Transcription initiation of the copy-number control and better-thanrandom segregation genes of the broad-host-range and low-copynumber plasmid pSM19035 are subjected to repression by the autoregulated pSM19035-encoded product in Bacillus subtilis cells. The promoters of the copS (Pcop1 and Pcop2), ␦ (P␦), and (P) genes have been mapped. These promoters are embedded in a set of either seven copies of a 7-bp direct repeat or in a block consisting of two 7-bp direct repeats and one 7-bp inverted repeat; the blocks are present either two or three times. The cooperative binding of protein to the repeats on the Pcop1, Pcop2, P␦, and P promoters represses transcription initiation by a mechanism that does not exclude A RNAP from the promoters. These results indicate that protein regulates plasmid maintenance by controlling the copy number on the one hand and by regulating the amount of proteins required for better-than-random segregation on the other hand.plasmid replication ͉ Gram-positive bacteria ͉ protein-DNA interactions ͉ transcriptional repressor
pSM19035 of the pathogenic bacterium Streptococcus pyogenes is a low-copy-number plasmid carrying erythromycin resistance, stably maintained in a broad range of gram-positive bacteria. We show here that the --operon of this plasmid constitutes a novel proteic plasmid addiction system in which the and genes encode an antitoxin and toxin, respectively, while plays an autoregulatory function. Expression of toxin Zeta is bactericidal for the gram-positive Bacillus subtilis and bacteriostatic for the gram-negative Escherichia coli. The toxic effects of gene expression in both bacterial species are counteracted by proper expression of . The -toxin-antitoxin cassette stabilizes plasmids in E. coli less efficiently than in B. subtilis.Bacterial plasmids are generally inherited in a very stable manner and independently of the cell chromosome. The specific mechanisms of stable plasmid maintenance have been studied mainly for plasmids replicating in gram-negative bacteria (23,44). In the majority of high-copy-number plasmids, the copy number and cell division control in combination with the multimer resolution system ensure a very low frequency of plasmid loss. For low-copy-number plasmids, mechanisms exist which enable their maintenance during cell growth in nonselective conditions. While the active partitioning process precisely distributes plasmid copies to each daughter cell at division (33, 64), plasmid addiction systems (also called toxinantitoxin TA cassettes) kill or reduce the growth of plasmidfree descendant cells (1, 35). The molecular basis of the postsegregational killing (PSK) requires the existence of at least two plasmid genes: one specifying a stable toxic agent and another coding for an unstable factor which prevents the lethal action of the toxin. While the toxin is always a protein, the antidote is either antisense RNA (which inhibits the translation of toxin mRNA) or a protein (35). Many such systems and their chromosomal analogues have been described for different gram-negative bacteria (6,24,28,45). Antisense RNA-regulated stabilization systems constitute a well-conserved group called the hok-sok family (the name reflecting the functions of the host killing and suppression of killing genes from plasmid R1) (22).Some common features can be indicated for proteic plasmid addiction systems (PPAS): organization in operons, autoregulation, formation of the antidote-toxin complex, and different decay rates of the two proteins involved (23,29,67). In contrast to the hok-sok family, there is no significant sequence similarity among PPAS genes. The specific mechanisms leading to the noxious effects of the toxin are known for few systems only. The first identified and best understood is ccd of Escherichia coli F factor (34). The CcdB toxin is a gyrase poison able to bind the free GyrA subunit and to trap cleaved DNA-gyrase complex, leading to the induction of SOS response and subsequent cell death (3). Gyrase is also the target for the ParE toxin of the broad-host-range RK2 plasmid parDE system (37), although t...
Plasmid pIPO2 is a cryptic, conjugative, broad-host-range plasmid isolated from the wheat rhizosphere. It efficiently self-transfers between α, β and γ Proteobacteria and has a mobilizing/retromobilizing capacity for IncQ plasmids. The complete nucleotide sequence of pIPO2 is presented on the basis of its mini-Tn5 ::luxABtet-tagged derivative, pIPO2T. The pIPO2 sequence is 39 815 bp long and contains at least 43 complete ORFs. Apart from a suite of ORFs with unknown function, all of the genes carried on pIPO2 are predicted to be involved in plasmid replication, maintenance and conjugative transfer. The overall organization of these genes is different from previously described plasmids, but is similar to the genetic organization seen in pSB102, a conjugative plasmid recently isolated from the bacterial community of the alfalfa rhizosphere. The putative conjugative transfer region of pIPO2 covers 23 kb and contains the genes required for DNA processing (Dtr) and mating pair formation (Mpf). The organization of these transfer genes in pIPO2 is highly similar to the genetic organization seen in the environmental plasmid pSB102 and in pXF51 from the plant pathogen Xylella fastidiosa. Plasmids pSB102 and pXF51 have recently been proposed to form a new family of environmental broad-host-range plasmids. Here it is suggested that pIPO2 is a new member of this family. The proposed Mpf system of pIPO2 shares high amino acid sequence similarity with equivalent VirB proteins from the type IV secretion system of Brucella spp. Sequence information was used to design primers specific for the detection of pIPO2. Environmental DNA from a range of diverse habitats was screened by PCR with these primers. Consistently positive signals for the presence of pIPO2 were obtained from a range of soil-related habitats, including the rhizospheres of young wheat plants, of field-grown oats and of grass (all gramineous plants), as well as from the rhizosphere of tomato plants. These data add to the growing evidence that plasmids carry advantageous genes with as yet undefined functions in plant-associated communities.
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