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

Abstract

Background: 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.

Results: 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.

Conclusion: 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 sigma70-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.

Figure 1

Alignment of the ParA and ParB proteins. Comparison of the ParA (left) and ParB (right) aminoacid sequences of M. bovis BCG (MbBCG), M. smegmatis (Msm), M. tuberculosis (Mtb), M. leprae (Mlep) and S. coelicolor (Sc). The alignment was carried out using CLUSTAL W (1.83) . Conserved amino acids are indicated with asterisk below the alignment; "-" represents gaps, ":" indicate conserved substitutions and "." semi-conserved substitutions. The A (VL/FTIANQKGGVGKT), A' (GLKTLVIDLDP) and B (FDYVFV/ID) boxes typical of the Walker-ATPases and the C motif (LGLLTINALVAAPEVM/L) of ParA proteins are highlighted on grey. The Helix-turn-Helix motif (HDELAARIGRSRPLITNMIR) involved in DNA-protein interactions of ParB is also highlighted on grey [3]. As noted, mycobacterial ParA have a longer N-terminal domain (between 67 to 91 aa) than other bacterial-ParA proteins.

Figure 2

Transcriptional pattern of the M. bovis BCG orf60K-parB region. (A): Schematic representation of the M. bovis BCG orf60K-parB region showing the position of the transcriptional start sites (TSSs). The parS sequences are represented by solid grey rectangles. Cotranscripts identified by RT-qPCR are shown as horizontal bold arrows. TSSs are showed as bent arrows. The position of the TSSs mapped are in parenthesis and it localization is related to the start of the gene immediately downstream. (B): Transcriptional fusions to gfp and measurement of the fluorescence emission. Recombinant plasmids were obtained by cloning of PCR fragments (white rectangles) upstream of the gfp. The coordinates (5' and 3' ends with respect to the start codon of the gene being evaluated), of the cloned fragments are shown in parenthesis together with the plasmid name. The length (in bp) of the cloned fragments is indicated within the white rectangles and the grey arrows represent the cloning direction and the gfp gene. Promoter activity was measured by fluorimetry as Relative Fluorescent Units (RFU) in M. smegmatis corrected by subtracting pFPV27 mediated background fluorescence.The bars on the graphic represent RFU (means ± SE of at least three independently experiments) during stationary phase of growth. (C): Mapping of the mRNA 5' termini on the jag-gidB-parA-parB region of M. bovis BCG by primer extension. The mRNA 5'-ends or TSSs using specific oligos are indicated (T1jag, transcription start site for the promoter 1 of gene jag, etc.). Sequencing reaction with the same primers is shown alongside. The ParA1B primer was annealed to total RNA at 48°C. The highlighted boxed region defines the -35 and -10 promoter sequences identified upstream of each TSS; the numbers in parenthesis indicate the position to the TSS according to the start codon of the gene locate immediately downstream. Start codon for jag, gidB, parA and parB is shown in bold and the putative parS sequence located upstream gidB is highlighted with grey.

Figure 3

Transcriptional pattern of the M. smegmatis orf60K-parB region. (A): Schematic representation of the M. smegmatis orf60K-parB region showing the position of the transcriptional start sites (TSSs). The parS sequences are represented by solid grey rectangles. Cotranscripts identified by RT-qPCR are shown as horizontal bold arrows. TSSs are showed as bent arrows. The position of the TSSs mapped are in parenthesis and it localisation is related to the start of the gene immediately downstream. (B): Transcriptional fusions to gfp and measurement of the fluorescence emission. Recombinant plasmids were obtained by cloning of PCR fragments (white rectangles) upstream of the gfp. The coordinates (5' and 3' ends with respect to the start codon of the gene being evaluated), of the cloned fragments are shown in parenthesis together with the plasmid name. The length (in bp) of the cloned fragments is indicated within the white rectangles and the grey arrows represent the cloning direction and the gfp gene. Promoter activity was measured by fluorimetry as Relative Fluorescent Units (RFU) in M. smegmatis corrected by subtracting pFPV27 mediated background fluorescence. The bars on the graphic represent RFU (means ± SE of at least three independently experiments) during stationary phase of growth. (C): Mapping of the mRNA 5' termini on the jag-gidB-parA-parB region of M. smegmatis by primer extension. The mRNA 5'-ends or TSSs using specific oligos are indicated (T1jag, transcription start site for the promoter 1 of gene jag, etc.). Sequencing reactions with the same primers is shown alongside. The highlighted boxed region defines the -35 and -10 promoter sequences identified upstream of each TSS; the numbers in parenthesis indicate the position to the TSS according to the start codon of the gene locate immediately downstream. Start codon for jag, gidB, parA and parB is shown in bold and the putative parS sequence located upstream gidB is highlighted with grey.

Figure 4

parA and parB mRNA synthesis during growth of Mycobacteria in broth culture. (A): Total RNA isolated from exponential (7 days) and stationary (21 days) cultures of M. bovis BCG. (B): Total RNA isolated from exponential (OD_{595 nm} = 0.9), late exponential (OD_{595 nm} = 1.2) and stationary (OD_{595 nm} = 3.0) cultures of M. smegmatis. At the indicated time, bacterial RNA was extracted and transcript levels of parA (black bars) and parB (white bars) were analysed by real-time PCR; 16S rRNA levels were used for normalization. The error bars show the mean (± SD) of at least two separate determinations made with different batches of total RNA.