Download Control of the acetamidase gene of Mycobacterium smegmatis by

FEMS Microbiology Letters 221 (2003) 131^136
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Control of the acetamidase gene of Mycobacterium smegmatis by
multiple regulators
Gretta Roberts, D.G. Niranjala Muttucumaru, Tanya Parish
Department of Medical Microbiology, Barts and the London, Queen Mary’s School of Medicine and Dentistry, Turner Street, London E1 2AD, UK
Received 6 December 2002; received in revised form 18 February 2003; accepted 26 February 2003
First published online 20 March 2003
Abstract
The acetamidase of Mycobacterium smegmatis is an inducible enzyme which enables the organism to utilise several amides as sole
carbon sources. The acetamidase structural gene (amiE) is located downstream of four other genes, of which three form a probable operon
with amiE; the fourth (amiC) is divergently transcribed. We constructed deletion mutants in two of these genes in order to determine their
role in acetamidase expression. Both AmiC and AmiD were shown to be positive regulators of acetamidase expression required for
induction. Combinations of regulatory gene deletions were made which revealed that AmiC interacts with the previously characterised
negative regulator AmiA, whereas AmiD does not.
1 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords : Acetamidase ; Gene regulation; Promoter ; Mycobacterium smegmatis
1. Introduction
The acetamidase of Mycobacterium smegmatis is an inducible enzyme which enables the organism to utilise several amides, including acetamide and formamide, as sole
carbon sources [1]. The enzyme is expressed to a low level
under non-induced conditions, but is induced 100-fold in
the presence of a suitable substrate such as acetamide [1^
3].
Much interest has been focussed on this system for its
potential use in mycobacterial genetic studies. The availability of an inducible promoter which functions well in
mycobacteria including the important human pathogen
Mycobacterium tuberculosis would be extremely useful.
The acetamidase system has been used to over-express
proteins in mycobacteria [4,5] and to generate strains
which conditionally express heterologous genes [6]. However, the system is currently imperfect as the basal level of
expression seen with this system means that genes under
its control are always expressed to a low level. In order to
improve upon the current system and also to gain further
* Corresponding author. Tel. : +44 (20) 7377 7000 ext. 2961;
Fax : +44 (20) 7377 7259.
E-mail address : [email protected] (T. Parish).
insight into how M. smegmatis utilises amides, we have
further investigated the regulation of the enzyme. In particular we were interested in the role of the previously
proposed regulators.
The acetamidase structural gene (amiE) is located downstream of four other genes, of which three form a probable
operon with amiE (the fourth is divergently transcribed;
Fig. 1) [2,3]. Three of these genes were originally identi¢ed
as regulators based on sequence homologies [2,3], and one
of them (amiA) has subsequently been shown to play a
direct role in the regulation of this operon [7]. The fourth
gene (amiS) is likely to be one component of an ABC
transporter.
Previous work has shown that both positive and negative control elements are involved in the regulation of the
acetamidase. Induction occurs at the transcriptional level
with increased amounts of acetamidase mRNA detectable
after 1 h [3]. Four promoters have been identi¢ed using a
reporter gene, three of these drive transcription towards
the acetamidase and the fourth drives expression of amiC
(Fig. 1). In these experiments small regions upstream of
each open reading frame were used and regulatory sites
may have been missing from the constructs, so that the
level of promoter induction may not completely re£ect the
natural situation. The promoters identi¢ed include Pc ,
which drives the expression of amiC and is active at a
0378-1097 / 03 / $22.00 1 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
doi:10.1016/S0378-1097(03)00177-0
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G. Roberts et al. / FEMS Microbiology Letters 221 (2003) 131^136
Lemco powder, 5 g l31 NaCl, 10 g l31 Bacto peptone)
with 0.05% w/v Tween 80 (liquid) or 15 g l31 Bacto agar
(solid). Kanamycin was added to 20 Wg ml31 , hygromycin
to 100 Wg ml31 , gentamicin to 10 Wg ml31 and sucrose to
5% where appropriate. Minimal media [2] contained 0.05%
w/v Tween 80 and carbon sources (acetamide or succinate)
at 0.02% w/v.
2.2. Construction of deletion strains
Fig. 1. Vector constructs. A: Arrangement of genes in the M. smegmatis
chromosome. amiA, negative regulator of acetamidase expression ; amiC
and amiD are proposed regulators, amiS encodes one component of a
putative ABC transporter. amiE is the acetamidase gene. Promoter regions previously identi¢ed are marked underneath with arrows indicating the direction of transcription. The annotated proteins are deposited
in GenBank (Accession number BK001051). B: The fragments used to
generate the delivery vectors for construction of the deletion mutants
are shown, regions deleted are marked in black. C: The regions used to
complement the mutants.
low, but constitutive level. P2 , upstream of amiD, is a
stronger promoter which shows a two-fold induction in
the presence of acetamide. P1 , upstream of amiA, has
the highest promoter activity and is constitutive. P3 is a
very weak promoter found immediately upstream of amiE
itself. P3 could allow the transcription of a monocistronic
message encoding the acetamidase only, whereas P1 and
P2 are more likely to be responsible for polycistronic messages. Northern blotting has previously shown that a 1.2
kb acetamidase transcript is indeed present (and induced)
in the cells [3]. This could either arise from P3 or from
processing of mRNA transcribed from P1 or P2 . Additional studies have mapped two possible transcriptional start
sites located upstream of P1 and P3 [8] and demonstrated
the presence of a polycistronic message, but this work
could not distinguish between true start sites and mRNA
processing. P2 is proposed to be the site of action of the
negative regulator AmiA, since the promoter is derepressed in an AmiA mutant [7].
The aim of this work was to determine the role of the
two other proposed regulators (AmiC and AmiD) and to
investigate how these regulators combine in order to control the expression of the acetamidase enzyme.
2. Materials and methods
2.1. Media
M. smegmatis was grown in Lemco medium (5 g l31
Suicide (non-replicating) delivery vectors were constructed to generate amiC and amiD mutants using a rapid
cloning system [9] (Fig. 1). For the amiC deletion construct a 1.4-kb BamHI^HindIII region was cloned into
p2NIL and the central 0.4-kb XhoI fragment was deleted
to give pURR55. For the amiD deletion construct polymerase chain reaction (PCR) primers were used to amplify
a 1.2-kb fragment from the 5P £anking region and a 1.1-kb
fragment from the 3P £anking region. Primers were designed to introduce KpnI sites into the 5P region and BamHI sites into the 3P region. The fragments were cloned into
the appropriate sites in p2NIL to make pURR209. The
sacB and hyg marker genes were then cloned in as a PacI
fragment from pGOAL15 [9] into pURR55 and pURR209
to give pURR551 and pURR211, respectively. These reporter genes confer sucrose sensitivity and hygromycin
resistance, respectively, and the ¢nal delivery vectors
also had a kanamycin resistance gene. The amiA deletion
vector (pURR541) has previously been described [7].
M. smegmatis Mad (mycobacterial acetamidase deletion)
strains were constructed using a two-step strategy [9]. The
delivery vectors were pretreated with UV light and electroporated into M. smegmatis mc2 155. Single crossovers were
selected on hygromycin, kanamycin plates. One such
transformant was streaked out to allow the second recombination event to occur. Double crossovers were identi¢ed
by selecting for sucrose resistance and screening for kanamycin and hygromycin sensitivity. PCR analysis and
Southern blotting was used to determine which of the
double crossovers had the wild-type genotype restored
and which had the deletions in the chromosome. To generate double and triple mutants, the same procedure was
carried out sequentially in the single and double deletion
mutant strains.
2.3. Complementation studies
To make the complementing construct for AmiC, PCR
was used to amplify the indicated region (Fig. 1). The
PCR product was cloned into pGEM-Easy T (Promega)
and subsequently excised as an EcoRI fragment and subcloned into the integrating vector pINT3 (which carries
the mycobacteriophage L5 integrase and attachment sites
and a gentamicin resistance gene) to make pAGAN304.
The AmiA complementing vector pAGAN303 has previously been described [7]. Mad strains were electroporated
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133
Fig. 2. Acetamidase expression in Mad strains. Cell-free extracts were run on a 10% acrylamide gel and proteins visualised with Coomassie brilliant
blue. The acetamidase band at 47 kDa is indicated. S: minimal media with succinate. A: minimal media with acetamide and succinate.
with these plasmids and transformants selected on gentamicin.
2.4. Preparation of cell-free extracts and sodium dodecyl
sulfate^polyacrylamide gel electrophoresis
(SDS^PAGE)
Strains were grown overnight in 5 ml Lemco broth and
used to inoculate 100 ml of minimal media plus either
acetamide and succinate or succinate alone and incubated
for 24 h. Bacteria were harvested, washed and resuspended
in 1 ml of 10 mM Tris^HCl, pH 8. An equal volume of 0.1
mm glass beads was added and the suspensions subjected
to 2U1 min pulses in the MiniBead Beater (Biospec Products). Cell debris was removed by spinning at 13 000Ug.
Cell-free extracts were diluted to 1.5 mg ml31 . A 30 Wl
sample was mixed with 5 Wl of SDS^PAGE sample bu¡er
(10% w/v SDS, 37% v/v glycerol, 20% v/v L-mercaptoethanol, 0.3 M Tris^HCl pH 6.8) and heated to 100‡C
for 5 min. Samples were applied to a 200U160U1.5 mm
12% SDS^PAGE gel (Protean II XL, Bio-Rad), immersed
in electrophoresis running bu¡er (25 mM Tris, 250 mM
glycine, 0.1% w/v SDS) and proteins resolved at 35 mA
over 6 h at 15‡C. Gels were ¢xed in 2% v/v acetic acid,
40% v/v methanol for 1 h and proteins visualised by staining with a colloidal Coomassie brilliant blue solution (16%
v/v colloidal concentrate, 20% v/v methanol) for 16 h. The
gels were then washed in 30% v/v methanol, 5% v/v acetic
acid and destained in 30% methanol for 30 min before
photography.
3. Results and discussion
We have previously shown that AmiA is involved in the
negative regulation of acetamidase expression, as AmiA
mutants show a constitutively high-level expression of
AmiE [7]. We extended these studies to determine if
AmiC and AmiD also have a role to play in AmiE
expression. We constructed individual deletion mutants
of amiC and amiD and looked at acetamidase expression
under non-induced and induced conditions. Fig. 1 shows
the deletion constructs used to generate the mutants
by homologous recombination. Mutants obtained were
con¢rmed by PCR and Southern analysis (data not
shown).
3.1. The role of AmiC and AmiD
We constructed an unmarked amiCv mutant strain carrying a deletion of the central 0.4-kb XhoI fragment by
homologous recombination. The e¡ect of this deletion on
AmiE expression was assessed by SDS^PAGE analysis of
cell-free extracts from cultures grown in induced (acetamide medium) and non-induced (succinate medium) conditions. As can be seen in Fig. 2 there was no induction of
the acetamidase in the amiC deletion strain (Mad2). Wildtype extracts showed clear induction of the 47-kDa acetamidase protein in the presence of acetamide. Complementation with a functional copy of amiC (Mad2:pAGAN304) restored the ability of the cells to upregulate
the acetamidase in the presence of an inducer. Since the
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G. Roberts et al. / FEMS Microbiology Letters 221 (2003) 131^136
acetamidase is not induced in the absence of amiC, we
conclude that it is a positive regulator of expression.
We also constructed an amiD deletion strain (Mad4)
and looked for acetamidase expression under the same
conditions as for Mad2. In this case, no induction of the
acetamidase was seen. This was not due to deletion of a
promoter region as the identi¢ed promoters were all
present in the deletion strain [7]. Since amiD is immediately downstream of amiA, we were unable to complement
this strain solely with a copy of amiD. However, the likelihood of polar e¡ects causing the lack of induction is
small as the gene downstream (amiS) is a proposed transporter and there is another promoter (P3 ) upstream of
amiE itself. Thus, AmiD is also a positive regulator of
acetamidase expression.
3.2. Interactions between regulatory proteins
In order to characterise the interaction between the regulators and dissect the system more fully, we constructed
double and triple deletions by sequentially deleting the
genes (Table 1). Mutant strains were characterised by
SDS^PAGE analysis as before. The deletion of all three
regulatory genes (amiC, amiA, amiD; Mad6) indicates that
the ‘default’ for acetamidase expression in the absence of
regulation is high-level constitutive expression. This ¢ts in
with the observation that there are two strong promoters
(P1 and P2 ) upstream of the acetamidase which could drive
high-level expression in the absence of any further control
mechanism [3,7].
In the current model of regulation, AmiA binds to the
P2 promoter region in the absence of acetamide and prevents transcription of the three genes downstream. In the
presence of acetamide, AmiA no longer binds to the P2
promoter region and transcription can then proceed at a
higher level. We have previously suggested that AmiC,
which has a probable acetamide-binding domain, binds
to both acetamide and AmiA, thereby preventing its interaction with the promoters [7]. The data from the amiAC
strain (Mad3, which was also high-level constitutive) con¢rm that AmiC is not required for acetamidase induction
in the absence of AmiA. This is supported by the fact that
AmiC is required for AmiE induction in the presence of
AmiA, since the amiCv strain (Mad2) was non-inducible.
Thus the data from the mutants are compatible with a
model of control where AmiC interacts directly with
AmiA.
Partial complementation of the amiAC deletion with
either amiA (Mad3:303) or amiC (Mad3:304) gave unexpected results. The amiACv, amiAþ strain showed constitutive expression, albeit to a lower level than the parental
amiACv strain. Thus the reintroduction of amiA was not
su⁄cient to repress the system completely. This may re£ect the stoichiometry of the system; in the complemented
strain there is only one copy of amiA, but two copies of P2
(and Pc , to which AmiA may also bind). If AmiA binding
to P2 is responsible for the repression only half of the P2
sites would be blocked, resulting in the derepression seen.
The amiACv, amiCþ strain showed normal induction
rather than the derepression seen in the amiAv strain previously. Again this may re£ect the fact that the complementing construct has P1 as well as Pc and amiC, so the
complemented strain has extra copies of potential DNAbinding sites. These results demonstrate the complexity
and tight stoichiometry of this system in terms of the
interaction between proteins and DNA.
The amiA, amiD mutant (Mad5) showed no induction
of the acetamidase, but the amiA, amiD, amiC deletion
Table 1
Plasmids and strains
Description
Plasmids
p2NIL
pGOAL15
pURR541
pURR211
pURR551
pINT3
pAGAN303
pAGAN304
Strains
Mad2
Mad3
Mad4
Mad5
Mad6
Mad2:304
Mad3:303
Mad3:304
Genotype
cloning vector, oriE, kan
gene cassette vector, hyg, Phsp60 sacB, lacZ
amiA deletion delivery vector
amiD deletion construct delivery vector
amiC deletion construct delivery vector
integrating vector; attP, int, Gm
amiA complementing vector attP, int, Gm
amiC complementing vector attP, int, Gm
amiC deletion
amiA, amiC double deletion
amiD deletion
amiA, amiD double deletion
amiC, amiA, amiD triple deletion
complemented amiCv mutant
partially complemented amiCAv mutant
partially complemented amiCAv mutant
Source
[9]
[9]
[7]
This study
This study
[7]
[7]
This study
amiCv
amiCv, amiAv
amiDv
amiAv amiDv
amiAv amiDv amiCv
amiCv [amiC+, Gm]
amiCv, amiA [amiA+, Gm]
amiCv, amiA [amiC+, Gm]
This
This
This
This
This
This
This
This
study
study
study
study
study
study
study
study
kan, kanamycin resistance gene; hyg, hygromycin resistance gene ; Gm, gentamicin resistance gene; int, mycobacteriophage L5 integrase; attP, mycobacteriophage L5 attachment site.
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135
strain (Mad6) was derepressed. This con¢rms that the promoter for acetamidase expression had not been deleted in
the amiD mutant (Mad4). In contrast to the situation with
AmiC, the absence of AmiA does not remove the requirement for AmiD for induction (the amiAD deletion combination was non-inducible). This indicates that there is no
direct interaction between AmiA and AmiD. AmiD has
been proposed as a DNA-binding protein and would
most probably be required to activate one of the promoters (possibly P3 ).
AmiC interacts with AmiA and prevents binding to P2 ,
thus allowing transcription of amiD to occur. AmiD is a
second positive regulator, which either activates P3 directly
or by relieving repression, and this leads to increased transcription of amiE. Further work to investigate the DNAbinding activity of these regulators should help to identify
the operator sites within this complex operon.
3.3. Conclusion and current model
G.R. was funded by the GlaxoSmithKline Action TB
Programme. D.G.N.M. was funded by the Link Applied
Genomics Programme.
The mode of regulation of the M. smegmatis acetamidase is complex, involving three regulatory proteins and
four promoters. The construction of several deletion
strains missing di¡erent combinations of the acetamidase
regulatory genes has enabled us to con¢rm that AmiA, C
and D are all involved in the regulation of this operon.
AmiC is a positive regulator which interacts directly with
acetamide and AmiA, whereas AmiA and AmiD are proposed DNA-binding proteins controlling the activity of
the four promoters in the operon. Previous work has suggested that there is a promoter immediately upstream of
amiE (P3 ) which may require activation by a regulator [7].
Several other bacteria have inducible amidase enzymes
and the genes of the M. smegmatis operon show similarity
to other regulatory genes [2,3]. We have previously shown
that AmiC has homology to other regulatory proteins,
including AmiC of Pseudomonas aeruginosa and NhhC
and NhlC of Rhodococcus rhodochrous [3]. Homology
has also been noted with FmdD of Methylophilus methylotrophus [10]. Both the Ps. aeruginosa AmiC and Me.
methylotrophus FmdD bind to amides, although the former is a regulatory protein and the latter is a transport
protein, whilst NhhC and NhlC are regulators of their
own operons [11,12]. Based on these similarities, the previously predicted role of AmiC was as a regulatory protein
which binds acetamide as a sensory mechanism [7]. Ps.
aeruginosa AmiC controls the regulation of amidase by
interaction with an anti-terminator AmiR [13]. There is
no evidence for anti-termination in M. smegmatis, which
does not possess a homologue of amiR, but the data
shown here suggest that AmiC does control expression
via protein^protein interaction with AmiA. In Me. methylotrophus, expression of the amidase is controlled by
FmdB, which has homology to AmiD, and this protein
has been proposed as a DNA-binding regulatory protein
[14]. Our data con¢rm the regulatory role of AmiD,
although a direct role in DNA-binding has not yet been
shown.
The data presented here allow us to re¢ne the previously
proposed model of regulation; AmiA is responsible for
repressing transcription of amiD from P2 in the absence
of acetamide by binding to the promoter and preventing
RNA polymerase access. In the presence of acetamide
Acknowledgements
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