Development and application of molecular tools in the study of IncN-related plasmids from lakewater sediments

Homology to IncN, P, Q and W inc regions was investigated amongst 114 Hg(2+)-resistant or antibiotic-resistant bacteria isolated from lakewater sediments. No hybridisation signals were found with Inc P, Q and W probes, and only one plasmid, pLV1402, hybridised to the IncN probe. PCR primers designed to conserved regions in the replicon of the IncN plasmid pCU1 and the related beta replicon from pGSH500 were used to amplify a 978-bp fragment from pLV1402, with sequence analysis showing a close relationship (99.2% identity) between their replication genes. A 387-bp region from the pLV1402 rep gene was used to re-screen the isolates and identified another related plasmid, pLV1403. A 3.7-kb probe containing the alpha replicon from pGSH500 hybridised to both pLV1402 and pLV1403, suggesting that both are multi-replicon plasmids. The PCR primers and probes described will be useful in future studies of plasmid diversity.     

Cloning of a plasmid region specifying the N transfer system of bacterial conjugation in Escherichia coli

HindIII restriction sites were created artificially by the insertion of the transposon Tn.5 into the IncN plasmid pCU1 near a presumptive end of its conjugal transfer region (tra). This allowed cloning of an entire and continuous 19.4-kb region of this plasmid that specifies the N transfer system. The cloning vector was the nonconjugative plasmid pACYC184. The recombinant plasmid was as efficient in transfer as the parental N plasmid. Other clones and deletions extending into the tra region allowed localization of a 11.2-kb segment of this region that determines sensitivity to the N-specific bacteriophages IKe and PRD1. It could also be concluded that the ability of pCU1 to promote the killing of Klebsiella pneumoniae requires a 2-kb region that is not part of, but adjacent to the tra region.     

Physical and genetic organization of the IncN-group plasmid pCU1

A restriction endonuclease-cleavage map of the IncN group plasmid pCU1 was constructed. Deletion mutants of the plasmid were obtained by in vivo or in vitro methods. Comparison of the restriction maps of these mutants to that of pCU1 enables one to assign the known functions of the plasmid to particular regions on the plasmid DNA. For different enzymes, the number and distribution of restriction sites on pCU1 is compared to that of other IncN and related plasmids.     

The incN plasmid replicon: two pathways of DNA polymerase I-independent replication.

The 2,053-bp broad-host-range incompatibility group N replicon of plasmid pCU1 has two components: a region of 1,200 bp that is sufficient for its replication in Escherichia coli PolA+ and PolA- hosts and a regulatory region called the group I iteron region that contains 13 39-bp iterons. Within the 1,200-bp region, there are three replication origins, two of which, called oriB and oriS, function in PolA+ and PolA- hosts and a third, called oriV, which functions only in PolA+ hosts. The region also specifies a protein called RepA. We now show that both oriB and oriS can function in a delta polA strain but that in such a strain, only oriB has an absolute requirement for RepA. oriS can function without RepA and polymerase I provided that the iteron region is deleted and that in this circumstance, it is the only origin, the usage of which is detected. The requirements for oriB usage can thus be distinguished from those for oriS usage. The oriB region can be recovered as a plasmid only if RepA is provided in trans. These complex features of this replicon are also shown to be shared by the IncN replicons of other antibiotic resistance plasmids. Functionally distinguishable origins in a small replicon may be a way of endowing such a replicon with a broad host range.     

Distribution and Replication of the Pathogenicity Plasmid pPATH in Diverse Populations of the Gall-Forming Bacterium Pantoea agglomerans

Pantoea agglomerans has been transformed from a commensal bacterium into two related gall-forming pathovars by acquisition of pPATH plasmids containing a pathogenicity island (PAI). This PAI harbors an hrp/hrc gene cluster, type III effectors, and phytohormone biosynthetic genes. DNA typing by pulsed-field gel electrophoresis revealed two major groups of P. agglomerans pv. gypsophilae and one group of P. agglomerans pv. betae. The pPATH plasmids of the different groups had nearly identical replicons (98% identity), and the RepA protein showed the highest level of similarity with IncN plasmid proteins. A series of plasmids, designated pRAs, in which the whole replicon region (2,170 bp) or deleted derivatives of it were ligated with nptI were generated for replicon analysis. A basic 929-bp replicon (pRA6) was sufficient for replication in Escherichia coli and in nonpathogenic P. agglomerans. However, the whole replicon region (pRA1) was necessary for expulsion of the pPATH plasmid, which resulted in the loss of pathogenicity. The presence of direct repeats in the replicon region suggests that the pPATH plasmid is an iteron plasmid and that the repeats may regulate its replication. The pPATH plasmids are nonconjugative but exhibit a broad host range, as shown by replication of pRA1 in Erwinia, Pseudomonas, and Xanthomonas. Restriction fragment length polymorphism analyses indicated that the PAIs in the two groups of P. agglomerans pv. gypsophilae are similar but different from those in P. agglomerans pv. betae. The results could indicate that the pPATH plasmids evolved from a common ancestral mobilizable plasmid that was transferred into different strains of P. agglomerans.     

Analysis of pCU1 replication origins: dependence of oriS on the plasmid-encoded replication initiation protein RepA

The broad-host-range replicon of the plasmid pCU1 has three origins of vegetative replication called oriB, oriS, and oriV. In the multi-origin replicon, individual origins can distinguish among replication factors provided by the host. It has been found that during replication in Escherichia coli polA(-) host, oriS was the only active origin of a mutant pCU1 derivative bearing a mutation in the gene encoding replication initiation protein RepA. To further investigate the capacity of oriS to function in an E. coli polA(-) host we constructed a number of clones of the basic replicon of pCU1 containing oriS as the only replication origin. An oriS construct created with pUC18 could transform the polA(-) strain when RepA was supplied in trans. When the oriS region (between nucleotides 290 and 832) was ligated to an antibiotic resistance Omega fragment, the construct could be recovered as a plasmid from polA(+) strain if functional RepA was provided in trans. Our results therefore indicate that the basic replicon of pCU1, containing oriS as the sole origin, does require RepA to initiate plasmid replication in E. coli     

Three clustered origins of replication in a promiscuous-plasmid replicon and their differential use in a PolA+ strain and a delta PolA strain of Escherichia coli K-12.

A 1,197-bp region of the broad-host-range plasmid pCU1 is adequate for its replication. Analysis of replicating molecules containing this region reveals three clustered origins of vegetative replication and replication proceeds bidirectionally from each in a theta mode. In an Escherichia coli polymerase I deletion mutant, utilization of one of these three origins was not detected. The potentiality for origin utilization may therefore be a determinant of replicon host range.     

Characterization of a replicon of the moderately promiscuous plasmid, pGSH5000, with features of both the mini-replicon of pCU1 and the ori-2 of F

The dominant, polA1-independent replicon of pGSH500, repβ (1.8 kb), consists of a cis-acting oriV region of 245 bp; a repB gene that is essential for autonomous replication and 18, 30 to 36 bp iterons which constitute the inc/cop region. The molecular organization of repβ resembles that of mini-pCU1 (IncN). Furthermore, there is a 58% identity between the Rep proteins of these replicons. RepB also shows a 31% identity with RepE of mini-F. In addition, an 80% identity over 200 bp was identified between the cis-acting βoriV region and the equivalent region of ori-2 (mini-F). Replicons with deletions of repB could be complemented by Rep (pCU1) and RepE (mini-F) in trans, supporting the hypothesis that repβ is a natural hybrid between a pCU1-like and F-like replicon.     

Functional Analysis of Three Plasmids from Lactobacillus plantarum

Lactobacillus plantarum WCFS1 harbors three plasmids, pWCFS101, pWCFS102, and pWCFS103, with sizes of 1,917, 2,365, and 36,069 bp, respectively. The two smaller plasmids are of unknown function and contain replication genes that are likely to function via the rolling-circle replication mechanism. The host range of the pWCFS101 replicon includes Lactobacillus species and Lactococcus lactis, while that of the pWCFS102 replicon also includes Carnobacterium maltaromaticum and Bacillus subtilis. The larger plasmid is predicted to replicate via the theta-type mechanism. The host range of its replicon seems restricted to L. plantarum. Cloning vectors were constructed based on the replicons of all three plasmids. Plasmid pWCFS103 was demonstrated to be a conjugative plasmid, as it could be transferred to L. plantarum NC8. It confers arsenate and arsenite resistance, which can be used as selective markers.     

The integron In1 in plasmid R46 includes two copies of the oxa2 gene cassette

The sequence of the insert region of the integron In1 found in the IncN plasmid R46 was completed. The insert region is 2929 bases long and includes four gene cassettes, two of which are identical copies of the oxa2 gene cassette flanking an aadA1 cassette. The fourth cassette encodes an open reading frame orfD. From comparison of these data with published maps and sequences it is argued that the integrons found in the IncN plasmids pCU1 and R1767 and in the transposon Tn2410 are closely related to In1 from R46. Both site-specific gene insertion and recA-dependent recombination are likely to have contributed to the evolution of these integrons.     

The oriT region of the conjugative transfer system of plasmid pCU1 and specificity between it and the mob region of other N tra plasmids.

The oriT region of the conjugative IncN plasmid pCU1 has been localized to a 669-bp sequence extending from pCU1 coordinates 8.48 to 9.15 kb. The nucleotide sequence of this region was determined. The region is AT-rich (69% AT residues), with one 19-bp and one 81-bp sequence containing 79% or more AT residues. Prominent sequence features include one set of thirteen 11-bp direct repeats, a second set of two 14-bp direct repeats, six different inverted repeat sequences ranging from 6 to 10 bp in size, and two sequences showing 12 of 13 nucleotides identical to the consensus integration host factor binding sequence. Specificity between this oriT and mobilization (mob) functions encoded by the N tra system was demonstrated. This specificity is encoded by the region lying clockwise of the BglII site at coordinate 3.3 on the pCU1 map. Two N tra plasmids isolated in the preantibiotic era were unable to mobilize recombinant plasmids carrying the oriT region of pCU1 or to complement transposon Tn5 mutations in the mob region of the closely related plasmid pKM101.     

The complete genome sequences of four new IncN plasmids from wastewater treatment plant effluent provide new insights into IncN plasmid diversity and evolution

The dissemination of antibiotic resistance genes among bacteria often occurs by means of plasmids. Wastewater treatment plants (WWTP) were previously recognized as hot spots for the horizontal transfer of genetic material. One of the plasmid groups that is often associated with drug resistance is the incompatibility group IncN. The aim of this study was to gain insights into the diversity and evolutionary history of IncN plasmids by determining and comparing the complete genome sequences of the four novel multi-drug resistance plasmids pRSB201, pRSB203, pRSB205 and pRSB206 that were exogenously isolated from the final effluent of a municipal WWTP. Their sizes range between 42,875 bp and 56,488 bp and they share a common set of backbone modules that encode plasmid replication initiation, conjugative transfer, and plasmid maintenance and control. All plasmids are transferable at high rates between Escherichia coli strains, but did not show a broad host range. Different genes conferring resistances to ampicillin, streptomycin, spectinomycin, sulfonamides, tetracycline and trimethoprim were identified in accessory modules inserted in these plasmids. Comparative analysis of the four WWTP IncN plasmids and IncN plasmids deposited in the NCBI database enabled the definition of a core set of backbone genes for this group. Moreover, this approach revealed a close phylogenetic relationship between the IncN plasmids isolated from environmental and clinical samples. Phylogenetic analysis also suggests the existence of host-specific IncN plasmid subgroups. In conclusion, IncN plasmids likely contribute to the dissemination of resistance determinants between environmental bacteria and clinical strains. This is of particular importance since multi-drug resistance IncN plasmids have been previously identified in members of the Enterobacteriaceae that cause severe infections in humans.     

Unique Helicase Determinants in the Essential Conjugative TraI Factor from Salmonella enterica Serovar Typhimurium Plasmid pCU1

The widespread development of multidrug-resistant bacteria is a major health emergency. Conjugative DNA plasmids, which harbor a wide range of antibiotic resistance genes, also encode the protein factors necessary to orchestrate the propagation of plasmid DNA between bacterial cells through conjugative transfer. Successful conjugative DNA transfer depends on key catalytic components to nick one strand of the duplex DNA plasmid and separate the DNA strands while cell-to-cell transfer occurs. The TraI protein from the conjugative Salmonella plasmid pCU1 fulfills these key catalytic roles, as it contains both single-stranded DNA-nicking relaxase and ATP-dependent helicase domains within a single, 1,078-residue polypeptide. In this work, we unraveled the helicase determinants of Salmonella pCU1 TraI through DNA binding, ATPase, and DNA strand separation assays. TraI binds DNA substrates with high affinity in a manner influenced by nucleic acid length and the presence of a DNA hairpin structure adjacent to the nick site. TraI selectively hydrolyzes ATP, and mutations in conserved helicase motifs eliminate ATPase activity. Surprisingly, the absence of a relatively short (144-residue) domain at the extreme C terminus of the protein severely diminishes ATP-dependent strand separation. Collectively, these data define the helicase motifs of the conjugative factor TraI from Salmonella pCU1 and reveal a previously uncharacterized C-terminal functional domain that uncouples ATP hydrolysis from strand separation activity.     

Lethality and survival of Klebsiella oxytoca evoked by conjugative IncN group plasmids.

The transmission of plasmid pCU1 (or other IncN group plasmid) into a population of Klebsiella oxytoca cells reduces the viability of the population. A 2,400-bp region adjacent to traA is responsible for this phenotype and includes two regions, called kikA and kikC. Klebsiella cells which received this region and survived were found to acquire a chromosomal mutation which renders them immune to killing even after the plasmid is cured from the cells. To obtain insight into the mode of this apparent lethality, an appropriate pCU1lacZ derivative was constructed. It could be introduced with high efficiency into Klebsiella cells. Analyses of the resultant colonies indicate that the loss of viability is not a consequence of the death of plasmid-free segregants. On the contrary and unlike postsegregational killing by plasmids, cells survived by losing the plasmid or by acquiring, secondarily, a chromosomal mutation which confers immunity to killing.     

IncC helper dependent plasmid-like replication of Salmonella Genomic Island 1

The Salmonella genomic island 1 (SGI1) and its variants are mobilized by IncA and IncC conjugative plasmids. SGI1-family elements and their helper plasmids are effective transporters of multidrug resistance determinants. SGI1 exploits the transfer apparatus of the helper plasmid and hijacks its activator complex, AcaCD, to trigger the expression of several SGI1 genes. In this way, SGI1 times its excision from the chromosome to the helper entry and expresses mating pore components that enhance SGI1 transfer. The SGI1-encoded T4SS components and the FlhDC-family activator proved to be interchangeable with their IncC-encoded homologs, indicating multiple interactions between SGI1 and its helpers. As a new aspect of this crosstalk, we report here the helper-induced replication of SGI1, which requires both activators, AcaCD and FlhDC_SGI1, and significantly increases the stability of SGI1 when coexists with the helper plasmid. We have identified the oriV_SGI1 and shown that S004-repA operon encodes for a translationally coupled leader protein and an IncN2/N3-related RepA that are expressed under the control of the AcaCD-responsive promoter P_S004. This replicon transiently maintains SGI1 as a 4–8-copy plasmid, not only stabilizing the island but also contributing to the fast displacement of the helper plasmid.     

pUCL287 plasmid from Tetragenococcus halophila (Pediococcus halophilus) ATCC 33315 represents a new theta-type replicon family of lactic acid bacteria

Abstract A cryptic plasmid, pUCL287, was isolated from Tetragenococcus halophila (Pediococcus halophilus) ATCC 33315. It had a theta-type mechanism of replication in its natural host. Its minimal replicon, Rep 281, was isolated on a 1.6-kb Eco RI fragment. The Rep 287 host range included the genera Pediococcus, Enterococcus, Lactobacillus and Leuconostoc but not genus Lactococcus . Plasmids hybridizing to pUCL287 are rare among lactic acid bacteria. As assessed by hybridization, Rep2Sl is dissimilar to pAMβ1, pIP50l and pUCL22, representatives of the most common theta-type replicon groups in Gram-positive bacteria. Therefore, pUCL287 appears to represent a new theta-type replicon family from lactic acid bacteria.     

The integron In1 in plasmid R46 includes two copies of the oxa2 gene cassette.

The sequence of the insert region of the integron In1 found in the IncN plasmid R46 was completed. The insert region is 2929 bases long and includes four gene cassettes, two of which are identical copies of the oxa2 gene cassette flanking an aadA1 cassette. The fourth cassette encodes an open reading frame orfD. From comparison of these data with published maps and sequences it is argued that the integrons found in the IncN plasmids pCU1 and R1767 and in the transposon Tn2410 are closely related to In1 from R46. Both site-specific gene insertion and recA-dependent recombination are likely to have contributed to the evolution of these integrons.     

Characterization and sequence analysis of a stable cryptic plasmid from Enterococcus faecium 226 and development of a stable cloning vector.

A small cryptic plasmid, pMBB1, isolated from Enterococcus faecium 226 was characterized. The plasmid contained an extremely stable replicon which has limited homology to the lactococcal plasmid pCI305. Sequence analysis of the replicon detected one open reading frame of 822 bp capable of encoding a 32-kDa protein. No detectable single-stranded intermediates were found for the replicon, suggesting that pMBB1 may be included in the same family as pCI305, although pCI305 exhibits a more narrow host range. A small stably maintained vector able to replicate in a variety of lactic acid bacteria, containing a large multiple cloning region, was constructed by using the pMBB1 replicon.     

A chimeric vector for efficient chromosomal modification in Enterococcus faecalis and other lactic acid bacteria

Aim: To construct a chimeric vector named pBVGh for quickly generating gene modifications in Enterococcus faecalis. Methods and Results: The constructed plasmid pBVGh carries the pG⁺host replicon (a thermosensitive (TS) derivative of pWV01), allowing a simple generation of mutants by growing colonies first at the permissive temperature and then switching the culture to the nonpermissive temperature. Additionally, this vector facilitates the screening of mutants by a rapid colorimetric blue‐white discrimination of plasmid‐free bacteria. Conclusions: The pBVGh vector allows a straightforward inactivation or modification of target genes as well as a fast selection of enterococcal mutant strains. Significance and Impact of the Study: The broad range of the TS replicon utilized in this plasmid permits the easy establishment and the efficient generation of food‐grade mutant strains in Ent. faecalis and several other Gram‐positive bacteria.