par genes in Mycobacterium bovis and Mycobacterium smegmatisare arranged in an operon transcribed from "SigGC" promoters
© Casart et al; licensee BioMed Central Ltd. 2008
Received: 06 August 2007
Accepted: 27 March 2008
Published: 27 March 2008
The ParA/Soj and ParB/Spo0J proteins, and the cis-acting parS site, participate actively in chromosome segregation and cell cycle progression. Genes homologous to parA and parB, and two putative parS copies, have been identified in the Mycobacterium bovis BCG and Mycobacterium smegmatis chromosomes. As in Mycobacterium tuberculosis, the parA and parB genes in these two non-pathogenic mycobacteria are located near the chromosomal origin of replication. The present work focused on the determination of the transcriptional organisation of the ~6 Kb orf60K-parB region of M. bovis BCG and M. smegmatis by primer extension, transcriptional fusions to the green fluorescence protein (GFP) and quantitative RT-PCR.
The parAB genes were arranged in an operon. However, we also found promoters upstream of each one of these genes. Seven putative promoter sequences were identified in the orf60K-parB region of M. bovis BCG, whilst four were identified in the homologous region of M. smegmatis, one upstream of each open reading frame (ORF).
Real-time PCR assays showed that in M. smegmatis, mRNA-parA and mRNA-parB levels decreased between the exponential and stationary phases. In M. bovis BCG, mRNA-parA levels also decreased between the exponential and stationary phases. However, parB expression was higher than parA expression and remained almost unchanged along the growth curve.
The majority of the proposed promoter regions had features characteristic of Mycobacterium promoters previously denoted as Group D. The -10 hexamer of a strong E. coli σ70-like promoter, located upstream of gidB of M. bovis BCG, overlapped with a putative parS sequence, suggesting that the transcription from this promoter might be regulated by the binding of ParB to parS.
Partitioning systems were first characterised in low copy number plasmids of Escherichia coli. In general, plasmid partition modules encode two trans-acting proteins and a cis-acting, centromere-like DNA sequence required for partitioning . E. coli plasmid P1 and F factor partitioning systems encode: i) homologous ATPases (ParA/SopA), characterised by a conserved 'deviant' Walker A motif ; and ii) site-specific DNA-binding proteins containing helix-turn-helix (HTH) motifs (ParB/SopB) . The centromere-like sites, parS and sopC, are located downstream of the genes encoding the trans-acting proteins [4, 5]. Chromosomal homologues of parA and parB (sometimes denoted as soj and spo0J, because of their involvement in sporulation), as well as parS, have been identified in a wide range of Gram-negative and Gram-positive bacteria, with the exception of certain γ-proteobacteria, including E. coli and Haemophilus influenzae [3, 6]. The par genes are commonly arranged in an operon, whose expression is autoregulated by par-encoded proteins [7–9]. In numerous bacteria, chromosomal par genes are located upstream of the dnaA-oriC region .
Two or more 16-bp parS inverted repeats, with a consensus sequence 5'-TGTTNCACGTGAAACA-3, are clustered near the origin of chromosome replication (oriC) region . In Bacillus subtilis, Spo0J binds to 8 of these 10 pseudo-palindromic 16-bp invert repeats in vivo. Furthermore, the presence of one of such site on an otherwise unstable plasmid stabilizes it in a Soj- and Spo0J dependent manner . In Streptomyces coelicolor, 20 of the 24 parS sequences are packed around oriC, and ParB binds to many of them in vitro and in vivo . Although the precise function of ParA and ParB is still unclear, it has been proposed that the recruitment of these proteins to parS sites may lead to the positioning of replicated chromosomal origins at opposite poles of the cell . The parAB genes are essential for the viability of Caulobacter crescentus , whereas in B. subtilis , Streptomyces coelicolor  and Pseudomonas putida , deletion of soj/parA and spo0J/parB is not lethal. spo0J mutants of B. subtilis display defects in chromosome segregation in both vegetative and sporulating cells [14, 17]. Deletion of parAB in S. coelicolor results in the production of significant numbers of anucleate spores, although no detectable defect is visible in vegetatively growing cells . In P. putida, whose cellular division occurs only by binary fission, anucleated-cells are only observed when mutants in these genes are grown in minimal medium or as they enter into stationary phase [16, 18]. The Par proteins are involved in other processes, such as chromosome replication, transcription, and a cell-cycle checkpoint that links chromosome segregation to cell division [13, 19, 20].
New insights about the role of Par proteins in chromosome segregation are emerging with the recent discovery of the bacterial cytoskeleton. A bacterial actin homolog, MreB, has been implicated in chromosome segregation. In the bacterial cells that have MreB, a membrane-associated coiled structure extends along the cell length . In C. crescentus, this structure may be used for transporting oriC rapidly towards the cell poles. MreB may bind to DNA via ParB forming a kinetocore-like complex, which might connect the oriC region to the MreB coil at the membrane, and thus may actively move this region toward the cell poles .
Tuberculosis (TB) is a major public health problem with one-third of the world's population infected by its etiologic agent, Mycobacterium tuberculosis. Over two million people die from TB each year . The tubercle bacilli can lie dormant for years, only to rise again when the immune system weakens due to old age, malnutrition or AIDS. M. tuberculosis is a non-capsulate and non-spore forming bacterium with a relatively simple life cycle. Despite the medical importance of this human pathogen, very little is known about the molecular mechanisms controlling its cell cycle.
An interesting problem in M. tuberculosis biology is therefore to understand how this intracellular pathogen regulates progression of its cell cycle during the stages of TB infection, including the dormant state. The dormant state may be considered in some ways analogous to sporulation, and some genes related to sporulation in B. subtilis and S. coelicolor are found in the genome of M. tuberculosis . Nevertheless, the dormant state may also be considered a special physiological state during which mycobacteria grow slowly, but are not sporulated.
Studies based on experimentally-mapped transcriptional start sites have provided a consensus sequence for several mycobacterial promoters [25–27]. Group A includes the σA and σB Mycobacterium promoters, which share homology to the E. coli σ70 consensus sequence. The Group D or "SigGC" Mycobacterium promoters, with -10 (C90R70C50C50M70S90) and -35 (T90G50S80C50S90T30) GC rich-hexamers, are likely to be unique to mycobacteria [27, 28]. However, it is still unknown which of the 13 sigma factors described in Mycobacterium actually drive transcription from these promoters [26, 27].
In order to understand their possible role in mycobacterial cell cycle, in this work we examined the genetic regulation of the parA and parB partitioning genes, by analysing the transcription of these genes in Mycobacterium bovis BCG and Mycobacterium smegmatis, two non-pathogenic mycobacteria, belonging respectively to the slow and fast-growing groups of the Mycobacterium genus.
Nucleotide sequence of the jag-parB region and conservation of the parSsites near the chromosomal origin of replication
Analysis of the complete genome sequence indicates that the ParA and ParB proteins of M. tuberculosis H37Rv have high sequence identity (50–60%) with the chromosomal partitioning Soj/ParA and SpoJ/ParB proteins of S. coelicolor, P. putida and C. crescentus . Genes homologous to parA and parB were also identified in the close relatives Mycobacterium leprae , Mycobacterium bovis  and M. smegmatis  and like in M. tuberculosis they are located near the chromosomal origin of replication (oriC).
Eight ORFs could be identified in the 6 Kb region upstream of the dnaA gene in M. tuberculosis, M. bovis BCG and M. smegmatis (see Additional file 1). All eight ORFs were divergently oriented in relation to the dnaA gene and included the parA and parB genes along with several other conserved genes, following a similar gene order to that found in other Gram-positive and -negative bacteria .
M. tuberculosis ParA and ParB proteins had sequences that were 99% and 100% identical to the homologous proteins in M. bovis BCG, and 77% and 71% identical to the homologous proteins in M. smegmatis, respectively. In M. tuberculosis and M. bovis BCG, the stop and start codons of gidB, parA and parB genes overlapped, suggesting that these genes could be part of a single operon. In M. smegmatis, the stop and start codons of gidB and parA genes overlapped, while the parA and parB genes were separated by 59 nucleotides, suggesting that promoters localized in the parA-parB intergenic region could initiate the transcription of the M. smegmatis parB gene. Lin and Grossman  identified a 16 bp perfect palindrome (5'-TGTTTCACGTGAAACA-3') identical to the parS sequence of B. subtilis, at two sites in the M. tuberculosis chromosome, located at ~1.1 Kb and ~2 Kb upstream of the parB gene. A Blast search of this sequence revealed that two putative parS sites seemed to be conserved in M. bovis BCG and M. smegmatis genomes at similar positions, 1.761 Kb and 0.9 Kb upstream of the start codon of parB for M. bovis BCG, and 1.749 Kb and 0.984 Kb upstream of the start codon of parB for M. smegmatis. No additional parS sequences were found in these mycobacterial chromosomes.
Promoter activity in the parA and parBregulatory regions
Plasmids used in this work
Reference or source
Kmr, shuttle vector for operon and gene fusion to gfp gene
261 bp PCR fragment from M. bovis BCG containing the upstream region of the gene jag
148 bp PCR fragment from M. bovis BCG containing part of the coding region of the orf60K
114 bp PCR fragment from M. bovis BCG containing the upstream region of the gene jag
205 bp PCR fragment from M. bovis BCG containing the upstream region of the gene gidB
116 bp PCR fragment from M. bovis BCG containing the coding region of the gene jag
113 bp PCR fragment from M. bovis BCG containing the upstream region of the gene gidB
214 bp PCR fragment from M. bovis BCG containing the upstream region of the gene parA
113 bp PCR fragment from M. bovis BCG containing part of the coding region of the gene parA
229 bp PCR fragment from M. bovis BCG containing the upstream region of the gene parB
320 bp PCR fragment from M. smegmatis containing the upstream region of the gene jag
159 bp PCR fragment from M. smegmatis containing the upstream region of the gene jag
256 bp PCR fragment from M. smegmatis containing part of the coding region of the gene jag
217 bp PCR fragment from M. smegmatis containing part of the coding region of the gene parA cloned in the direction of parA gene
217 bp PCR fragment from M. smegmatis containing part of the coding region of the gene parA cloned in the reverse direction of parA gene
120 bp PCR fragment from M. smegmatis containing part of the coding region of the gene gidB
200 bp PCR fragment from M. smegmatis containing part of the coding region of the gene gidB
475 bp PCR fragment from M. smegmatis containing the upstream region of the gene parB
122 bp PCR fragment from M. smegmatis containing the upstream region of the gene parB
M. smegmatis cells emitted fluorescence when they bore plasmids containing the orf60K-jag (pD19B) and jag-gidB (pB5B) intergenic regions, as well as plasmids containing the 3'-end coding region of the gidB (pA2B) and parA (pE1B) genes of M. bovis BCG (Figure 2B), suggesting that jag, gidB, parA and parB genes of M. bovis BCG may be transcribed from promoters localised immediately upstream of each one of these genes. The parA and parB genes of M. smegmatis could also be transcribed from their own promoters, because substantial fluorescence was detected when the cells had the GFP transcriptional fusion to the orf60K-jag (pJ3M), jag-gidB (pG2M plasmid) and parA-parB (pB16Ms plasmid) intergenic regions as well as to the 3'-end of the gidB gene (pB1M plasmid) (Figure 3B). Unexpectedly, we found that a 217 bp fragment containing the parS motif localised in the 5'-end of the gidB gene of M. smegmatis (pC18Ms and pC11Ms plasmids) showed fluorescence emission independently of the clone direction, suggesting divergent promoter activity in this region.
When we deleted 89 bp of the 3'-end (pA15B) or 92 bp of the 5'-end (pB3B) from pB5B, the fluorescence emission was practically abolished, showing that the entire 205 bp region of pB5B was necessary in order to have the activity observed with this transcriptional fusion (Figure 2B). Finally, the fluorescence of M. smegmatis bearing some constructs (pA3B, pA15B, pB3B, pC5B, pJ3M and pG2M) was not detectable during the exponential phase of growth (data not shown), suggesting that the promoters contained in these fragments were weak and their expression could be detected only after enough GFP have accumulated during growth.
Mapping the transcription start sites in the jag-parBregion
Promoter sequences for jag, gidB, parA and parB genes of M. bovis BCG (Mb) and M. smegmatis (Ms)
P1 jag (Mb)
P2 jag (Mb)
P1 gidB (Mb)
P2 gidB (Mb)
P parA (Mb)
P1 parB (Mb)
P2 parB (Mb)
P jag (Ms)
P gidB (Ms)
P parA (Ms)
P parB (Ms)
In contrast, we found just a single TSS upstream of the jag, gid, parA and parB genes in M. smegmatis (Figures 3A and 3C) and upstream of the parA gene in M. bovis BCG (Figure 2A and 2C). This implied the presence of only one promoter for each one of these genes.
The -10 (AAACAT) hexamer associated to the T1gidB of M. bovis BCG overlapped with a putative parS sequence (Figure 2C), suggesting that ParB could be regulating the transcription from P1gidB by competing for the same region with the RNA polymerase.
Dicistronic transcripts in the jag-parBregion
Sequences of PCR primers used for RT-qPCR¶
Coordinates (5', 3')
Coordinates (5', 3')
Co-transcription in the jag-parB region
Cotranscription region (cDNA copies/16S × 10-6)
M. bovis BCG
Exponential (7 days)
9.16 ± 5.4
16.40 ± 1.4
17.92 ± 2.2
1.42 ± 0.1
Stationary (14 days)
43.45 ± 5.9
108.70 ± 20.4
65.85 ± 23.9
10.39 ± 0.1
Early Exponential (OD585nm = 0.6)
58.27 ± 5.6
4.29 ± 1.2
14.95 ± 0.1
Late Exponential (OD585nm = 1.2)
35.61 ± 4.5
3.39 ± 0.5
12.36 ± 1.9
Stationary (OD585nm = 2.0)
0.28 ± 0.0
1.65 ± 0.1
Quantification of parA and parBmRNA levels during mycobacterial growth
We found evidence that the chromosomal parA and parB genes of M. bovis BCG and M. smegmatis are expressed from multiple promoters. To identify the promoter sequences that regulate the expression of the par genes, we mapped the transcription start sites of the par-mRNAs by primer extension and confirmed the activity of the identified promoters by transcriptional fusions to a fluorescent reporter. We also demonstrated that in M. bovis BCG the parA and parB genes are differentially expressed during the exponential and stationary growth phases.
In all microorganisms studied thus far, plasmid and chromosome-encoded partitioning genes are arranged in an operon. Transcription of the par genes is driven by one (in F and R1 plasmids, P1 prophage and C. crescentus) or two (in S. coelicolor) promoters located upstream of the gene encoding the ATPase (parA or sopA) [5, 7, 13, 15, 32]. The jag, gidB, parA and parB genes of M. bovis BCG and M. smegmatis shared orientation and close spacing, suggesting that they may be co-transcribed. However, we identified at least one promoter sequence for each of these genes (Figures 2 and 3 and Table 2). RT-qPCR (Table 4) and Northern blot hybridisation (data not shown) demonstrated that the parA-parB, gidB-parA and orf60-jag gene pairs were also transcribed as dicistronic operons; however, co-transcription between the jag-gidB region was only detected in M. bovis BCG (Table 3).
Most of the putative promoter sequences identified (Table 2) had features of the Mycobacterium promoters denoted as Group D. Only two of the promoter sequences found belonged to Group A Mycobacterium promoters. We were unable to identify promoter sequences for σ factors different from σA (or σB) and "SigGC" in the jag-parB region of both mycobacterial species, probably due to the exiguous data accumulated regarding DNA sequences recognized by RNA polymerases containing other σ factors. Nevertheless, no variation in the parA and parB gene expression has been observed in M. tuberculosis knockout mutants of σE , σH , σF , σC , σD , σL  or σM, suggesting that none of these σ factors were involved in the parAB expression.
Based on our results, we propose that in both M. bovis BCG and M. smegmatis, the parA and parB genes comprise an operon. Therefore, the expression of parB may be derived from three promoters in M. bovis BCG – two Group D and one Group A promoters – whereas parB transcription in M. smegmatis seems to be driven from only two promoters, both belonging to the Group D of Mycobacterium promoters (Figures 2 and 3 and Tables 2 and 4).
Results also indicated that the parA and parB genes in M. bovis BCG and M. smegmatis were differentially expressed (Figure 4), possibly due to the differential quantity and activity that each promoter contributed to transcribe the gidB parA and parB genes in each mycobacteria. It has been suggested that mycobacterial promoters homologous to E. coli σ70 have a higher activity than the Group D Mycobacterium promoters . In agreement with these observations, we found that the TSSs in Group D mycobacterial promoter sequences (T2gidB and T2parB) showed weaker signals in comparison with those preceded by Group A (T1gidB and T1parB) of Mycobacterium promoters (Figure 2C).
The decrease of the mRNAs for parA and parB observed during the transition from exponential to stationary phase in M. smegmatis (Figure 3B) may be in agreement with the assumption that genes involved in replication and cell division must be down regulated during the stationary phase. In keeping with this interpretation, the expression of these genes decreases when M. tuberculosis is cultured under starvation . The parB gene expression in M. bovis BCG seems to be differently regulated, because one Group A Mycobacterium promoter as well as two "SigGC" promoters appeared to contribute to parB expression in this mycobacterial species (Figure 2 and Table 2). The expression of E. coli σ70-like promoters (P1parB) appears to be particularly important for parB, because the transcription from P2parB (T2parB in Figure 2C) as well as from parA (Table 4) did not account for the mRNA-parB levels observed at the stationary growth phase (Figure 4A). Since during stationary growth, the levels of σA decrease  whilst σB expression increases [41, 42], we proposed that transcription from P1parB may be driven by σB, the principal-like sigma factor.
On the other hand, it has been suggested that the correct stoichiometry of the Par proteins is important for partition of plasmids [43, 44] and the bacterial chromosome [9, 45], and that therefore the par loci must be under strict regulation. Recently, it has been suggested that modulation of the chromosomal parAB expression may be mediated by the binding of ParB to parS sites located near promoter sequences . Here, one putative parS site was identified in the regulatory region of the gidB gene of M. bovis BCG, which overlapped with the -10 sequence of one Group A promoter (Figure 2C), suggesting that the binding of the ParB protein to the parS sequence may obstruct the access of the RNA Polymerase and negatively regulate the gidB expression. The other putative parS sequence identified was located within the coding region of the parA gene (Figure 2A). This suggests that ParB protein may also affect the expression of the parA gene in M. bovis BCG by blocking transcription initiated from TparA or the translation of the mRNA-parA. Thus, the regulation of the gidBparA genes and the parA expression by ParB binding to the parS sequences might contribute to maintain appropriate levels of the Par proteins.
Transcriptional analysis demonstrated that the par genes in M. bovis BCG and M. smegmatis had a dicistronic arrangement in which parA and parB were mainly expressed from weak "SigGC" promoters. However, additional Group A promoters were found upstream of parB and gidB in M. bovis BCG. Furthermore, the presence of multiple promoters for genes related to cell cycle as parAB, which may be regulated by different sigma factors, might be responsible of the differential regulation of these genes.
Media, bacterial strains and growth conditions
E. coli XL1-blue cultures were grown in Luria-Bertani (LB) broth or on LB agar plates at 37°C. M. smegmatis mc2155  and M. bovis BCG Pasteur (ATCC 35734) were grown at 37°C using Middlebrook 7H9 broth or 7H10 agar supplemented with 0.5 % (v/v) glycerol and 10 % (v/v) Middlebrook OADC (Difco). To avoid clumping, Tween 80 (0.05 %) was added to liquid media. The following concentrations of antibiotics were added when appropriate: Carbenicillin (Cb, 50 μg ml-1) or Kanamycin (Km, 50 μg ml-1 for E. coli, 25 μg ml-1 for mycobacteria).
Transcriptional fusion to gfpand fluorescence measurement
The nucleotide sequences of the orf60k-parB regions were obtained in a Blast search [29, 30]. Fragments of variable length containing the upstream region of the genes parA and parB from M. smegmatis and M. bovis BCG were inserted into the shuttle plasmid pFPV27  to obtain the transcriptional fusions to gfp. The fragments were the products of PCR amplification using specific primers and chromosomal DNA as template. Plasmids digestions with restriction endonucleases and sequencing confirmed the direction of the inserts. The plasmids generated (Table 1) were electroporated in M. smegmatis mc2155 and grown at 37°C in 7H9 medium containing Km. Aliquots of the cultures were taken at exponential (OD595 nm = 0.8 – 1.3) and stationary (OD595 nm > 1.6) growth phases for fluorescence measurements. Fluorescence was determined from 150 μl of culture using a fluorimeter (Tecan GENius) and the appropriate filter combinations for GFP. The specific promoter activities were expressed as relative fluorescence units (RFU) corrected by subtracting the fluorescence emission of M. smegmatis bearing the promoterless plasmid pFPV27.
RNA extraction and primer extension analysis
RNA was isolated from M. smegmatis and M. bovis BCG by cell disruption as previously described . For primer extension experiments, at least six synthetic oligonucleotides complementary to the mRNA strand of the upstream jag-gidB-parA-parB sequences were 5' end labeled with [γ-32P] ATP and T4 polynucleotide kinase. Each labeled primer (100 fmol) was annealed to 5–20 μg of total RNA at 52°C for 30 min. After cooling at room temperature, the primer extension reactions were carried out with AMV reverse transcriptase (Promega) at 42°C for 45 min. The extension products were separated on an 8% polyacrylamide/urea gel, alongside the sequencing reaction generated using the PCR fragments corresponding to the analysed sequence and the oligonucleotide used in the primer extension reaction as primer .
Detection of mRNA by quantitative RT-PCR
Total RNA was treated with DNAseI (Promega) during 45 min at 37°C and the absence of DNA was checked before reverse transcription by PCR amplification. The number of amplicons was measured by real-time PCR using gene-specific primers and SYBR Green. A standard curve was obtained for each set of primers by performing four different PCRs in parallel, using 10-fold dilutions of known amounts of M. bovis BCG or M. smegmatis chromosomal DNA (1,000, 10,000, 100,000, and 1,000,000 theoretical copies) alongside the uncharacterized samples. The melting curve of each amplicon was determined at the end of each experiment. Each measurement was performed at least in duplicate and repeated twice using independent RNA preparations from different cultures. In each sample 500 ng (or as indicated) of RNA and 0.5 μg of random hexamers (total concentration of 1 μM) were mixed in a total volume of 12 μl, heated to 65°C for 10 min and immediately chilled in ice-water for at least 5 min. Subsequently, 1 × PCR Buffer (10 mM Tris-Cl pH 8.3; 50 mM KCl), 5 mM MgCl2, 40 U of RNase inhibitor (RNasin Plus, Promega), 200 U of M-MLV (Moloney murine leukemia virus; Invitrogen) or AMV (Avian myeloblastosis virus; Promega) reverse transcriptase (RT) and all four deoxynucleoside triphosphates (final concentration of 1 mM each) were added. The reverse transcription reaction was performed at 42°C for 60 min. In all cases, a duplicate sample was prepared without RT as a control to measure DNA carryover. The enzyme was inactivated by heating at 99°C for 5 minutes.
Amplifications were performed in the DNA Engine Opticon (MJ Research) with sampling during elongation. Reactions were performed in 20 μl volume consisting of 0.25 μM concentration of each primer (Table 3), 10 μl of 2 × SG1Master mix (DyNAmo SYBR Green qPCR Kit. FINNZYMES) and 2 μl of the cDNA previously obtained. A control without RT was included in each run. The samples were subjected to 40 cycles of amplification (96°C denaturation for 10 s, specific annealing temperature for 15 s and 72°C extension for 20 s) in sealed strip tubes with optical caps; followed by incubation at 72°C for 5 min. To ensure that the fluorescent levels detected were due to the amplification of a specific product, a melting curve followed the final extension step, from 60°C to 95°C, with readings every 0.2°C.
Other molecular techniques
Digestions, ligations, filling of protruding ends and plasmid DNA isolation were performed according to standard procedures. Amplified fragments and plasmid DNAs were sequenced with USB Sequenase 2.0 (USB, Amersham) and [α-35S]dATP or with a dye terminator cycle sequencing kit and an ABI377 sequencer (PE Biosystem), using the appropriate primers.
This work was supported by grants from the Fondo Nacional de Investigaciones Científicas y Tecnológicas – Venezuela (FONACIT-S1-2001000706), and the European Union through its INCO program (ICA4-CT-2002-10063). JG-M received support from COFAA, EDI and SIP-20071141, IPN, Mexico, and CONACyT (Grant SEP-2004-C01-46404). We are grateful to A. Sánchez for technical support. We thank K. Rodriguez-Clark and I. Beacham for reading the manuscript and making helpful suggestions.
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