par genes in Mycobacterium bovis and Mycobacterium smegmatisare arranged in an operon transcribed from "SigGC" promoters

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 [1]. E. coli plasmid P1 and F factor partitioning systems encode: i) homologous ATPases (ParA/SopA), characterised by a conserved 'deviant' Walker A motif [2]; and ii) site-specific DNA-binding proteins containing helix-turn-helix (HTH) motifs (ParB/SopB) [3]. 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 [10].

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 [11]. 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 [11]. 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 [12]. 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 [11]. The parAB genes are essential for the viability of Caulobacter crescentus [13], whereas in B. subtilis [14], Streptomyces coelicolor [15] and Pseudomonas putida [16], 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 [15]. 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 [21]. 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 [22].

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 [23]. 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 [24]. 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 σ^A and σ^B 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.

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 σ^A (or σ^B) 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 σ^E [33], σ^H [34], σ^F [35], σ^C [36], σ^D [37], σ^L [38] or σ^M[39], 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 [27]. 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 [40]. 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 σ^A decrease [41] whilst σ^B expression increases [41, 42], we proposed that transcription from P1parB may be driven by σ^B, 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 [9]. 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.