0.01
MSMEG_6883
hypothetical protein
0.74
0.02
MSMEG_6892
replicative DNA helicase
12.62
0.04
MSMEG_6898
hypothetical protein
0.78
0.01
MSMEG_6913
putative transcriptional regulatory protein
1.28
0.03
MSMEG_6917
leucyl-tRNA synthetase
0.77
0.01
MSMEG_6919
proline-rich 28 kDa antigen
0.38
< 0.01
MSMEG_6920
hypothetical protein
0.54
0.01
MSMEG_6921
conserved hypothetical protein
1.35
0.02
MSMEG_6923
conserved hypothetical protein
1.28
0.03
MSMEG_6941
R3H domain-containing protein
1.20
0.03
XXV