Autoregulation of the Synthesis of the MobM Relaxase Encoded by the Promiscuous Plasmid pMV158

Abstract

bThe streptococcal promiscuous plasmid pMV158 (5,540 bp) replicates by the rolling-circle mechanism and can be mobilized among a wide number of Gram-positive and -negative bacteria. The plasmid region involved in its conjugative transfer includes the mobM gene, which encodes the MobM relaxase, and the cis-acting origin of transfer (oriT). MobM initiates transfer by cleavage of supercoiled pMV158 DNA at a specific dinucleotide within oriT. In the present work, we have performed a detailed transcriptional analysis to assess the role of MobM in the control of its own gene expression. By in vivo and in vitro approaches, we demonstrated that mobM transcription in Escherichia coli was mostly initiated from a promoter (Pmob2) different from the one (Pmob1) used in Lactococcus lactis. Whereas promoter Pmob1 was embedded within the oriT sequence, promoter Pmob2 was placed apart from but adjacent to oriT. Further, MobM was able to repress the expression of its own gene from both promoters. Given the promiscuity of pMV158, the organization of the mobM promoter region suggests a strategy of the plasmid to cope with different transcription machineries of the hosts it colonizes.

Bringing them together: Plasmid pMV158 rolling circle replication and conjugation under an evolutionary perspective

“…3), the generation of cruciform secondary structures by one of them would hinder the formation of the other, indicating that the target DNA accessibility by the relaxase could depend on the plasmid DNA superhelicity (Fernández-López et al, 2014). Our in vitro analysis demonstrated that MobM binds specifically to ssDNA encompassing IR1/3 with high affinity (Lorenzo-Díaz et al, 2011), which allowed the MobM protein to repress its own synthesis (Lorenzo-Díaz et al, 2012). Functional relevance of the IRs in the different steps of the conjugative process is currently under exploration.…”

Mobilizable Rolling-Circle Replicating Plasmids from Gram-Positive Bacteria: A Low-Cost Conjugative Transfer

“…We have shown that MobM is able to negatively regulate the transcription of its own gene (76). This does not seem to be the case of several other plasmids, which require an accessory protein for transcriptional regulation of the relaxase synthesis (77).…”

“…Two promoters placed nearly in tandem, and termed P mob1 and P mob2 , are used by the host RNAP: the latter is preferentially used in E. coli (76), whereas the former is used in L. lactis (51). Whereas promoter P mob1 was mapped to reside within oriT pMV158 , promoter P mob2 was found to be adjacent to it; and yet both promoters were subjected to self-regulation by MobM in E. coli (76). Given the relative positions of RNAP and MobM proteins in the region encompassing the two promoters (Fig.…”

“…The region of pMV158 that includes the Pmob1 and Pmob2 promoters of the mobM gene is shown. In E. coli, mobM transcription is mostly initiated from promoter Pmob2 (in blue) (76). Promoter Pmob1 (in black), which is located within the oriT sequence, is used in L. lactis ((51).…”

“…Transcription start sites are also indicated with arrows. The regions recognized by MobM and E. coli RNA polymerase (RNAP) were determined by DNase I foot printing assays using linear double-stranded DNAs (42, 76). MobM is able to repress in vivo the transcription initiated from both promoters, P mob1 and P mob2 (76).…”