Download Novel regulatory roles of cAMP receptor proteins in

Microbiology (2015), 161, 648–661
DOI 10.1099/mic.0.000015
Novel regulatory roles of cAMP receptor proteins in
fast-growing environmental mycobacteria
Htin Lin Aung,1,2 Laura L. Dixon,3 Laura J. Smith,3 Nathan P. Sweeney,4
Jennifer R. Robson,1 Michael Berney,1,5 Roger S. Buxton,4 Jeffrey Green3
and Gregory M. Cook1,2
Correspondence
Greg M. Cook
[email protected]
1
Department of Microbiology and Immunology, Otago School of Medical Sciences,
University of Otago, Dunedin, New Zealand
2
Maurice Wilkins Centre for Molecular Biodiscovery, The University of Auckland,
Private Bag 92019, Auckland 1042, New Zealand
3
The Krebs Institute for Biomolecular Research, Department of Molecular Biology and
Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, UK
4
Division of Mycobacterial Research, MRC National Institute for Medical Research, Mill Hill,
London NW7 1AA, UK
5
Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx,
NY 10461, USA
Received 12 December 2014
Accepted 15 December 2014
Mycobacterium smegmatis is a fast-growing, saprophytic, mycobacterial species that contains
two cAMP-receptor protein (CRP) homologues designated herein as Crp1 and Crp2.
Phylogenetic analysis suggests that Crp1 (Msmeg_0539) is uniquely present in fast-growing
environmental mycobacteria, whereas Crp2 (Msmeg_6189) occurs in both fast- and slowgrowing species. A crp1 mutant of M. smegmatis was readily obtained, but crp2 could not be
deleted, suggesting it was essential for growth. A total of 239 genes were differentially regulated
in response to crp1 deletion (loss of function), including genes coding for mycobacterial energy
generation, solute transport and catabolism of carbon sources. To assess the role of Crp2 in M.
smegmatis, the crp2 gene was overexpressed (gain of function) and transcriptional profiling
studies revealed that 58 genes were differentially regulated. Identification of the CRP promoter
consensus in M. smegmatis showed that both Crp1 and Crp2 recognized the same consensus
sequence (TGTGN8CACA). Comparison of the Crp1- and Crp2-regulated genes revealed
distinct but overlapping regulons with 11 genes in common, including those of the succinate
dehydrogenase operon (MSMEG_0417-0420, sdh1). Expression of the sdh1 operon was
negatively regulated by Crp1 and positively regulated by Crp2. Electrophoretic mobility shift
assays with purified Crp1 and Crp2 demonstrated that Crp1 binding to the sdh1 promoter was
cAMP-independent whereas Crp2 binding was cAMP-dependent. These data suggest that Crp1
and Crp2 respond to distinct signalling pathways in M. smegmatis to coordinate gene expression
in response to carbon and energy supply.
INTRODUCTION
cAMP receptor proteins (CRPs) are members of the CRP–
FNR (fumarate nitrate reductase regulator) superfamily of
transcription factors. The ~370 family members control a
Abbreviations: CCR, carbon catabolite repression; CRP, cAMP-receptor
protein; ED pathway; Entner–Doudoroff pathway; EMP pathway,
Embden–Meyerhof–Parnas pathway; EMSA, electrophoretic mobility
shift assay; FNR, fumarate nitrate reductase regulator; qRT-PCR,
quantitative real-time PCR.
Three supplementary tables and six supplementary figures are available
with the online Supplementary Material.
648
wide range of physiological functions in a diverse set
of bacteria. Thus, different members of the family regulate
carbon, sulfur and nitrogen metabolism, denitrification,
nitrogen fixation, aerobic and anaerobic respiration and
virulence gene expression in response to a range of environmental and metabolic signals (Green et al., 2001, 2014;
Kolb et al., 1993; Shimada et al., 2011). Members of the
CRP–FNR family are homodimeric proteins with a sensory
domain and a helix–turn–helix DNA-binding domain
located at the N- and C-terminal regions, respectively, of
each protomer (Weber & Steitz, 1987). The archetypal CRP
fold is a versatile structure and its N-terminal domain has
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Roles of CRPs in Mycobacterium smegmatis
evolved to accommodate different sensory modules to respond
to a diverse range of signals (Green et al., 2001; Körner et al.,
2003).
In Escherichia coli, CRP controls gene expression in response
to changes in intracellular cAMP concentration. Carbon
catabolite repression (CCR) is a vital part of a global control
system in bacteria to achieve hierarchical carbon usage
(Kovárová-Kovar & Egli, 1998). During this process,
depletion of glucose leads to activation of adenylyl cyclase
by the phosphorylated form of the EIIA protein of the
phosphotransferase system (PTS) (Saier & Reizer, 1994). As
a result of this activation, the levels of cAMP increase, which
in turn binds to CRP, resulting in the formation of the
cAMP–CRP binary complex (Saier & Reizer, 1994). The
cAMP–CRP complex binds at promoters containing a
specific DNA sequence (consensus 59-TGTGAN6TCACA39) to regulate expression of the downstream genes (Berg &
von Hippel, 1988). In addition to mediating CCR, a recent
study demonstrated that cAMP–CRP signalling is involved
in coordinating the expression of catabolic proteins with
biosynthetic (anabolic) and ribosomal proteins in response
to cellular metabolic demands (You et al., 2013). There are
currently estimated to be between 378 and ~500 E. coli genes
under the control of cAMP–CRP, including those encoding
the transporters and catabolism of glucose and the process of
aerobic respiration, demonstrating that CRP is a global
regulator of gene expression in E. coli (Perrenoud & Sauer,
2005; Shimada et al., 2011).
Most mycobacterial genomes harbour 10 or more genes
encoding purported adenylyl cyclases in contrast to the single
adenylyl cyclase found in E. coli (Shenoy & Visweswariah,
2006). For example, Mycobacterium tuberculosis harbours 16
genes encoding adenylyl cyclases (McCue et al., 2000; Shenoy
et al., 2004). Consequently, intracellular cAMP concentrations in mycobacterial cells are considered to be ~100-fold
higher than those of other bacteria (Dass et al., 2008; Padh &
Venkitasubramanian, 1976, 1980), but the significance of
this is not known. Secretion of cAMP directly into host
macrophages after infection has been reported and is thought
to be important in tuberculosis pathogenesis (Agarwal et al.,
2009; Bai et al., 2009; Lowrie et al., 1975). M. tuberculosis
H37Rv contains a single CRP–FNR homologue encoded by
the gene Rv3676 (Cole et al., 1998), which is 32 % identical to
the E. coli CRP over 189 amino acid residues. Most of the
amino acid residues contributing to cAMP binding and DNA
binding in E. coli CRP are conserved in M. tuberculosis CRP
(Bai et al., 2005; Rickman et al., 2005). M. tuberculosis CRP
has been reported to regulate expression of genes required to
control metabolism and growth under hypoxic and starvation conditions (Bai et al., 2005). Furthermore, deletion of
Rv3676 (crp) resulted in impaired growth in macrophage cell
lines and in mice, indicative of a role of CRP (Rv3676) in
tuberculosis pathogenesis (Hunt et al., 2008; Rickman et al.,
2005).
Unlike M. tuberculosis, the genome of the fast-growing
environmental saprophyte Mycobacterium smegmatis mc2155
http://mic.sgmjournals.org
encodes two purported CRPs, MSMEG_0539 (Crp1) and
MSMEG_6189 (Crp2) (http://cmr.jcvi.org/). The presence of
multiple copies of CRP in bacterial genomes is unusual and
warrants investigation. In this communication, we show that
Crp1 and Crp2 of M. smegmatis have distinct roles in
controlling transport and catabolism of carbon sources and
components of the electron transport chain for energy
generation.
METHODS
Bacterial strains and cultivation conditions. E. coli DH10B was
grown in Luria–Bertani (LB) (Sambrook & Russell, 2001) medium at
37 uC with agitation at 200 r.p.m. or on LB agar plates (Table S1,
available in the online Supplementary Material). M. smegmatis
mc2155 (Snapper et al., 1990) and derived strains (Table S1) were
grown in LB supplemented with 0.05 % (w/v) Tween 80 (Sigma
Aldrich) (LBT), Hartmans-de Bont (HdB) minimal medium
supplemented with 10 mM glycerol and 0.05 % (w/v) Tween 80
unless otherwise stated or on LBT agar plates. All M. smegmatis
strains were inoculated to an initial optical density of 0.005 and
grown at 37 uC with agitation at 200 r.p.m. Samples (2 ml) to
measure b-galactosidase expression were taken (see below), the
optical density was measured and samples were then stored at
220 uC. All solid media contained 1.5 % agar and liquid media
contained ampicillin (100 mg ml21), kanamycin (50 mg ml21 for E.
coli; 20 mg ml21 for M. smegmatis), hygromycin B (200 mg ml21 for
E. coli; 50 mg ml21 for M. smegmatis) or tetracycline (20 ng ml21 for
M. smegmatis). The optical density was measured at 600 nm (OD600)
in a Jenway 6300 spectrometer. All strains, plasmids and primers used
in this study are listed in Table S1.
Phylogenetic analysis. Protein sequences of crp homologues were
imported into MEGA 5 software (Tamura et al., 2011) from
MicrobesOnline (Dehal et al., 2010) and the Comprehensive
Microbial Resource (Peterson et al., 2001). Evolutionary history was
reconstructed using the neighbour-joining method with a bootstrap
test (1000 replicates) to determine the percentage of replicate trees in
which the associated taxa clustered together. Branch lengths were
represented in the same units as those of the evolutionary distances
used to infer the phylogenetic tree. Phylogenetic analyses were
performed using MEGA 5 (Tamura et al., 2011).
Construction of M. smegmatis mutants and complementation
vector. All molecular biology techniques were performed according
to standard procedures (Sambrook & Russell, 2001). Restriction
enzymes and other molecular biology reagents were obtained from
Roche Diagnostics or New England Biolabs. Genomic DNA of M.
smegmatis was isolated as described previously (Gebhard et al., 2006).
To create a markerless deletion of MSMEG_0539 (Crp1), a 932 bp
fragment upstream of Crp1 including a 115 bp coding sequence was
amplified with primers HLA34 and HLA35, and a 1014 bp fragment
downstream of Crp1 including a 96 bp coding sequence was
amplified with primers HLA36 and HLA37. These two PCR products
were used as a template for overlap extension PCR (Ho et al., 1989)
and the product was then cloned into the SpeI site of the pX33 vector
(Gebhard et al., 2006), the pPR23-derived vector (Pelicic et al., 1997),
resulting in pHLA13, and transformed into M. smegmatis mc2155.
The crp1 gene was then deleted using the two-step method for
integration and excision of the plasmid as described previously (Tran
& Cook, 2005). Similarly, a 1012 bp fragment upstream of Crp2
including a 156 bp coding sequence was amplified with primers
HLA39 and HLA40, and a 926 bp fragment downstream of Crp2
including a 102 bp coding sequence was amplified with primers
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H. L. Aung and others
HLA40 and HLA41. The construct for making a crp2 deletion strain
was made as described above, resulting in pHLA14, and deletion was
performed as described above. In addition, a fragment containing a
500 bp coding sequence of crp2 was amplified and cloned into the
SpeI site of the pX33 vector, resulting in pHLA15. The deletion of
genes was confirmed by Southern analysis using the Amersham Gene
Images AlkPhos Direct Labelling and Detection System with CDPStar detection reagent (GE Healthcare) according to the manufacturer’s protocol.
To complement the crp1 deletion mutant with the crp1 gene, crp1 was
amplified from genomic DNA using the primers HLA54 and HLA55
(Table S1) and the Phusion High-Fidelity PCR kit (New England
Biolabs). The product was then cloned into the multiple cloning site
of E. coli–mycobacteria shuttle vector pMV261(Stover et al., 1991)
using restriction sites EcoRI and HindIII to create pHLA21. The
plasmid was then used to transform E. coli DH10B for amplification,
and the sequence of the plasmid was checked using the PCR primers
described above. The correct construct was used to transform the M.
smegmatis crp1 mutant strain. In addition, the vector pMV261 was
used to transform the M. smegmatis wild-type and mutant strains.
Construction of tetracycline inducible construct. To create the
Crp2 tetracycline inducible expression construct, a 678 bp PCR
product of the crp2 gene was amplified from M. smegmatis mc2155
using primers HLA52, containing a consensus ribosome-binding site
sequence GGAGG upstream of crp2, and HLA53. The product was
digested with NdeI and SpeI and ligated into the tetracycline inducible
vector pMind (Blokpoel et al., 2005), digested with the same restriction enzymes. The resulting construct was confirmed via sequencing
and was designated pHLA20. M. smegmatis mc2155 strains HLA126
and HLA107, harbouring plasmids pMind and pHLA20, respectively,
were grown in HdB minimal medium supplemented with 10 mM
glycerol and 0.05 % (w/v) Tween 80 to an OD600 of 0.3, and expression was then induced with tetracycline (20 ng ml21). All cultures
were supplemented with hygromycin B.
Construction of promoter transcriptional fusions. To measure
promoter activity, crp1–lacZ, MSMEG_0420–lacZ and sdhB–lacZ
fusion constructs were created using the primers listed in Table S1.
The 500 bp upstream of the crp1 translational start site, 300 bp
upstream of the crp2 translational start site, 227 bp upstream of the
MSMEG_0420 translational start site or 441 bp upstream of the sdhB
translational start site were PCR amplified with the primers listed in
Table S1 and ligated into the BamHI and KpnI restriction sites of the
vector pJEM15 to create plasmids pHLA25, pHLA5, pJEM07 and
pJEM113, respectively. All the promoter regions amplified by PCR
were confirmed by DNA sequencing. The plasmids were used to
transform M. smegmatis mc2155 by electroporation. b-Galactosidase
assays were performed as described previously (Gebhard et al., 2006).
Carbon source concentration assay. Cultures were grown in HdB
with glycerol and glucose, glycerol and pyruvate or glucose and
pyruvate. Supernatant samples were taken after initial inoculation to
determine the starting carbon concentration of each individual flask
and at specified time points during growth. All culture samples
were centrifuged (13 000 r.p.m. 16 000 g, 5 min) to obtain cell-free
supernatant for use in carbon source concentration assay and stored
at 220 uC. Glycerol concentration was measured by detecting NADH
oxidation (A340) using the methods described by Pinter et al. (1967).
The assay solution contained: 0.5 U glycerokinase ml21 from E. coli
(Sigma Aldrich), 0.5 U pyruvate kinase ml21 from rabbit muscle
(Sigma Aldrich), 1 U lactic dehydrogenase from ml21 from rabbit
muscle (Roche), 50 mM Tris/HCl (pH 8), 2 mM MgCl2, 0.25 mM
NADH, 3 mM phosphoenolpyruvate and 3 mM ATP. Approximately
100 ml of each sample was added to 1 ml of assay solution, mixed and
incubated at 37 uC for 15 min to allow the enzyme assay to reach an
650
end point. Similarly, glucose levels were determined using a
hexokinase-based assay (Bergmeyer et al., 1983). The assay solution
contained: 25 ml of a 50 mM Tris/HCl (pH 7.5) solution, 20 mg
MgCl2, 10 mg ATP, 10 mg NADP and 75 ml of a hexokinase/glucose 6phosphate dehydrogenase (G6P-DH) enzyme mix (Roche) that is
equivalent to approximately 25 U hexokinase activity and 13 U glucose
6-phosphate dehydrogenase activity. Approximately 150 ml of each
sample was added to 1 ml of assay solution, mixed and incubated at
25 uC for 30 min to allow the enzyme assay to reach an end point.
Pyruvate levels were determined by detecting NADH oxidation (A340)
using a modified version of the method of Pinter et al. (1967). The
assay solution contained: 1 U lactic dehydrogenase ml21 from rabbit
muscle (Roche), 50 mM Tris/HCl (pH 8), 2 mM MgCl2, 0.25 mM
NADH and 3 mM ATP. Approximately 100 ml of each sample was
added to 1 ml of assay solution, mixed and incubated at 37 uC for
15 min to allow the enzyme assay to reach an end point. The A340 of
each sample was measured and a standard curve of absorbance versus
carbon source concentration was analysed with each set of samples,
which was prepared from either 10 mM glycerol, 10 mM glucose or
10 mM pyruvate stock.
RNA extraction, microarray analysis and quantitative real-time
PCR (qRT-PCR). To extract total RNA, cells were resuspended in
TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. Cells were lysed with two rounds of bead beating in a MiniBeadbeater (Biospec) at 5000 r.p.m. for 30 s. DNA was removed from
the RNA preparation by treatment with 2 U RNase-free DNase using
the TURBO DNA-free kit (Ambion) according to the manufacturer’s
instructions. The quality of RNA was checked on a 1 % agarose gel
and the concentration was determined using a NanoDrop ND-1000
spectrophotometer. Microarray analysis was performed as described
previously (Berney & Cook, 2010; Berney et al., 2012) using arrays
provided by the Pathogen Functional Genomics Research Centre
funded by the National Institute of Allergy and Infectious Diseases
using protocols SOP# M007 and M008 from The Institute of Genomic
Research. Gene expression was calculated from the normalized signal
intensities from replicate spots within a single slide and averaged for
each set of biological replicates before expression ratios were calculated.
The results from four biological replicates, which included two dye
swaps, were then subjected to a t-test without false discovery correction
in The Institute of Genomic Research MeV software (version 4.3.02).
The mean value to be tested against was set to 1 with a critical P-value
of 0.05. The analysis was used as a ranking method. For a general
overview, genes with expression ratios ¢1.4 and ¡0.7 and a P-value of
¡0.05 were used for data interpretation. All data have been deposited
at Gene Expression Omnibus (NCBI) with accession number
GSE56057. qRT-PCR was performed as described previously (Berney
& Cook, 2010). The sigA gene was used as an internal standard and the
DDCT method was used for calculation of gene expression ratios. Error
bars represent standard deviations from three biological replicates.
Recombinant expression and purification of Crp1 and Crp2
from M. smegmatis mc2155. The crp1 gene sequence of M.
smegmatis mc2155 was amplified by PCR using primers HLA64 and
HLA65 and cloned into pQE80L, which contains an N-terminal Histag (Qiagen), using KpnI and HindIII restriction sites (Table S1). The
resulting plasmid pHLA27 was used to transform the expression
strain JRG5876 E. coli BL21(lDE3Dcya), which is unable to synthesize
cAMP (Stapleton et al., 2010). The authors validated that the
recombinant protein was not bound to cAMP (Stapleton et al., 2010).
Cultures were grown in 2 l flasks with 500 ml LB medium
supplemented with 100 mg ampicillin ml21 at 37 uC with agitation
at 200 r.p.m. until an OD600 of approximately 0.5 was reached.
Expression was then induced by the addition of IPTG (1 mM final
concentration) prior to an additional 4 h of growth. Cells were
harvested by centrifugation (7000 r.p.m. 7519 g, 4 uC, 15 min),
washed and resuspended in lysis buffer [150 mM Tris/HCl, 2 mM
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Microbiology 161
Roles of CRPs in Mycobacterium smegmatis
MgCl2, 1 % glycerol, one Complete Mini protease inhibitors cocktail
tablet (Roche) per 7.5 ml and 5 mg DNase (Roche)] prior to cell
disruption. Cells were disrupted by three passages through a French
pressure cell (American Instrument Company) at 20 000 p.s.i. (138 MPa).
Unbroken cells were then removed by centrifugation (10 000 r.p.m.
11 872 g, 10 min, 4 uC) and the cell-free supernatant was collected
from the membranes by ultracentrifugation (45 000 r.p.m. 207 871 g,
45 min, 4 uC). The supernatant containing cytoplasmic proteins was
then loaded at a flow rate of 0.5 ml min21 onto a 1 ml HisTrap (GE
Healthcare) equilibrated with 10 column volumes of buffer A [20 mM
Tris (pH 7.2), 500 mM NaCl, 20 mM imidazole, 10 % glycerol (v/v),
one Complete Mini protease inhibitors cocktail tablet (Roche) per
7.5 ml]. Unbound samples were removed by washing with five column
volumes of buffer A. The column was eluted with a gradient of buffer B
[20 mM Tris (pH 7.2), 500 mM NaCl, 400 mM imidazole, 10 % glycerol
(v/v), one Complete Mini protease inhibitors cocktail tablet (Roche) per
7.5 ml] at a flow rate of 1 ml min21 over 30 column volumes, reaching
100 % B. Eluted fractions were analysed by SDS-PAGE (12.5 %) and
visualized with Simply blue stain (Invitrogen). Elution fractions
containing hexa-histidine-tagged Crp1 were pooled and concentrated
using a centrifugal filter with a 10 kDa molecular mass cut-off filter
(Amicon). Protein concentration was determined using a BCA protein
assay kit (Pierce). The presence of the 66 His tag was confirmed by
immunoblotting using a horseradish peroxidase-linked anti-His polyclonal antibody (AbCam) and visualized using chemiluminescence
(SuperSignal West Pico chemiluminescent substrate; Thermo Scientific).
The identification of Crp1 was further confirmed by MALDI TOF/TOF
MS on a 4800 MALDI TOF/TOF analyser (AB Sciex).
For Crp2 expression, the expression plasmid pNS24, a pET28a
(Invitrogen) derivative encoding a His6–MSMEG_6189 fusion
protein, was constructed using primers PRNS_17F and PRNS_17R
(Table S1). The plasmid was used to transform electro-competent E.
coli strain JRG5876 (Stapleton et al., 2010) and the Crp2 protein was
typically overproduced by culturing the resulting strain in a 2 l flask
containing 500 ml Lennox Broth (5 g NaCl, 5 g yeast extract, 10 g
tryptone) with 200 mg ampicillin ml21, inoculated 1 : 100 from an
overnight culture. The cultures were grown aerobically at 37 uC with
shaking (250 r.p.m.) until an OD600 of ~0.6 was reached. IPTG was
then added (up to 120 mg ml21 final concentration). Cultures were
then incubated at 37 uC with shaking (250 r.p.m.) for 2–3 h before
collecting the bacteria by centrifugation at 11 900 g for 30 min at
4 uC. Cell pellets were resuspended in 10 ml of binding buffer
(20 mM sodium phosphate, 0.5 M NaCl, pH 7.4) and lysed by two
passages through a French pressure cell at 16 000 p.s.i. (110 MPa). The
soluble and insoluble fractions were separated by centrifugation at
39 190 g for 15 min at 4 uC. The soluble fraction was used immediately
for purification of His-tagged Crp2 by nickel affinity chromatography.
Purification was performed using the His-tag purification program for
a 1 ml HiTrap chelating column of the AKTA prime machine (GE
Healthcare) according to the manufacturer’s instructions. The bound
His-tagged Crp2 protein was eluted by applying an imidazole gradient
(0–0.5 M in 20 mM sodium phosphate, 0.5 M NaCl, pH 7.4). The
fractions collected were stored at 4 uC and samples were analysed by
SDS-PAGE to locate the target protein.
Electrophoretic mobility shift assays. Purified M. smegmatis Crp1
was used in electrophoretic mobility shift assays (EMSAs) using a 2nd
Generation DIG Gel-Shift kit (Roche) to 39-end-label target DNA
with DIG. The 227 and 431 bp probes designated sdh1 and sdh2 were
obtained using the primers described in Table S1. Binding reactions
were performed by incubating 0.4 ng DIG-labelled DNA with Crp1 in
the presence or absence of 0.2 mM cAMP in 10 ml reaction volumes.
The gel-shift reactions were then loaded into a 6 % native acrylamide
gel (37.5 : 1 acrylamide/bis) and were electrophoresed in 0.56 TBE
(44.5 mM Tris, 44.4 mM borate and 10 mM EDTA) at 300 V for
20 min. Protein-bound DIG-labelled DNA was then blotted using an
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Xcell II Blot Module (Invitrogen) and fixed to a nylon N+
membrane (GE Healthcare) and was detected by a chemiluminescent
immunoassay and visualized using an ODYSSEY Fc Dual Mode
Imaging system (Licor).
To assess Crp2 binding, promoter regions were radiolabelled by
addition of [a-32P]-dCTP [1 mCi (37 GBq) at ~3000 Ci mmol21] to
PCRs containing M. smegmatis genomic DNA and the desired
primers (Table S1). Amplified radiolabelled DNA was purified using a
Qiagen PCR purification kit. EMSAs were carried out with
radiolabelled promoter DNA (200 bp sdh1 and 200 bp shd2) and
varying concentrations of Crp2. The Crp2 protein was pre-incubated
at 20 uC for 30 min, with or without cAMP as indicated, in 100 mM
NaCl, 50 mM Tris (pH 7.5), 10 mM MgCl2, 0.1 mM EDTA, 1 mM
DTT and 0.25 mg BSA ml21, before the addition of radiolabelled
DNA (~3 ng) for a further 10 min (total reaction volume of 15 ml).
Calf thymus DNA (~1 mg) was used as a non-specific competitor in
all assays. After incubation, loading buffer (2 ml of 50 % glycerol,
0.25 % bromophenol blue) was added to the samples immediately
before applying to 6 or 10 % polyacrylamide gels buffered with 0.56
TBE. Electrophoresis was carried out at 30 mA for ~30 min with
0.56 TBE running buffer. The gels were then dried and visualized by
autoradiography.
RESULTS AND DISCUSSION
Fast-growing environmental mycobacteria
possess two functional CRPs
The CRP protein from E. coli is the founding member of
the CRP–FNR superfamily of transcriptional regulators.
Crp1 and Crp2 of M. smegmatis are 34 and 32 % identical
to E. coli CRP over 189 aa, respectively, and possess several
conserved residues involved in both cAMP binding and
DNA recognition (Schultz et al., 1991) (Fig. 1). Crp1 and
Crp2 of M. smegmatis are, respectively, 76 and 97 % identical
to M. tuberculosis CRP (Fig. 1). Amino acid residues (from
Arg218 to Asn224) in the C-terminal region of Crp1 and
Crp2 form an additional helix (G-helix) that is absent in E.
coli CRP (Fig. 1). Multiple sequence alignment of CRP
from different bacterial species across different phyla demonstrated that the additional C-terminal helix is present
only in actinomycetes, including Mycobacterium, Rhodococcus, Gordonia, Corynebacterium and Streptomyces, but is
absent in non-actinomycetes, including E. coli, Vibrio
cholerae and Pseudomonas aeruginosa (Fig. S1). The role of
this helix remains to be elucidated in actinomycetes. In M.
tuberculosis, deletion of this C-terminal helix resulted in an
insoluble form of the protein, demonstrating that it was
required for correct folding of CRP, but making it difficult
for functional studies to be performed (Kumar et al., 2010).
To establish a phylogenetic relationship for Crp1 and Crp2
in M. smegmatis, the amino acid sequences of the CRPs from
other mycobacterial species, as well as from those belonging
to the genera Streptomyces, Corynebacterium and Escherichia
were compared using MEGA 5 (Tamura et al., 2011) (Fig. S2).
The resulting tree was calculated based on the similarity of
the CRP sequences and created by the neighbour-joining
method (Saitou & Nei, 1987). It is noteworthy that
only fast-growing environmental mycobacteria such as M.
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H. L. Aung and others
1
A
Crp2
MtbCRP
Crp1
EcoliCRP
6
7
8
B
*
*
D
9
10
E
117
104
117
111
*
*
F
Crp2
MtbCRP
Crp1
EcoliCRP
4
61
48
61
54
C
Crp2
MtbCRP
Crp1
EcoliCRP
3
1
1
1
1
5
Crp2
MtbCRP
Crp1
EcoliCRP
2
11
12
G
224
211
224
210
173
160
173
166
Fig. 1. The cAMP-binding and DNA-binding domains are conserved in E. coli CRP and M. smegmatis Crp1 and Crp2
(accession numbers WP_011727023 and WP_003897584, respectively). Amino acid sequence alignment of the product of
crp1 and crp2 of M. smegmatis with CRP of M. tuberculosis (NP_218193) and E. coli (YP_492074) was generated using
ClustalW2 and the conserved residues are visualized using BOXSHADE. Black and grey blocks indicate identical and similar
amino acids, respectively. *Residues involved in cyclic nucleotide binding in E. coli CRP that are conserved in M. smegmatis
Crp1 and Crp2. The amino acids directly involved in DNA recognition in E. coli CRP are depicted by a star. The amino acids that
are only present in mycobacterial species are depicted by a caret mark (‘). The cAMP-binding domain and DNA-binding
domain are represented by red and blue rectangles, respectively. Spirals indicate a-helices and arrows depict b-sheets.
smegmatis and Mycobacterium gilvum possessed two CRPs,
whilst slow-growing pathogenic mycobacteria had only one
CRP (Fig. S2). M. tuberculosis harbours an additional
cAMP-binding protein, designated Cmr (Rv1675c) (cAMP
and macrophage regulator) (Gazdik & McDonough, 2005;
Gazdik et al., 2009). Orthologues of Cmr are present in other
slow-growing mycobacteria and the Cmr regulon is distinct
from the M. tuberculosis CRP regulon (Gazdik et al., 2009).
Phylogenetic analysis revealed that Cmr from slow-growing
mycobacterial species forms a distinct cluster (Fig. S2) and
both Crp1 and Crp2 of M. smegmatis, as well as CRP of M.
tuberculosis, are only 26 % identical to M. tuberculosis Cmr.
These observations suggest that Crp1 found only in fastgrowing environmental mycobacteria is distinct from Cmr.
Mutational analyses of Crp1 and Crp2 in M.
smegmatis indicate an essential role for Crp2
To establish the expression profile of the crp1 and crp2
genes in M. smegmatis, we first constructed transcriptional
652
crp1–lacZ and crp2–lacZ fusions and studied gene expression throughout growth using HdB minimal medium
supplemented with 10 mM glycerol. Expression of crp1–
lacZ was constitutive throughout the growth cycle, with a
mean promoter activity of 67 MU. Similar to crp1 expression, that of crp2–lacZ was constitutive throughout the
growth cycle with a mean promoter activity of 153 MU. To
determine the physiological roles of Crp1 and Crp2 in M.
smegmatis, we set out to construct unmarked non-polar
deletions of each gene. A Dcrp1 mutant was readily
obtained (Fig. S3), but a Dcrp2 deletion mutant could
not be obtained using the same method. Southern
hybridization of putative Dcrp2 mutants was consistent
with the presence of wild-type crp2, even after several
attempts and screening a large number of colonies (131)
(Fig. S3). To further validate the essentiality of Crp2, we
employed a single cross-over strategy that would integrate
into the chromosome within the coding sequence of crp2,
resulting in disruption of the crp2 gene (Fig. S3f). We
hypothesized that if the crp2 gene was non-essential for
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Microbiology 161
Roles of CRPs in Mycobacterium smegmatis
Identification of genes under Crp1 and Crp2
control in M. smegmatis
Microarray analysis was performed to establish the Crp1
regulon using RNA extracted from exponential phase
(OD600 of ~0.5) cultures of M. smegmatis wild-type and
the Dcrp1 mutant grown on HdB minimal medium supplemented with 10 mM glycerol (Fig. S5a). Microarray analysis
revealed 239 genes that were differentially regulated,
including 170 that were upregulated (¢1.4-fold) and 69
that were downregulated (¡1.4-fold) in response to the loss
of crp1 function (P¡0.05) (Table S2). The M. smegmatis
Crp1 consensus sequence was identified as described by
Hümpel et al., 2010) and designated TGTGN8CACA. A
histogram of genes differentially expressed in response to
crp1 deletion within functional categories is shown in Fig.
S5(c).
Crp1 regulation of solute transport and carbon
metabolism in M. smegmatis
Microarray analysis revealed that 15 % of the genes that
were differentially regulated in response to crp1 deletion are
involved in the transport and catabolism of carbohydrates
(Table S2; Fig. S5c). Many of these proposed genes were
upregulated in response to deletion of crp1, suggesting
Crp1 repressed the expression of these genes (Table S2).
http://mic.sgmjournals.org
2.0
Arabinose
OD600
1.5
WT
Dcrp1
1.0
complement
0.5
0.0
0
10
20
30
40
50
40
50
40
50
Time (h)
(b)
2.0
Xylose
1.5
OD600
Next, the phenotypic effect of crp1 and crp2 was assessed
using HdB minimal medium supplemented with either
5 mM glucose or 10 mM glycerol under aerobic and
hypoxic growth conditions (Fig. S4). As crp2 could not be
deleted using conventional allelic exchange mutagenesis, we
overexpressed Crp2 using a tetracycline inducible plasmid
pMind (Blokpoel et al., 2005) and performed growth
analysis. Crp2 was induced with tetracycline (20 ng ml21)
in HdB minimal medium supplemented with either 5 mM
glucose or 10 mM glycerol at an OD600 of 0.3 and monitoring of growth. No significant phenotypic effect of crp2
overexpression was observed under aerobic growth conditions (Fig. S4a and b), whereas there was a strong growth
phenotype with the Dcrp1 mutant on glucose (Fig. 2c), but
not on glycerol (Fig. S5a). Growth analysis of both the crp1
deletion mutant and the crp2 overexpression mutant in HdB
minimal medium supplemented with 10 mM glycerol under
hypoxic conditions was also performed as described previously (Aung et al., 2014; Berney et al., 2012). However, no
profound phenotypic effect was observed under these
conditions (Fig. S4c and d).
(a)
WT
Dcrp1
1.0
complement
0.5
0.0
0
10
20
30
Time (h)
(c)
OD600
growth, a crp2 integrant would be obtained after the recombination event. No integrant was obtained despite prolonged
incubation at 37 uC for 10 days, suggesting that Crp2 was
essential for growth of M. smegmatis under the conditions
tested herein. In M. tuberculosis, Crp2 is non-essential for
growth, but Dcrp2 mutants show impaired growth in
macrophages and in mice (Bai et al., 2011; Rickman et al.,
2005).
1.5
Glucose
WT
1.0
Dcrp1
complement
0.5
0.0
0
10
20
30
Time (h)
Fig. 2. Growth curves of M. smegmatis wild-type with the vector
pMV261 (WT), crp mutant with the vector pMV261 (Dcrp1) and its
complemented strain in HdB minimal medium supplemented with
either (a) 6 mM arabinose, (b) 6 mM xylose or (c) 5 mM glucose at
37 6C under aerobic conditions. Results shown are means±SD of
three biological replicates.
These included operons for five carbon sugar transporters
(namely MSMEG_1370–1374, MSMEG_1704–1706, MSMEG_
1713–1715), a glucose, trehalose, N-acetylglucosamine transporter belonging to the sugar PTS family (MSMEG_2116–
2119) and a glycerol transporter of the PTS family encoded
by MSMEG_2121–2124 (Table S2) (functions reviewed by
Titgemeyer et al., 2007). To determine the physiological
effect of the de-repression of genes involved in transport and
catabolism of carbohydrates, growth analysis was performed
using HdB minimal medium supplemented with either
arabinose, xylose or glucose (Fig. 2) because the transporters
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653
H. L. Aung and others
for these sugars were upregulated in the Dcrp1 strain (Table
S2). The Dcrp1 mutant had a faster growth rate on xylose,
arabinose and glucose compared with the isogenic wild-type
strain (Fig. 2). The wild-type phenotype could be restored in
the Dcrp1 mutant by in trans complementation with crp1,
suggesting that the observed growth phenotype was due to
the deletion of crp1 (Fig. 2). Glycolysis is one of the main
pathways of central carbon metabolism in mycobacteria. A
limited number of genes encoding the enzymes involved in
glycolysis were downregulated in response to crp1 deletion.
These included glucose 6-phosphate isomerase, fructose
biphosphate aldolase, triosephosphate isomerase (tpiA) and
phosphoglycerate kinase (pgk) (Table S2). In addition, genes
involved in the transport of amino acids, polyamines and
spermidine were upregulated in response to crp1 deletion
(Table S2).
0.5
0
20
Time (h)
30
50
0.4
Glucose
Pyruvate
25
0.2
0
20
Time (h)
30
(e)
0.8
0.6
0.4
Glycerol
Pyruvate
25
0.2
0
0.0
0
10
20
Time (h)
30
40
OD600
0.8
75
0.6
50
0.4
Glucose
Pyruvate
25
0.2
0.0
10
20
Time (h)
30
40
(f)
1.0
50
40
1.0
0
100
75
30
100
40
% carbon remaining
10
20
Time (h)
0
0.0
0
% carbon remaining
OD600
% carbon remaining
0.8
0.6
10
(d)
100
75
0.5
0.0
0
1.0
(c)
Glycerol
Glucose
25
40
% carbon remaining
10
1.0
50
0
0.0
0
1.5
75
OD600
Glycerol
Glucose
25
2.0
100
1.0
100
0.8
75
0.6
50
0.4
Glycerol
Pyruvate
25
OD600
1.0
50
OD600
1.5
75
Dcrp1
(b)
2.0
% carbon remaining
Wild-type
100
OD600
% carbon remaining
(a)
The Entner–Doudoroff (ED) pathway is present in M.
smegmatis (Bai et al., 1976), and transcripts encoding enzymes
of this pathway [MSMEG_0314 (zwf), MSMEG_0313 (edd),
MSMEG_0312 (eda)] were upregulated in the Dcrp1 mutant
(Table S2). Prokaryotes often contain several glycolytic
pathways, of which the ED pathway is the most common
after the canonical Embden–Meyerhof–Parnas (EMP) glycolytic pathway. The microarray data suggest that Crp1 controls
the differential expression of the ED and EMP pathways. In
wild-type cells, Crp1 appears to repress the ED pathway to
ensure carbon is routed through the EMP pathway. Flamholz
et al. (2013) showed that the EMP pathway incurs a 3.5-fold
higher protein cost compared with the ED pathway, but this is
offset by the higher ATP yield of the EMP pathway versus the
ED pathway. CRP mediates the hierarchical utilization of
carbon sources in many bacteria, resulting in a diauxic pattern
0.2
0
0.0
0
10
20
Time (h)
30
40
Fig. 3. Growth and pattern of carbon source utilization by M. smegmatis wild-type (a, c, e) and Dcrp1 mutant (b, d, f) in HdB
medium supplemented with 2.5 mM glucose and 5 mM glycerol (a, b), 2.5 mM glucose and 5 mM pyruvate (c, d) or 5 mM
glycerol and 5 mM pyruvate (e, f). M. smegmatis wild-type and Dcrp1 mutant were grown in flasks at 37 6C for 39 h under
aerobic conditions. Quantification of substrates from culture supernatants was performed using an enzyme assay (see
Methods). Results shown are means±SD of three biological replicates.
654
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Microbiology 161
Roles of CRPs in Mycobacterium smegmatis
(a)
***
To further investigate Crp1 regulation of the sdh1 and sdh2
operons, sdh promoter activity was measured using
promoter–lacZ fusions in HdB minimal medium supplemented with 10 mM glycerol (Fig. 4b). Consistent with the
microarray and qRT-PCR data (Fig. 4b), there was a 5.0fold increase in promoter activity of the sdh1 construct in
the Dcrp1 strain, indicating that Crp1 was a repressor of
sdh1 expression (Fig. 4b). In contrast, the promoter activity
of sdh2 construct was approximately 1.4-fold lowered in
the Dcrp1 strain, suggesting Crp1 was an activator of sdh2
expression (Fig. 4b).
Crp2 regulon of M. smegmatis reveals diverse
gene functions
As described above, crp2 could not be deleted using
conventional allelic exchange mutagenesis, and we therefore overexpressed Crp2 using a tetracycline inducible
http://mic.sgmjournals.org
*
atpB
cydB
sdhB
**
Microarray
qPCR
(b)
100
80
Dcrp1
WT
**
***
60
40
20
0
Sdh2
pJEM15
10
***
0.1
sdhB
*
**
atpB
*
1
cydB
***
Msmeg_0420
(c)
Gene expression ratio
HLA102/WT
Microarray analysis revealed that 15 % of the differentially
regulated genes in the M. smegmatis Dcrp1 were involved in
energy generation, including genes that encode components
of the oxidative phosphorylation machinery (Table S2).
These included gene clusters for the F1FO-ATP synthase,
type I proton-translocating NADH-quinone oxidoreductase
(nuo), succinate dehydrogenase 1 (MSMEG_0417-0420,
Sdh1), succinate dehydrogenase 2 (MSMEG_1669-1672,
Sdh2) and the cytochrome bd terminal oxidase (Table S2).
To further validate this observation, qRT-PCR was
performed on selected genes encoding components of the
mycobacterial electron transport chain for the wild-type and
Dcrp1 mutant (Fig. 4a). These experiments confirmed the
regulatory patterns revealed by the microarray analysis of the
M. smegmatis Dcrp1 mutant.
*
Msm0420
0.1
Sdh1
Crp1 regulates respiratory energy metabolism in
M. smegmatis
*** ***
1
nuoC
Gene expression ratio
Dcrp1/WT
10
0.01
β-Galactosidase activity (MU)
of growth (Saier, 1989). The ability of M. smegmatis wild-type
and the Dcrp1 mutant to metabolize gluconate, a substrate of
the ED pathway, was assessed (Fig. S6a). Both the wild-type
and the Dcrp1 mutant were able to grow on gluconate and no
significant difference in growth rate was observed (Fig. S6a),
whereas growth analysis with arabinose and xylose revealed
differences in growth rate (Fig. S6b and c). Despite Crp1
controlling expression of genes involved in the glycolytic flux
through the EMP and ED pathways, Crp1 had no effect on
the pattern of sugar utilization in M. smegmatis (Fig. 3). For
example, when M. smegmatis (wild-type versus Dcrp1) was
grown on equimolar mixtures of glycerol and glucose (Fig. 3a
and b), glucose and pyruvate (Fig. 3c and d) or glycerol and
pyruvate (Fig. 3e and f) the same pattern of carbon source
utilization was noted between the wild-type strain and the
Dcrp1 mutant. In most cases the carbon sources were used
simultaneously and no diauxie was noted (Fig. 3).
Simultaneous use of carbon substrates and an apparent lack
of diauxic growth has been reported previously in M.
tuberculosis (de Carvalho et al., 2010).
Microarray
qPCR
0.01
Fig. 4. Crp1 and Crp2 regulation of electron transport chain
components in M. smegmatis. (a) Validation of Crp1 microarray
analysis by qRT-PCR. Results shown represent gene expression
ratio in response to deletion of crp1 from M. smegmatis and as
means±SD of four (microarray) or three (qRT-PCR) independent
biological replicates. *P,0.05, **P,0.01, ***P,0.001. (b)
Promoter activity of succinate dehydrogenase 1 (sdh1) and 2
(sdh2) in wild-type and the Dcrp1 mutant of M. smegmatis.
Promoter activity was measured as described in Methods.
pJEM15, empty vector. Results are shown as means±SD of three
biological replicates. **P,0.01, ***P,0.001. (c) Validation of Crp2
microarray analysis by qRT-PCR. Results shown represent gene
expression ratio in response to overexpression of crp2 from M.
smegmatis and as means±SD of four (microarray) or three (qRTPCR) independent biological replicates. *P,0.05, **P,0.01,
***P,0.001.
plasmid pMind (Blokpoel et al., 2005) and performed
transcriptional profiling analysis from RNA extracted from
exponential phase (OD600 of ~0.5) cultures grown in HdB
minimal medium supplemented with 10 mM glycerol to
determine the molecular response to the overexpression of
crp2 in M. smegmatis mc2155 (Fig. S5b). The analysis
confirmed that crp2 was upregulated 6.0-fold in response
to tetracycline induction (Table S3). The microarray results
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655
H. L. Aung and others
revealed 58 genes that were differentially regulated, including 49 that were upregulated (¢1.4-fold) and nine that were
downregulated (¡1.4-fold) in response to the overexpression of crp2 (P¡0.05). A Crp2 consensus sequence was also
identified as described above and designated TGTGN8CACA.
homologue of MSMEG_6041 in M. tuberculosis encoding
fadE34 is essential for growth on cholesterol (Griffin et al.,
2011). Whether MSMEG_6041 is essential for growth of M.
smegmatis is not known.
Eleven genes were found in both the Crp1 and Crp2
regulons (Table 1). These genes included the sdh1 operon
but not the sdh2 operon. To determine whether Crp2 also
regulates sdh1 expression, qRT-PCR was performed using
three genes coding for three different electron transport
chain components (MSMEG_0420) for Sdh1, sdhB for
Sdh2 and cydB for cytochrome bd oxidase, and atpB for the
F1Fo ATPase (Fig. 4c). Expression levels observed by qRTPCR correlated well with those from the microarray
analysis for Crp2 regulation.
A histogram of genes differentially expressed in response to
overexpression of crp2 within functional categories is
provided in Fig. S5(d). Microarray analysis showed that
Crp2 regulates genes with diverse biological functions,
including genes encoding Sdh1, the WhiB4 and WhiB6 of
WhiB family proteins, which have been implicated in many
cellular processes (Larsson et al., 2012), and RpfE, one of
five resuscitation-promoting factor proteins (Rpfs) that are
required for the resuscitation of dormant cells (Kana et al.,
2008) (Table S3). The M. tuberculosis genome encodes
seven WhiB proteins (WhiB1–7) and their homologues are
present in M. smegmatis. Recently, it was shown that cAMP
induced both whiB4 and whiB6 in M. tuberculosis (Larsson
et al., 2012). The authors also identified a putative CRPbinding site present in the 59 untranslated region of both
whiB4 and whiB6 and suggested that CRP may influence
the expression of these whiB genes (Larsson et al., 2012).
One of the whiB genes, whiB1, is directly regulated by CRP
in M. tuberculosis (Stapleton et al., 2010). A CRP-binding
site is also present in the promoter regions of both whiB4
and whiB6 genes in M. smegmatis, suggesting that Crp2
could directly regulate these whiB genes. M. tuberculosis
possesses five rpf genes, rpfA–E (Downing et al., 2004, 2005;
Kana et al., 2008). In M. tuberculosis, a CRP-binding site
was identified in the promoter region of rpfA, and rpfA
is directly regulated by CRP (Rickman et al., 2005). A
putative CRP-binding site was also identified in the
promoter region of rpfE in M. smegmatis, and Crp2 could
regulate rpfE. The reason why Crp2 appears to be essential
for growth of M. smegmatis remains unknown. Analysis of
the genes regulated by Crp2 (Table S3) reveals that a
Regulation of sdh1 and sdh2 expression by CRP
To determine whether Crp1 interacts directly with the promoters of the sdh1 and sdh2 operons, EMSAs were carried
out with purified Crp1 and sdh1 promoter DNA (227 bp
probe designated sdh1) and sdh2 promoter DNA (431 bp
probe designated sdh2). EMSAs demonstrated that Crp1
caused a mobility shift in both sdh promoters independently
of cAMP (Fig. 5a). The specificity of the Crp1 for binding
to both sdh promoters was demonstrated by using an
unlabelled PCR fragment (amtR) amplified from the
promoter region of amtR in the presence or absence of
cAMP (Fig. 5b, lanes 5 and 10; Fig. 5c, lanes 5 and 10). The
presence of the amtR product at 250-fold excess did not
affect the mobility shift of either sdh promoter, whereas the
addition of the same amount of unlabelled sdh promoters
abolished the ability of Crp1 to shift DIG-labelled sdh
promoters (Fig. 5b, lanes 4 and 9; Fig. 5c, lanes 4 and 9).
Taken together, these data demonstrate the specific DNAbinding ability of Crp1 to the sdh1 promoter in a cAMPindependent manner. To establish whether the regulation
Table 1. Genes that are common in both Crp1 and Crp2 regulons
Gene ID
MSMEG_0416
MSMEG_0417
MSMEG_0418
MSMEG_0419
MSMEG_0420
MSMEG_1837
MSMEG_2121
MSMEG_2122
MSMEG_2123
MSMEG_2124
MSMEG_5850
Gene symbol
dhaL
dhaK
Description
Succinate dehydrogenase subunit B
Succinate dehydrogenase
Succinate dehydrogenase flavoprotein subunit
Integral membrane protein
Succinate dehydrogenase subunit C
Secreted protein
Multiphosphoryl transfer protein
Dihydroxyacetone kinase, L subunit
Dihydroxyacetone kinase, DhaK subunit
Glycerol uptake facilitator, MIP major intrinsic
protein channel
Transcriptional regulator, TetR family protein
Crp1*
Crp2D
1.7
5.1
3.4
3.2
2.6
1.6
2.5
2.2
2.1
2.3
1.0
2.0
2.4
2.7
1.6
1.5
1.8
1.6
1.4
1.5
1.86
1.87
*Mean gene expression ratio (four biological replicates) in the crp1 mutant compared to the wild-type. Bold type indicates a ¢1.4-fold change in
gene expression, P¡0.05.
DMean gene expression ratio (four biological replicates) in crp2 overexpression compared to the wild-type. Bold type indicates a ¢1.4-fold change
in gene expression, P¡0.05.
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Microbiology 161
Roles of CRPs in Mycobacterium smegmatis
(a)
1
3
2
(b)
(a)
sdh2
sdh1
4
5
sdh1
6
1
sdh2
2
3
4
5
6
Sdh1
– cAMP
None
Crp1 Compt
None
+ cAMP
1
2
6 7
3
4
5
(b)
1
Crp1 Compt
8
cAMP
2
3
4
5 6
8
7
8
9 10
9 10
(c)
Crp2
1
(c)
7
2
3
4
5
6
9 10
Sdh2
– cAMP
Crp1 Compt
1 2
3 4
None
None
+ cAMP
Crp1 Compt
5 6 7 8
9 10
Fig. 5. EMSAs of Crp1 with the sdh1 and sdh2 promoters. (a)
The DIG-labelled MSMEG_0420 (sdh1) and sdhC (sdh2) DNA
probes were incubated in both the absence (lanes 1 and 4) and
the presence of Crp1 (3.8 mM) with or without cAMP (lanes 2 and
5, no cAMP; lanes 3 and 6, 0.2 mM cAMP). (b) DIG-labelled
sdh1 was incubated with increasing concentrations of Crp1 in the
presence of cAMP (0.2 mM) (lanes 1–5) and in the absence of
cAMP (lanes 6–10). Lanes 1 and 6, no protein; lanes 2 and 7,
3.8 mM Crp1; lanes 3 and 8, 7.7 mM Crp1; lanes 4 and 9, 250fold excess of unlabelled sdh1; lanes 5 and 10, 250-fold excess
of unlabelled amtR incubated with 7.7 mM Crp1. (c) DIG-labelled
sdh2 was incubated with increasing concentrations of Crp1 in the
presence of cAMP (0.2 mM) (lanes 1–5) and in the absence of
cAMP (lanes 6–10). Lanes 1 and 6, no protein; lanes 2 and 7,
3.8 mM Crp1; lanes 3 and 8, 7.7 mM Crp1; lanes 4 and 9, 250fold excess of unlabelled sdh1; lanes 5 and 10, 250-fold excess
of unlabelled amtR incubated with 7.7 mM Crp1. Compt,
competition.
of sdh1 by Crp2 is direct or indirect, EMSAs were
conducted using the promoter regions of sdh1 or sdh2
with purified Crp2 (Fig. 6a). The results revealed that Crp2
binds at the promoter of sdh1 in a cAMP-dependent
manner, but did not bind at the promoter of sdh2 (Fig. 6a),
http://mic.sgmjournals.org
Fig. 6. (a) EMSAs of Crp2 with radiolabelled M. smegmatis sdh1
(lanes 1–3) and sdh2 (lanes 4–6) promoter DNA. Promoter DNA
was incubated without Crp2 (lanes 1 and 4), with Crp2 (5.5 mM;
lanes 2 and 5) and with Crp2 plus cAMP (5.5 mM Crp2, 0.2 mM
cAMP; lanes 3 and 6). (b) EMSAs with the sdh1 promoter
incubated with CRP2 (3.6 mM) in the presence of increasing
concentrations of cAMP (lanes 1–8: 0.66, 1.33, 3.33, 6.7, 13.3,
33, 67, 133, 0 mM). The reaction in lane 9 has no cAMP. The
reaction in lane 10 lacks CRP2. (c) Binding of Crp2 at the wildtype sdh1 promoter. Promoter DNA was incubated at 25 6C for
5 min with Crp2 in the presence of 2 mM cAMP. Lane 1, no
protein; lanes 2–10, 0.001, 0.005, 0.01, 0.025, 0.05, 0.1, 0.25,
0.5 and 1 mM Crp2, respectively.
consistent with the gene expression profiles obtained by
microarray and qRT-PCR analyses (Fig. 4c). To further
validate this, EMSAs of Crp2 with the sdh1 promoter in the
presence of increasing concentrations of cAMP were
performed (Fig. 6b). Crp2 caused a mobility shift of the
sdh1 promoter at cAMP concentrations of 6.7 mM and
above, with a complete shift at 133 mM (Fig. 6b),
suggesting that binding is cAMP-dependent (Fig. 6b).
EMSAs of Crp2 at the sdh1 promoter with increasing
concentrations of Crp2 in the presence of cAMP were
further performed to validate that the specific binding of
Crp2 at the sdh1 promoter is cAMP-dependent (Fig. 6c).
The direct binding of CRP to sdh1 (Rv0249c–Rv0247c) in
M. tuberculosis has not yet been studied, but CRP is
computationally predicted to bind this promoter region
(Bai et al., 2005; Krawczyk et al., 2009).
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H. L. Aung and others
CONCLUSION
The CRPs of M. smegmatis play central roles in regulating
metabolic and respiratory activity and function as bona
fide DNA-binding proteins and regulators of gene expression (Fig. 7). We show that Crp1 regulates the electron
chain components of M. smegmatis, and for the sdh
operons, the binding of Crp1 was cAMP-independent. In
contrast, Crp2 bound to the sdh1 promoter in a cAMPdependent manner and no binding of Crp2 to the sdh2
promoter was detected. These data highlight the complex
regulation of the sdh operons by Crp1 and Crp2 in M.
smegmatis. The sdh operons in M. smegmatis are differentially regulated in response to energy limitation and
hypoxia (Berney & Cook, 2010; Pecsi et al., 2014), and the
involvement of CRP in this regulation points to Crp1 and
Crp2 being able to integrate diverse signals to control gene
expression. The involvement of CRP in regulation of aerobic
respiration has also been reported in E. coli (Shimada et al.,
2011), highlighting an important regulatory role for CRP in
prokaryotic energy metabolism. We have recently reported
that Crp1 regulates the expression of cydAB coding for the
terminal oxidase cytochrome bd in M. smegmatis in response
to hypoxia (Aung et al., 2014). The lack of the classical
Fnr and ArcBA regulation of respiratory metabolism in
mycobacteria is intriguing and suggests that CRP may have
been adapted for this function. Moreover, none of the
electron transport chain components in M. smegmatis is
under control of the hypoxic regulator DosR (Berney et al.,
2014), suggesting unique regulation of respiratory metabolism in mycobacteria.
Crp1 and Crp2 recognized the same consensus sequence
(TGTGN8CACA), and this CRP binding is similar to that
reported for Crp2 of M. tuberculosis (GTGN8CAC)
(Kahramanoglou et al., 2014). A cAMP-independent Crp1
DNA-binding activity suggests that Crp1 may be evolutionarily adapted to interact with DNA in the absence of cAMP,
given the high levels of cAMP reported in mycobacteria
(Dass et al., 2008; Padh & Venkitasubramanian, 1976, 1980).
Mycobacterial CRP lacks typical redox-sensing domains and
crp expression is not under the control of DosR, a regulator
of hypoxic gene expression in mycobacteria. In E. coli, the
traditional view of cAMP–CRP signalling has centred on
CCR to achieve hierarchical carbon usage (Kovárová-Kovar
& Egli, 1998; Saier, 1989; Saier & Reizer, 1994). Crp1 in M.
smegmatis had no effect on the pattern of carbon source
utilization, pointing to other roles in mycobacterial physiology. You et al. (2013) report that cAMP–CRP signalling
mediates more than CCR and coordinates the expression of
catabolic proteins with biosynthetic and ribosomal proteins
in response to cellular metabolic demands, implying that
CRP senses the anabolic demands of the cell and allocates
resources appropriately in response to growth rate. The M.
smegmatis Crp1 regulon and the growth rate (i.e. fast versus
slow) regulon in M. smegmatis (Berney & Cook, 2010) both
contain genes encoding components of the respiratory
chain, suggesting that as mycobacteria make the transition
H+
H+
F1Fo-ATP
syntase
NDH-1
MFS
ADP
+ Pi
NADH
ABC
NAD+ + H+
Glucose
Embden–Meyerhof–
Parnas pathway
ATP
TCA cycle
Succinate
SDH
Entner–Doudoroff pathway
Fumarate
Cytochrome bd
2H2O
O2
PTS
Fig. 7. Regulatory roles of Crp1 and Crp2 in M. smegmatis. Crp1 regulates three pathways of sugar transport (MFS, PTS and
ABC), the Entner–Doudoroff (ED) pathway for glycolysis, and major components of the electron transport chain and F1Fo-ATP
synthase. Regulation by Crp1 is indicated by grey shading. Both Crp1 and Crp2 regulate succinate dehydrogenase I and this is
indicated by absence of shading. ABC, ATP-binding cassette family; MFS, major facilitator superfamily; NDH-1, NADH-quinone
oxidoreductase; PTS, the phosphotransferase system; SDH, succinate dehydrogenase.
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Microbiology 161
Roles of CRPs in Mycobacterium smegmatis
from fast to slow growth rate, the cell adjusts its metabolic
machinery to match physiological demand for energy via
Crp1. This warrants further investigation to elucidate the
complex and unique molecular signalling network involving
Crp1 and Crp2 in M. smegmatis.
Cole, S. T., Brosch, R., Parkhill, J., Garnier, T., Churcher, C., Harris,
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S. S. (2008). Cyclic AMP in mycobacteria: characterization and
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ACKNOWLEDGEMENTS
The work at the University of Otago was supported by the Maurice
Wilkins Centre for Molecular Biodiscovery (H. L. A.) and a Marsden
Grant from the Royal Society of New Zealand (M. B.). G. M. C. was
supported by a James Cook Fellowship from the Royal Society of New
Zealand. The work at the University of Sheffield (L. J. S. and J. G.) was
supported by the Biotechnology and Biological Sciences Research
Council UK (project grant BB/K000071/1) and a University of
Sheffield PhD scholarship (L. L. D.). The work at the National
Institute for Medical Research was supported by the Medical Research
Council through grant U117585867 to R. S. B.
de Carvalho, L. P., Zhao, H., Dickinson, C. E., Arango, N. M., Lima,
C. D., Fischer, S. M., Ouerfelli, O., Nathan, C. & Rhee, K. Y. (2010).
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