BACKGROUND
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  <cit> .
e. coli plasmid p <dig> and f factor partitioning systems encode: i) homologous atpases , characterised by a conserved 'deviant' walker a motif  <cit> ; and ii) site-specific dna-binding proteins containing helix-turn-helix  motifs   <cit> .
the centromere-like sites, pars and sopc, are located downstream of the genes encoding the trans-acting proteins  <cit> .
chromosomal homologues of para and parb , 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  <cit> .
the par genes are commonly arranged in an operon, whose expression is autoregulated by par-encoded proteins  <cit> .
in numerous bacteria, chromosomal par genes are located upstream of the dnaa-oric region  <cit> .
two or more 16-bp pars inverted repeats, with a consensus sequence 5'-tgttncacgtgaaaca- <dig>  are clustered near the origin of chromosome replication  region  <cit> .
in bacillus subtilis, spo0j binds to  <dig> of these  <dig> 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  <cit> .
in streptomyces coelicolor,  <dig> of the  <dig> pars sequences are packed around oric, and parb binds to many of them in vitro and in vivo  <cit> .
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  <cit> .
the parab genes are essential for the viability of caulobacter crescentus  <cit> , whereas in b. subtilis  <cit> , streptomyces coelicolor  <cit>  and pseudomonas putida  <cit> , 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  <cit> .
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  <cit> .
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  <cit> .
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  <cit> .
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  <cit> .
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  <cit> .
tuberculosis  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  <cit> .
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  <cit> .
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  <cit> .
group a includes the σa and σb mycobacterium promoters, which share homology to the e. coli σ <dig> consensus sequence.
the group d or "siggc" mycobacterium promoters, with - <dig>  and - <dig>  gc rich-hexamers, are likely to be unique to mycobacteria  <cit> .
however, it is still unknown which of the  <dig> sigma factors described in mycobacterium actually drive transcription from these promoters  <cit> .
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.
RESULTS
nucleotide sequence of the jag-parb region and conservation of the pars sites 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  with the chromosomal partitioning soj/para and spoj/parb proteins of s. coelicolor, p. putida and c. crescentus  <cit> .
genes homologous to para and parb were also identified in the close relatives mycobacterium leprae  <cit> , mycobacterium bovis  <cit>  and m. smegmatis  <cit>  and like in m. tuberculosis they are located near the chromosomal origin of replication .
eight orfs could be identified in the  <dig> kb region upstream of the dnaa gene in m. tuberculosis, m. bovis bcg and m. smegmatis .
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  <cit> .
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  <dig> nucleotides, suggesting that promoters localized in the para-parb intergenic region could initiate the transcription of the m. smegmatis parb gene.
lin and grossman  <cit>  identified a  <dig> bp perfect palindrome  identical to the pars sequence of b. subtilis, at two sites in the m. tuberculosis chromosome, located at ~ <dig>  kb and ~ <dig> 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,  <dig>  kb and  <dig>  kb upstream of the start codon of parb for m. bovis bcg, and  <dig>  kb and  <dig>  kb upstream of the start codon of parb for m. smegmatis.
no additional pars sequences were found in these mycobacterial chromosomes.
para and parb proteins alignments were performed using the translated par sequences proposed for m. bovis bcg strain pasteur 1173p <dig>  <cit> , m. smegmatis mc <dig>  <cit> , m. tuberculosis h37rv  <cit>  and m. leprae  <cit> .
multiple amino acid sequence alignments showed that all the motifs identified in the chromosomal-coding par proteins were conserved in the mycobacterial para and parb proteins .
the high aa sequence homology at the n-terminal region of the mycobacterial paras – and the fact that possible rbs sequences were not identified further downstream of the proposed para start codons – suggest that in contrast to other chromosome-encoded para proteins, mycobacterial paras begin far upstream of the walker a-box motif.
therefore, the mycobacterial para proteins may have an unusually long n-terminal domain.
however, the helix-turn-helix  dna-binding motif present in this region of some plasmid para proteins homologues was not present  <cit> .
promoter activity in the para and parb regulatory regions
in order to locate the promoters responsible for the transcription of the para and parb genes, we cloned fragments of the orf60k-parb region of m. bovis bcg and m. smegmatis in the promoterless vector pfpv <dig>  upstream of the gfp reporter gene .
gfp stability produced the accumulation of the fluorescent protein inside the cell and therefore the fluorescence at stationary phase always was higher than at exponential phase.
in addition, the absence of a transcriptional terminator upstream of the cloning site in pfpv <dig> resulted in a relatively high and almost constant fluorescence background during the different growth phases studied, ranging from  <dig> to  <dig> rfu.
hence, the gfp fusions performed were not to evaluate cell growth-related expression, but to identify the promoter of each gene under study.
fluorescence > 18– <dig> % of the background was considered to be indicative of activity of the cloned promoter.
all the constructs were tested for fluorescence emission in m. smegmatis mc <dig>  the figures 2b and 3b show the fluorescence obtained during the stationary phase of growth for each transcriptional fusion corrected by subtracting the fluorescence emission of m. smegmatis bearing the plasmid pfpv <dig>  we found that m. bovis bcg promoter activities were well expressed in the heterologous host m. smegmatis.
m. smegmatis cells emitted fluorescence when they bore plasmids containing the orf60k-jag  and jag-gidb  intergenic regions, as well as plasmids containing the 3'-end coding region of the gidb  and para  genes of m. bovis bcg , 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 , jag-gidb  and para-parb  intergenic regions as well as to the 3'-end of the gidb gene  .
unexpectedly, we found that a  <dig> bp fragment containing the pars motif localised in the 5'-end of the gidb gene of m. smegmatis  showed fluorescence emission independently of the clone direction, suggesting divergent promoter activity in this region.
when we deleted  <dig> bp of the 3'-end  or  <dig> bp of the 5'-end  from pb5b, the fluorescence emission was practically abolished, showing that the entire  <dig> bp region of pb5b was necessary in order to have the activity observed with this transcriptional fusion .
finally, the fluorescence of m. smegmatis bearing some constructs  was not detectable during the exponential phase of growth , 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-parb region
in an attempt to precisely localise the transcriptional start sites  in the jag-parb region of m. bovis bcg and m. smegmatis, primer extension experiments were carried out using several specific primers and total rna isolated from exponentially growing mycobacteria .
analysis of the nucleotide sequence upstream of the identified tsss was performed in order to identify potential promoters.
published consensus promoter sequences as well as the distance between the - <dig> hexamer and the tss and the length of the spacer between the - <dig> and - <dig> regions were considered.
promoter sequences proposed according to our results are shown in table  <dig>  all but one of the tsss of all genes corresponded to a purine  and each one was very well associated to a recognised promoter sequence.
all the identified promoters in both m. smegmatis and m. bovis bcg belonged to the group d of mycobacterium promoter recognition sequences, with the exception of two possible e. coli σ70- like promoters located upstream of gidb  and parb  in m. bovis bcg.
we found two tsss upstream of the jag, gid and parb genes in m. bovis bcg .
they were close to each other, suggesting that two promoters may drive the expression of each one of these genes.
fragments containing only one of the proposed promoters for jag , gid  and parb  genes of m. bovis bcg showed fluorescence activity  corroborating the presence of two promoters upstream of each one of these genes.
length of the spacer between the - <dig> and - <dig> hexamers.
length of the spacer between the - <dig> hexamer and tss.
transcription start site  determined by primer extension.
consensus nucleotides are shown with capital letters.
in contrast, we found just a single tss upstream of the jag, gid, para and parb genes in m. smegmatis  and upstream of the para gene in m. bovis bcg .
this implied the presence of only one promoter for each one of these genes.
the - <dig>  hexamer associated to the t1gidb of m. bovis bcg overlapped with a putative pars sequence , 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-parb region
the primer extension, transcriptional fusions to gfp, and nucleotide sequence analysis together indicated that the gid, para and parb genes of both m. bovis bcg and m. smegmatis, seem to be transcribed independently from their own promoters.
however, the short or missing intergenic regions found in this study do not eliminate the possibility that gid and the two par genes can be part of a single transcript.
to ascertain whether the par genes had a dicistronic arrangement, rt-qpcr was performed using m. bovis bcg and m. smegmatis rnas.
specific primers were designed in order to obtain products encompassing from the 3'-end to the 5'-start of the orf60k-jag, jag-gidb, gidb-para and para-parb pair genes , which always excluded the contribution of the promoters located immediately upstream of each evaluated gene.
although the possible presence of transcriptional termination signals into the downstream gene cannot be discarded, our results suggested that all the transcripts, except the one for jag gene of m. smegmatis, were at least dicistronic .
¶normalization was performed using primers for 16s rrna amplification previously published  <cit>
quantification of para and parb mrna levels during mycobacterial growth
the levels of para and parb genes mrnas in m. bovis bcg and m. smegmatis were quantified by real-time rt-pcr  in exponential as well as in the stationary growth phase.
quantitative pcrs for para, parb and 16s-rrna were performed using the cdnas obtained from the same rt reaction.
the amount of mrna for each par gene was calculated and expressed in relation to the total rna and normalized by the 16s-rrna levels.
we detected mrna-para that was double of mrna-parb levels in m. smegmatis, although the mrnas of both genes decreased between the exponential and stationary phases.
in contrast, the mrna-parb levels in m. bovis bcg were very similar between the exponential and stationary phases, but mrna-para levels showed an important reduction in the stationary growth phase.
additionally, unlike the transcriptional pattern observed in m. smegmatis, the mrna-parb levels were higher that mrna-para in m. bovis bcg .
discussion
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  or two  promoters located upstream of the gene encoding the atpase   <cit> .
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 .
rt-qpcr  and northern blot hybridisation  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 .
most of the putative promoter sequences identified  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  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  <cit> , σh  <cit> , σf  <cit> , σc  <cit> , σd  <cit> , σl  <cit>  or σm <cit> , 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 .
results also indicated that the para and parb genes in m. bovis bcg and m. smegmatis were differentially expressed , 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 σ <dig> have a higher activity than the group d mycobacterium promoters  <cit> .
in agreement with these observations, we found that the tsss in group d mycobacterial promoter sequences  showed weaker signals in comparison with those preceded by group a  of mycobacterium promoters .
the decrease of the mrnas for para and parb observed during the transition from exponential to stationary phase in m. smegmatis  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  <cit> .
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 .
the expression of e. coli σ70-like promoters  appears to be particularly important for parb, because the transcription from p2parb  as well as from para  did not account for the mrna-parb levels observed at the stationary growth phase .
since during stationary growth, the levels of σa decrease  <cit>  whilst σb expression increases  <cit> , 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  <cit>  and the bacterial chromosome  <cit> , 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  <cit> .
here, one putative pars site was identified in the regulatory region of the gidb gene of m. bovis bcg, which overlapped with the - <dig> sequence of one group a promoter , 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 .
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.
CONCLUSIONS
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.

 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 p and f factor partitioning systems encode: i) homologous atpases, characterised by a conserved'deviant' walker a motif; and ii) site-specific dna-binding proteins containing helix-turn-helix motifs. the centromere-like sites, pars and sopc, are located downstream of the genes encoding the trans-acting proteins. chromosomal homologues of para and parb, 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. the par genes are commonly arranged in an operon, whose expression is autoregulated by par-encoded proteins. 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- are clustered near the origin of chromosome replication region. in bacillus subtilis, spo0j binds to of these 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, of the 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. 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. 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. 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 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. group a includes the σa and σb mycobacterium promoters, which share homology to the e. coli σ consensus sequence. the group d or "siggc" mycobacterium promoters, with - and - gc rich-hexamers, are likely to be unique to mycobacteria. however, it is still unknown which of the sigma factors described in mycobacterium actually drive transcription from these promoters. 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 pars sites 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 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. eight orfs could be identified in the kb region upstream of the dnaa gene in m. tuberculosis, m. bovis bcg and m. smegmatis. 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 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 bp perfect palindrome identical to the pars sequence of b. subtilis, at two sites in the m. tuberculosis chromosome, located at ~ kb and ~ 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, kb and kb upstream of the start codon of parb for m. bovis bcg, and kb and kb upstream of the start codon of parb for m. smegmatis. no additional pars sequences were found in these mycobacterial chromosomes. para and parb proteins alignments were performed using the translated par sequences proposed for m. bovis bcg strain pasteur 1173p, m. smegmatis mc, m. tuberculosis h37rv and m. leprae. multiple amino acid sequence alignments showed that all the motifs identified in the chromosomal-coding par proteins were conserved in the mycobacterial para and parb proteins. the high aa sequence homology at the n-terminal region of the mycobacterial paras – and the fact that possible rbs sequences were not identified further downstream of the proposed para start codons – suggest that in contrast to other chromosome-encoded para proteins, mycobacterial paras begin far upstream of the walker a-box motif. therefore, the mycobacterial para proteins may have an unusually long n-terminal domain. however, the helix-turn-helix dna-binding motif present in this region of some plasmid para proteins homologues was not present. promoter activity in the para and parb regulatory regions in order to locate the promoters responsible for the transcription of the para and parb genes, we cloned fragments of the orf60k-parb region of m. bovis bcg and m. smegmatis in the promoterless vector pfpv upstream of the gfp reporter gene. gfp stability produced the accumulation of the fluorescent protein inside the cell and therefore the fluorescence at stationary phase always was higher than at exponential phase. in addition, the absence of a transcriptional terminator upstream of the cloning site in pfpv resulted in a relatively high and almost constant fluorescence background during the different growth phases studied, ranging from to rfu. hence, the gfp fusions performed were not to evaluate cell growth-related expression, but to identify the promoter of each gene under study. fluorescence > 18– % of the background was considered to be indicative of activity of the cloned promoter. all the constructs were tested for fluorescence emission in m. smegmatis mc the figures 2b and 3b show the fluorescence obtained during the stationary phase of growth for each transcriptional fusion corrected by subtracting the fluorescence emission of m. smegmatis bearing the plasmid pfpv we found that m. bovis bcg promoter activities were well expressed in the heterologous host m. smegmatis. m. smegmatis cells emitted fluorescence when they bore plasmids containing the orf60k-jag and jag-gidb intergenic regions, as well as plasmids containing the 3'-end coding region of the gidb and para genes of m. bovis bcg, 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, jag-gidb and para-parb intergenic regions as well as to the 3'-end of the gidb gene. unexpectedly, we found that a bp fragment containing the pars motif localised in the 5'-end of the gidb gene of m. smegmatis showed fluorescence emission independently of the clone direction, suggesting divergent promoter activity in this region. when we deleted bp of the 3'-end or bp of the 5'-end from pb5b, the fluorescence emission was practically abolished, showing that the entire bp region of pb5b was necessary in order to have the activity observed with this transcriptional fusion. finally, the fluorescence of m. smegmatis bearing some constructs was not detectable during the exponential phase of growth, 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-parb region in an attempt to precisely localise the transcriptional start sites in the jag-parb region of m. bovis bcg and m. smegmatis, primer extension experiments were carried out using several specific primers and total rna isolated from exponentially growing mycobacteria. analysis of the nucleotide sequence upstream of the identified tsss was performed in order to identify potential promoters. published consensus promoter sequences as well as the distance between the - hexamer and the tss and the length of the spacer between the - and - regions were considered. promoter sequences proposed according to our results are shown in table all but one of the tsss of all genes corresponded to a purine and each one was very well associated to a recognised promoter sequence. all the identified promoters in both m. smegmatis and m. bovis bcg belonged to the group d of mycobacterium promoter recognition sequences, with the exception of two possible e. coli σ70- like promoters located upstream of gidb and parb in m. bovis bcg. we found two tsss upstream of the jag, gid and parb genes in m. bovis bcg. they were close to each other, suggesting that two promoters may drive the expression of each one of these genes. fragments containing only one of the proposed promoters for jag, gid and parb genes of m. bovis bcg showed fluorescence activity corroborating the presence of two promoters upstream of each one of these genes. length of the spacer between the - and - hexamers. length of the spacer between the - hexamer and tss. transcription start site determined by primer extension. consensus nucleotides are shown with capital letters. in contrast, we found just a single tss upstream of the jag, gid, para and parb genes in m. smegmatis and upstream of the para gene in m. bovis bcg. this implied the presence of only one promoter for each one of these genes. the - hexamer associated to the t1gidb of m. bovis bcg overlapped with a putative pars sequence, 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-parb region the primer extension, transcriptional fusions to gfp, and nucleotide sequence analysis together indicated that the gid, para and parb genes of both m. bovis bcg and m. smegmatis, seem to be transcribed independently from their own promoters. however, the short or missing intergenic regions found in this study do not eliminate the possibility that gid and the two par genes can be part of a single transcript. to ascertain whether the par genes had a dicistronic arrangement, rt-qpcr was performed using m. bovis bcg and m. smegmatis rnas. specific primers were designed in order to obtain products encompassing from the 3'-end to the 5'-start of the orf60k-jag, jag-gidb, gidb-para and para-parb pair genes, which always excluded the contribution of the promoters located immediately upstream of each evaluated gene. although the possible presence of transcriptional termination signals into the downstream gene cannot be discarded, our results suggested that all the transcripts, except the one for jag gene of m. smegmatis, were at least dicistronic. ¶normalization was performed using primers for 16s rrna amplification previously published quantification of para and parb mrna levels during mycobacterial growth the levels of para and parb genes mrnas in m. bovis bcg and m. smegmatis were quantified by real-time rt-pcr in exponential as well as in the stationary growth phase. quantitative pcrs for para, parb and 16s-rrna were performed using the cdnas obtained from the same rt reaction. the amount of mrna for each par gene was calculated and expressed in relation to the total rna and normalized by the 16s-rrna levels. we detected mrna-para that was double of mrna-parb levels in m. smegmatis, although the mrnas of both genes decreased between the exponential and stationary phases. in contrast, the mrna-parb levels in m. bovis bcg were very similar between the exponential and stationary phases, but mrna-para levels showed an important reduction in the stationary growth phase. additionally, unlike the transcriptional pattern observed in m. smegmatis, the mrna-parb levels were higher that mrna-para in m. bovis bcg. discussion 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 or two promoters located upstream of the gene encoding the atpase. 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. rt-qpcr and northern blot hybridisation 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. most of the putative promoter sequences identified 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 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. results also indicated that the para and parb genes in m. bovis bcg and m. smegmatis were differentially expressed, 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 σ 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 showed weaker signals in comparison with those preceded by group a of mycobacterium promoters. the decrease of the mrnas for para and parb observed during the transition from exponential to stationary phase in m. smegmatis 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. the expression of e. coli σ70-like promoters appears to be particularly important for parb, because the transcription from p2parb as well as from para did not account for the mrna-parb levels observed at the stationary growth phase. since during stationary growth, the levels of σa decrease whilst σb expression increases, 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 and the bacterial chromosome, 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 - sequence of one group a promoter, 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. 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. 
genes homologous to para and parb, and two putative pars copies, have been identified in the mycobacterium bovis bcg and mycobacterium smegmatis chromosomes. 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. the para/soj and parb/spo0j proteins, and the cis-acting pars site, participate actively in chromosome segregation and cell cycle progression. however, parb expression was higher than para expression and remained almost unchanged along the growth curve. the present work focused on the determination of the transcriptional organisation of the ~ kb orf60k-parb region of m. bovis bcg and m. smegmatis by primer extension, transcriptional fusions to the green fluorescence protein and quantitative rt-pcr. in m. bovis bcg, mrna-para levels also decreased between the exponential and stationary phases. the majority of the proposed promoter regions had features characteristic of mycobacterium promoters previously denoted as group d. the - 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. real-time pcr assays showed that in m. smegmatis, mrna-para and mrna-parb levels decreased between the exponential and stationary phases. as in mycobacterium tuberculosis, the para and parb genes in these two non-pathogenic mycobacteria are located near the chromosomal origin of replication. the parab genes were arranged in an operon. however, we also found promoters upstream of each one of these genes.
 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 ~ kb orf60k-parb region of m. bovis bcg and m. smegmatis by primer extension, transcriptional fusions to the green fluorescence protein 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. 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 - 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. 