Isolation and characterization of pLS 1 plasmid mutants with increased copy numbers

Abstract Streptococcus pneumoniae genetic systems designed for isolation of plasmid mutants with copy-up phenotypes have been developed. The target plasmids have the pLS1 replicon, and two different strategies have been followed: (i) selection of clones exhibiting augmented resistance to antibiotics, or (ii) obligatory co-existence of incompatible plasmids. We have isolated 23 plasmid mutants exhibiting increased number of copies. All the mutations corresponded to four different alleles of the copG gene of plasmid pLS1. These strategies could be used with other plasmids.     

Mathematical model for the control of ColE1 type plasmid replication

A mathematical model for the molecular events controlling replication of ColE1 type plasmids is described. All the model parameters can be evaluated independently. The model simulates plasmid replication and accurately predicts the copy-number of ColE1 plasmids carrying a variety of regulatory mutations. The model is used to test the plausibility of hypotheses concerning the interactions of regulatory elements involved in the replication apparatus. The model favorably supports the mechanism proposed by Tomizawa and co-workers concerning the nature of RNA-RNA interactions and that the Rom protein increases the binding between the two RNA species. The hypothesis that the interactions of RNA I-II increases the susceptibility of RNA II to the action of endonucleases is not a plausible mechanism.     

In vivo definition of the functional origin of leading strand replication on the lactococcal plasmid pFX2

The lactococcal plasmid pFX2 belongs to a family of plasmids, whose prototype is the streptococcal plasmid pMV158, that replicates by the rolling circle mechanism. Determination of the nucleotide sequence of the repX gene of pFX2 allowed us to make some minor corrections in the published sequence, and to show that the repX gene is identical to the rep gene of plasmid pWV01. We have established pFX2 in Escherichia coli and in Streptococcus pneumoniae. In the latter host, we have defined in vivo the nick site introduced by the RepX protein. Plasmid pFX2 and the pMV158 derivative pLS1 exhibit a moderate degree of incompatibility in S. pneumoniae. Cloning of the double strand origin (dso) of pFX2 into a high-copy-number plasmid that is compatible with the pMV158 replicon led to an increase in incompatibility toward pLS1. Plasmids pFX2 and pLS1 exhibit homologies in their Rep proteins and in their dso sequences, but not in their negative control elements. Thus, the observed incompatibility indicates that cross-recognition of Rep proteins and dso takes place. Received: 25 May 1998 / Accepted: 8 July 1998     

Mobility of the Native Bacillus subtilis Conjugative Plasmid pLS20 Is Regulated by Intercellular Signaling

Horizontal gene transfer mediated by plasmid conjugation plays a significant role in the evolution of bacterial species, as well as in the dissemination of antibiotic resistance and pathogenicity determinants. Characterization of their regulation is important for gaining insights into these features. Relatively little is known about how conjugation of Gram-positive plasmids is regulated. We have characterized conjugation of the native Bacillus subtilis plasmid pLS20. Contrary to the enterococcal plasmids, conjugation of pLS20 is not activated by recipient-produced pheromones but by pLS20-encoded proteins that regulate expression of the conjugation genes. We show that conjugation is kept in the default “OFF” state and identified the master repressor responsible for this. Activation of the conjugation genes requires relief of repression, which is mediated by an anti-repressor that belongs to the Rap family of proteins. Using both RNA sequencing methodology and genetic approaches, we have determined the regulatory effects of the repressor and anti-repressor on expression of the pLS20 genes. We also show that the activity of the anti-repressor is in turn regulated by an intercellular signaling peptide. Ultimately, this peptide dictates the timing of conjugation. The implications of this regulatory mechanism and comparison with other mobile systems are discussed.     

Segregation of the mutator property of plasmid R46 from its ultraviolet-protecting property

Summary Plasmid R46 (an R factor conferring resistance to ampicillin, sulfonamides, streptomycin and tetracycline) reduces the bactericidal effect of UV irradiation but increases its mutagenic effect (reversion of hisG46), and raises the frequency of spontaneous reversion (mutator effect). Putative deletion mutants of R46 were obtained by transduction of the plasmid, then two successive conjugal transfers. Plasmids of five of six deletion classes, each with a different combination of drug resistance traits, retained conjugative ability and the UV-protecting, mutagenesis-enhancing and mutator effects of R46. (pKM101, used in the Ames system to enhance responsiveness to chemical mutagens, is one such mutant of R46.) Plasmids of a sixth class, represented by pKM115, conferred resistance only to streptomycin and were non-conjugative. All of several such plasmids (of independent origin) had a much stronger mutator effect than did R46, but lacked UV-protecting ability and did not enhance the mutagenic effect of UV irradiation. We infer that R46 possesses: (i) a gene, uvp, which increases capacity for error-prone repair of UV-damaged DNA, and thus causes both UV protection and enhancement of UV mutagenesis; (ii) gene(s) whose action in the absence of gene uvp greatly increases the frequency of spontaneous reversion of hisG46. A plasmid of another incompatibility group, pLS51, has UV-protecting and mutagenesis-enhancing effect but lacks the mutator property; introduction of pLS51 into a clone of hisG46 carrying a pKM115-type plasmid greatly reduced its spontaneous reversion rate, as expected if pLS51 also has a uvp gene able to modulate the mutator effect of R46-derived gene(s) in the pKM115-type plasmid.     

RNA1 is sufficient to mediate plasmid ColE1 incompatibility in vivo

The multicopy plasmid ColE1 specifies a small RNA designated RNA1 that has been implicated in copy number control and incompatibility. We have inserted a 148 base-pair ColE1 DNA fragment containing a promoter-less RNA1 gene into a plasmid vector downstream from the tryptophan promoter of Serratia marcesens. The ColE1 RNA1 produced by this plasmid is not functional in vivo due to the presence of 49 nucleotides appended to the 5′-terminus of the wild-type RNA1 sequence. Deletions of these sequences by Bal3I nuclease in vitro and genetic selection for ColE1 incompatibility function in vivo permitted isolation of a plasmid expressing wild-type ColE1 RNA1 initiated properly from the S. marcesens trp promoter. These experiments demonstrate that RNA1 is sufficient to mediate ColE1 incompatibility in vivo. In addition, several plasmids were isolated that contain altered RNA1 genes. These alterations consist of additions or deletions of sequences at the 5′-terminus of RNA1. Analysis of the ability of these altered RNA1 molecules to express incompatibility in vivo suggests that the 5′-terminal region of RNA1 is crucial for its function.     

Physical and genetic analysis of the ColD plasmid.

The plasmid ColD-CA23, a high-copy-number plasmid of 5.12 kilobases, encodes colicin D, a protein of approximately 87,000 daltons which inhibits bacterial protein synthesis. Colicin D production is under the control of the Escherichia coli SOS regulatory system and is released to the growth medium via the action of the lysis gene product(s). A detailed map of the ColD plasmid was established for 10 restriction enzymes. Using in vitro insertional omega mutagenesis and in vivo insertional Tn5 mutagenesis, we localized the regions of the plasmid responsible for colicin D activity (cda), for mitomycin C-induced lysis (cdl), and for colicin D immunity (cdi). These genes were all located contiguously on a 2,400-base-pair fragment similar to a large number of other Col plasmids (A, E1, E2, E3, E8, N, and CloDF). The ColD plasmid was mobilizable by conjugative transfer by helper plasmids of the IncFII incompatibility group, but not by plasmids belonging to the groups IncI-alpha or IncP. The location of the mobilization functions was determined by deletion analysis. The plasmid needs a segment of 400 base pairs, which is located between the mob genes and the gene for autolysis, for its replication.     

Copy-number of broad host-range plasmid R1162 is regulated by a small RNA.

We have shown previously [Kim, K. and Meyer, R.J. (1985) J. Mol. Biol. 185,755-767] that copy-number of the broad host-range plasmid R1162 is controlled by the amounts of two proteins, encoded by cotranscribed genes comprising a region of the plasmid called RepI. We have now demonstrated that expression of RepI is negatively regulated by a 75 base RNA that is complementary to a segment of the RepI message. Increased intracellular amounts of RNA molecules that include this segment relieve the inhibition of RepI gene expression, suggesting that the target for regulation is the mRNA itself. A mutation decreasing the amount of the 75 base RNA results in elevated plasmid copy-number. Thus, consistent with our previous observations, regulation of the expression of the RepI genes is a factor in controlling plasmid copy-number.     

Isolation and characterization of a higher-copy-number mutant of plasmid R6K

A stable copy-number mutant (pNH601) of plasmid R6K was isolated by selection for increased resistance to ampicillin determined by this plasmid. The size of the mutant plasmid was found to be unchanged (26 Mg/mol) but it is present in 27 copies of pNH601 perE. coli K-12 chromosome which represents a two-fold increase of R6K copy number value. The following genetic properties of pNH601 are reported and compared with those of R6K: conjugative transfer, fertility inhibition of plasmids belonging to other incompatibility groups, incompatibility with plasmid R485 under both non-selective and selective conditions and the integrative suppression of thednaA ts mutation. The mutant plasmid pNH601 was found to be different from the original R6K in most of these properties.     

Multiple roles for DNA polymerase I in establishment and replication of the promiscuous plasmid pLS1

The polymerase activity of DNA polymerase I is important for the establishment of the pLS1 replicon by reconstitutive assembly in Streptococcus pneumoniae after uptake of exogenous pLS1 plasmid DNA. In polA mutants lacking the polymerase domain, such establishment was reduced at least 10-fold in frequency. Chromosomally facilitated establishment of pLS1-based plasmids carrying DNA homologous to the host chromosome was not so affected. However, both types of plasmid transfer gave mostly small colonies on initial selection, which was indicative of a defect in replication and filling of the plasmid pool. Once established, the pLS1-based plasmids replicated in polA mutants, but they showed segregational instability. This defect was not observed in strains with the wild-type enzyme or in an S. pneumoniae strain that encodes the polymerase and exonuclease domains of the enzyme on separate fragments. The role of DNA polymerase I in stably maintaining the plasmids depends on its polymerizing function in three separate steps of rolling-circle replication, as indicated by the accumulation of different replication intermediate forms in polA mutants. Furthermore, examination of the segregational stability of the pLS1 replicon in an Escherichia coli mutant system indicated that both the polymerase and the 5′-to-3′ exonuclease activities of DNA polymerase I function in plasmid replication.