Host growth temperature and a conservative amino acid substitution in the replication protein of pPS10 influence plasmid host range.

pPS10 is a replicon isolated from Pseudomonas syringe pv. savastanoi that can be established at 37 degrees C efficiently in Pseudomonas aeruginosa but very inefficiently in Escherichia coli. The establishment of the wild-type pPS10 replicon in E. coli is favored at low temperatures (30 degrees C or below). RepA protein of pPS10 promotes in vitro plasmid replication in extracts from E. coli, and this replication depends on host proteins DnaA, DnaB, DnaG, and SSB. Mutant plasmids able to efficiently replicate in E. coli at 37 degrees C were obtained. Three of four mutants whose mutations were mapped show a conservative Ala-->Val change in the amino-terminal region of the replication protein RepA. Plasmids carrying this mutation maintain the capacity to replicate in P. aeruginosa and have a fourfold increase in copy number in this host. The mutation does not substantially alter the autoregulation mediated by RepA. These results show that the physiological conditions of the host as well as subtle changes in the plasmid replication protein can modulate the host range of the pPS10 replicon.     

RepFIC, a basic replicon of IncFI plasmids that has homology with a basic replicon of IncFII plasmids

It was found that a DNA segment containing genes for autonomous replication and its control (basic replicon) present in the IncFI plasmid P307 has homology with RepA, a basic replicon present in IncFII plasmids. The basic replicon in P307 is referred to as RepFIC and the homologous basic replicon in IncFII plasmids is referred to as RepFIIA. In 11 other IncFI plasmids studied a region that has homology with RepFIC and RepFIIA was demonstrated. Thus, of the several basic replicons present in IncFI plasmids, RepFIC is evolutionarily related to a basic replicon of IncFII plasmids.     

Cloning vectors, derived from a naturally occurring plasmid of Pseudomonas savastanoi, specifically tailored for genetic manipulations in Pseudomonas

A minimal replicon of 1.8 kb isolated from a 10-kb plasmid of Pseudomonas savastanoi, pPS10, has been used to obtain a collection of small vectors specific for Pseudomonas (P. savastanoi, P. aeruginosa and P.putida). In addition, shuttle vectors that can be established both in Pseudomonas and Escherichia coli have been constructed by adding a pMB9 replicon. The vectors permit cloning of DNA fragments generated by a variety of restriction enzymes using different antibiotic resistance markers for selection and offer the possibility to screen recombinants by insertional inactivation. This cloning system can be used to establish recombinant plasmids in Pseudomonas either at low or high copy number. pPS10 derivatives are compatible with other Pseudomonas vectors derived from broad-host-range replicons of the incompatibility groups P1, P4/Q and W. Introduction and expression of the iaaMH operon in a P. savastanoi mutant deficient in the production of indoleacetic acid has been achieved using one of these vectors.     

Characterization and mobilization of nonconjugative plasmids encoding resistance to streptomycin and sulfanilamide

Most of nonconjugative streptomycin (Sm)- and sulfanilamide (Su)- resistance of clinical isolates belonging to various species of Enterobacteriaceae and Pseudomonas were encoded by an Inc Q plasmid, molecular size of which was 5.5 Md. The SmSu plasmids were efficiently mobilized by Inc P plasmids between E. coli strains. Inc I group and Inc F group plasmids could mobilize the Inc Q plasmids at lower efficiencies. The Inc Q plasmid was also mobilized to various species of Enterobacteriaceae at high frequencies without accompanying the conjugative Inc P plasmid; as a result, most of the SmSu-resistant transconjugants were nontransferable. The above results may explain the wide distribution of nonconjugative SmSu strains among clinical isolates.     

Isolation of a new broad-host-range IncQ-like plasmid, pTC-F14, from the acidophilic bacterium Acidithiobacillus caldus and analysis of the plasmid replicon

A moderately thermophilic (45 to 50 degrees C), highly acidophilic (pH 1.5 to 2.5), chemolithotrophic Acidithiobacillus caldus strain, f, was isolated from a biooxidation process used to treat nickel ore. Trans-alternating field electrophoresis analysis of total DNA from the A. caldus cells revealed two plasmids of approximately 14 and 45 kb. The 14-kb plasmid, designated pTC-F14, was cloned and shown by replacement of the cloning vector with a kanamycin resistance gene to be capable of autonomous replication in Escherichia coli. Autonomous replication was also demonstrated in Pseudomonas putida and Agrobacterium tumefaciens LBA 4404, which suggested that pTC-F14 is a broad-host-range plasmid. Sequence analysis of the pTC-F14 replicon region revealed five open reading frames and a replicon organization like that of the broad-host-range IncQ plasmids. Three of the open reading frames encoded replication proteins which were most closely related to those of IncQ-like plasmid pTF-FC2 (amino acid sequence identities: RepA, 81%; RepB, 78%; RepC, 74%). However, the two plasmids were fully compatible and pTC-F14 represents a new IncQ-like plasmid replicon. Surprisingly, asymmetrical incompatibility was found with the less closely related IncQ plasmid R300B derivative pKE462 and the IncQ-like plasmid derivative pIE1108. Analysis of the pTC-F14 oriV region revealed five direct repeats consisting of three perfectly conserved 22-bp iterons flanked by iterons of 23 and 21 bp. Plasmid pTC-F14 had a copy number of 12 to 16 copies per chromosome in both E. coli, and A. caldus. The rep gene products of pTC-F14 and pTF-FC2 were unable to functionally complement each other's oriV regions, but replication occurred when the genes for each plasmid's own RepA, RepB, and RepC proteins were provided in trans. Two smaller open reading frames were found between the repB and repA genes of pTC-F14, which encode proteins with high amino acid sequence identity (PasA, 81%; PasB, 72%) to the plasmid addiction system of pTF-FC2. This is the second time a plasmid stability system of this type has been found on an IncQ-like plasmid.     

Microarray-based analysis of IncA/C plasmid-associated genes from multidrug-resistant Salmonella enterica

In the family Enterobacteriaceae, plasmids have been classified according to 27 incompatibility (Inc) or replicon types that are based on the inability of different plasmids with the same replication mechanism to coexist in the same cell. Certain replicon types such as IncA/C are associated with multidrug resistance (MDR). We developed a microarray that contains 286 unique 70-mer oligonucleotide probes based on sequences from five IncA/C plasmids: pYR1 (Yersinia ruckeri), pPIP1202 (Yersinia pestis), pP99-018 (Photobacterium damselae), pSN254 (Salmonella enterica serovar Newport), and pP91278 (Photobacterium damselae). DNA from 59 Salmonella enterica isolates was hybridized to the microarray and analyzed for the presence or absence of genes. These isolates represented 17 serovars from 14 different animal hosts and from different geographical regions in the United States. Qualitative cluster analysis was performed using CLUSTER 3.0 to group microarray hybridization results. We found that IncA/C plasmids occurred in two lineages distinguished by a major insertion-deletion (indel) region that contains genes encoding mostly hypothetical proteins. The most variable genes were represented by transposon-associated genes as well as four antimicrobial resistance genes (aphA, merP, merA, and aadA). Sixteen mercury resistance genes were identified and highly conserved, suggesting that mercury ion-related exposure is a stronger pressure than anticipated. We used these data to construct a core IncA/C genome and an accessory genome. The results of our studies suggest that the transfer of antimicrobial resistance determinants by transfer of IncA/C plasmids is somewhat less common than exchange within the plasmids orchestrated by transposable elements, such as transposons, integrating and conjugative elements (ICEs), and insertion sequence common regions (ISCRs), and thus pose less opportunity for exchange of antimicrobial resistance.     

Small, stable shuttle vectors based on the minimal pVS1 replicon for use in gram-negative, plant-associated bacteria

The minimal replicon of the Pseudomonas plasmid pVS1 was genetically defined and combined with the Escherichia coli p15A replicon, to provide a series of new, oligocopy cloning vectors (5.3 to 8.3 kb). Recombinant plasmids derived from these vectors were stable in growing and nongrowing cells of root-colonizing P. fluorescens strains incubated under different environmental conditions for more than 1 month.     

An oligonucleotide microarray to characterize multidrug resistant plasmids

Many of the Enterobacteriaceae carry multiple drug resistance (MDR) genes on large plasmids of replicon type Inc A/C and Inc H1. It is important to understand the transmission of these MDR plasmids because the genes they carry can affect the outcome of antimicrobial therapy. The aim of this study was to design a microarray with oligonucleotide probes for every gene in the six Inc A/C and one Inc H1 plasmids of interest while representing all redundant sequences only once. The microarray is printed in triplicate with 493 unique oligonucleotide probes 70 nucleotides in length. Salmonella enterica and Escherichia coli control strains and test plasmids (in the parent strain and transformed into a known E. coli background strain) were hybridized to the plasmid microarray. This hybridization arrays presents a rapid and cost effective method for high-density screening of isolates to evaluate the gene content of Inc A/C and H1 plasmids and will show how plasmids can change content with transmission.     

Nucleotide sequence based characterizations of two cryptic plasmids from the marine bacterium Ruegeria isolate PR1b

Two plasmids, 76 and 148 kb in size, isolated from Ruegeria strain PR1b were entirely sequenced. These are the first plasmids to be characterized from this genus of marine bacteria. Sequence analysis revealed a biased distribution of function among the putative proteins encoded on the two plasmids. The smaller plasmid, designated pSD20, encodes a large number of putative proteins involved in polysaccharide biosynthesis and export. The larger plasmid, designated pSD25, primarily encodes putative proteins involved in the transport of small molecules and in DNA mobilization. Sequence analysis revealed uncommon potential replication systems on both plasmids. pSD25, the first repABC-type replicon isolated from the marine environment, actually contains two repABC-type replicons. pSD20 contains a complex replication region, including a replication origin and initiation protein similar to iteron-containing plasmids (such as pSW500 from the plant pathogen Erwinia stewartii) linked to putative RepA and RepB stabilization proteins of a repABC-type replicon and is highly homologous to a plasmid from the phototrophic bacterium Rhodobacter sphaeroides. Given the nature of the putative proteins encoded by both plasmids it is possible that these plasmids enhance the metabolic and physiological flexibility of the host bacterium, and thus its adaptation to the marine sediment environment.     

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.     

Isolation and sequencing of the replication region of plasmid pBFp1 isolated from a marine biofilm

A 24 kb plasmid, pBFp1, encoding mercury resistance was previously isolated from a marine biofilm. Isolation and sequencing of a 4280 bp DNA fragment containing the plasmid replicon (rep-pBFp1) revealed a putative open reading frame encoding a RepA protein and an oriV-like region containing an A+T rich sub-region, iterons, and DnaA boxes. Sequence comparisons showed significant similarities to the incW plasmid pSa both for the RepA amino acid sequence and in the iteron DNA sequence. Plasmid pBFp1 was also shown to be incompatible with pSa in standard incompatibility testing. A probe from the repA gene of pBFp1 was further made and tested on a collection of plasmids exogenously isolated from marine habitats in a previous study.     

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.     

Molecular classification of IncP-9 naphthalene degradation plasmids

A large collection of naphthalene-degrading fluorescent Pseudomonas strains isolated from sites contaminated with coal tar and crude oil was screened for the presence of IncP-9 plasmids. Seventeen strains were found to carry naphthalene catabolic plasmids ranging in size from 83 to 120 kb and were selected for further study. Results of molecular genotyping revealed that 15 strains were closely related to P. putida, one to P. fluorescens, and one to P. aeruginosa. All catabolic plasmids found in these strains, with the exception of pBS216, pSN11, and p8909N-1, turned out to belong to IncP-9 beta-subgroup. Plasmids pBS216, pSN11, and p8909N-1 were identified as members of IncP-9 delta-subgroup. One plasmid, pBS2, contains fused replicons of IncP-9beta and IncP-7 groups. RFLP analyses of the naphthalene catabolic plasmids revealed that organisation of the replicon correlates well with the overall plasmid structure. Comparative PCR studies with conserved oligonucleotide primers indicated that genes for key enzymes of naphthalene catabolism are highly conserved among all studied plasmids. Three bacterial strains, P. putida BS202, P. putida BS3701, and P. putida BS3790, were found to have two different salicylate hydroxylase genes one of which has no similarity to the "classic" enzyme encoded by nahG gene. Discovery of a large group of plasmid with unique nahR suggested that the regulatory loop may also represent a variable part of the pathway for catabolism of naphthalene in fluorescent Pseudomonas spp.     

In vivo cloning of Pseudomonas aeruginosa genes with mini-D3112 transposable bacteriophage.

The transposition properties of the Pseudomonas aeruginosa mutator bacteriophage D3112 were exploited to develop an in vivo cloning system. Mini-D replicon derivatives of D3112 were constructed by incorporating broad host range plasmid replicons between short terminal D3112 sequences. These elements were made with small replication regions from the RK2, Sa, and pVS1 plasmids and selectable genes for tetracycline, carbenicillin, kanamycin, and gentamicin resistance. Some of the mini-D replicons also contain the RK2 oriT origin-of-transfer sequence, which allows them to be mobilized by conjugation to many different species of gram-negative bacteria. These elements were used to clone DNA by preparing lysates from P. aeruginosa cells harboring an inducible D3112 cts prophage and a mini-D replicon plasmid. These lysates were used to infect sensitive P. aeruginosa recipients and select recombinant plasmids as drug-resistant transductant colonies. These transductants form a gene library from which particular clones can be selected, such as by their ability to complement specific mutations. This system was used to clone nine different genes from the PAO chromosome. The ability of this system to precisely identify a gene was demonstrated by isolating clones of the argF+ and cys-59+ genes. Restriction maps of clones of these genes, which have different amounts of flanking DNA, located the positions of these genes. The sizes of the chromosomal DNA segments from 10 individual clones examined ranged from 6 to 21 kilobases (kb), with an average of about 10 kb. This is consistent with the approximately 40-kb DNA-packaging size of the D3112 phage.     

Molecular interactions of a replication initiator protein, RepA, with the replication origin of the enterococcal plasmid p703/5

We previously identified the origin of replication of p703/5, a small cryptic plasmid from the KBL703 strain of Enterococcus faecalis. The origin of replication contains putative regulatory cis-elements required for replication and a replication initiator (RepA) gene. The replicon of p703/5 is similar in its structural organization to theta-type plasmids, and RepA is homologous to a family of Rep proteins identified in several plasmids from Gram-positive bacteria. Here, we report molecular interactions between RepA and the replication origin of p703/5. DNase I footprinting using recombinant RepA together with electrophoretic mobility shift assays confirmed the binding of RepA to the replication origin of p703/5 via iterons and an inverted repeat. We also demonstrated the formation of RepA dimers and the different binding of RepA to the iteron and the inverted repeat using gel filtration chromatographic analysis, a chemical crosslinking assay, and electrophoretic mobility shift assays in the presence of guanidine hydrochloride. Our results suggest that RepA plays a regulatory role in the replication of the enterococcal plasmid p703/5 via mechanisms similar to those of typical iteron-carrying theta-type plasmids.     

Features of the replicon of plasmid pAM10.6 of Pseudomonas fluorescens

We describe features of the basic replicon of the 10.6-kb medium-copy-number plasmid pAM10.6. pAM10.6 was able to replicate in various Pseudomonas strains but was maintained in Escherichia coli only after the p15A origin of replication was inserted. Deletion analysis suggests that the pAM10.6 origin of replication is located in a 0.5-kb region that includes inverted and direct repeats upstream of the repA gene. RepA (204 aa) has a clear homology to plasmid replication proteins of some other gram-negative bacteria. The pas (plasmid addiction system) (genes encoded in the region of 480-bp) stabilizes plasmid maintenance in P. putida cells under nonselective conditions for at least 200 generations. A 3.75-kb PstI fragment of pAM10.6 joined to a Km(r) gene was shown to be a minimal plasmid unit maintained in P. putida as a monomer. Further deletions of this 3.75-kb fragment caused a drive to form stable head-to-tail dimeric plasmids in P. putida.     

Plasmid-Encoded Anthranilate Synthase (TrpEG) in Buchnera aphidicola from Aphids of the Family Pemphigidae

Buchnera aphidicola is an obligate intracellular symbiont of aphids. One of its proposed functions is the synthesis of essential amino acids, nutrients required by aphids but deficient in their diet of plant phloem sap. The genetic organization of the tryptophan pathway in Buchnera from proliferous aphids of the family Aphididae has previously been shown to reflect a capacity to overproduce this essential amino acid (C.-Y. Lai, L. Baumann, and P. Baumann, Proc. Natl. Acad. Sci. USA 91:3819–3823, 1994). This involved amplification of the genes for the first enzyme in the pathway, anthranilate synthase (TrpEG), on a low-copy-number plasmid. Here we report on the finding and molecular characterization of TrpEG-encoding plasmids in Buchnera from aphids of the distantly related family Pemphigidae. Buchnera from Tetraneura caerulescens contained a 3.0-kb plasmid (pBTc2) that carried a single copy of trpEG and resembled trpEG plasmids of Buchnera from the Aphididae. The second plasmid (pBPs2), isolated from Buchnera of Pemphigus spyrothecae, contained a different replicon. It consisted of a putative origin of replication containing iterons and an open reading frame, designated repAC, which showed a high similarity to the gene encoding the replication initiation protein RepA of the RepA/C replicon from the broad-host-range IncA/C group of plasmids. The plasmid population was heterogeneous with respect to the number of tandem repeats of a 1.8-kb unit carrying repAC₁, trpG, and remnants of trpE. The two principal forms consisted of either five or six copies of this repeat and a single-copy region carrying repAC₂, the putative origin of replication, and trpE. The unexpected finding of elements of the RepA/C replicon in previously characterized trpEG plasmids from Buchnera of the Aphididae suggests that a replacement of replicons has occurred during the evolution of these plasmids, which may point to a common ancestry for all Buchnera trpEG amplifications.     

Characterization of the replicon of a 51-kb native plasmid from the gram-positive bacterium Leifsonia xyli subsp. cynodontis

The 4992-bp replicon of a large cryptic plasmid in the gram-positive bacterium Leifsonia xyli subsp. cynodontis was identified and sequenced. The replicon encoded two proteins essential for plasmid replication and stability. The putative replication protein (RepA) is homologous to that of the plasmids in mycobacterial pLR7 family, while the putative ParA protein immediately downstream of RepA is significantly homologous to the Walker-type ATPase required for partition of plasmid and chromosome of the gram-positive bacteria. These two proteins and other ORFs are clustered with the putative promoters and other regulatory sequences, illustrating an efficient organization of the replicon for this novel plasmid.