Isolation and characterization of an R-prime plasmid from Rhizobium meliloti.

Using a simple enrichment procedure, we isolated an R-prime derivative of plasmid R68.45 carrying a 17.8-megadalton segment of the Rhizobium meliloti 41 chromosome. The chromosomal segment carried on this plasmid (pGY1) includes the markers cys-24+, cys-46+, and att16-3. Plasmid pGY1 mobilized the chromosome in a polarized way starting from the region of homology, but cannot promote chromosome transfer from other sites. The att16-3 site on pGY1 allowed the integration of phage 16-3 into pGY1, and a composite plasmid of 91.8 megadaltons was formed. This vector (pGY2) is suitable for the introduction of Rhizobium bacteriophage 16-3 into other gram-negative bacteria.     

Sequence and Structure Analysis of Cryptic Plasmid pN30 from Oil-Oxidizing Strain <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis> 30

Nucleotide sequence of cryptic plasmid pN30 from a Rhodococcus erythropolis 30 soil isolate was determined. Plasmid DNA consists of 5403 nucleotide pairs and contains about 62% GC pairs, which is typical of Rhodococcus DNA. No significant homology was determined between the pN30 DNA sequence and those of known plasmids. Computer-aided analysis of pN30 sequence revealed open reading frames that encode proteins strongly homologous to replicative proteins encoded by small cryptic plasmids of different actinomycetes.     

Two Rep Genes in Small Cryptic Plasmid pKST21 of Escherichia coli

The complete nucleotide sequence of a small cryptic plasmid pKST21 from Escherichia coli was determined. This plasmid is 1,460 bp long with an overall GC content of 51 %. Based on sequence analysis, the presence of two segments with different average GC density was observed. The segment with higher GC content revealed 98–90 % similarity to several small plasmids of E. coli and to pCR1 from Gram-positive Corynebacterium renale. Plasmid pKST21 possesses two conversely oriented open reading frames encoding proteins with a high degree of amino acid identity to Rep proteins involved in replication. ORF1 encodes replication protein similar to RepA protein of Bartonella tribocorum or Bacillus cereus plasmids or to the putative plasmid Rep protein from ecologically close Selenomonas ruminantium. ORF2 similarly encodes a replication protein, which shares 97 % homology with Rep protein from C. renale. Genetic diversity observed in plasmid pKST21 indicates a mosaic structure of the plasmid with different segments acquired from different sources. Deletion analysis showed that both fragments carrying the repA and repB genes are necessary for the replication of pKST21 in E. coli. The presence of plasmid with the same gene composition was revealed in 14 % of tested E. coli isolates from the rumen of sheep. All these strains produced identical ERIC-PCR profiles indicating isogenic origin of the strain and lack of horizontal gene transfer of pKST21 plasmid.     

Nucleotide sequence and structural analysis of cryptic plasmid pBL90 from <Emphasis Type="Italic">Brevibacterium lactofermentum</Emphasis>

The nucleotide sequence of cryptic plasmid (designated as pBL90) detected in the cells of Brevibacterium lactofermentum DSM 1412 was determined. The length of plasmid DNA is 67826 bp. Comparison of the nucleotide sequence of pBL90 with known plasmid sequences showed no long regions of significant homology. Computer analysis of the plasmid DNA revealed 29 open reading frames (ORFs). The amino acid sequences of 15 ORFs (approximately 25% of plasmid length) have a high (>70%) level of identity to proteins from different plasmids of Corynebacterium representatives, including replicative proteins. Unusual in pBL90 is the presence of replicative genes from two different families and types of replication.     

Characterization,expression and regulation of a third gene encoding glutathione S-transferase from the fission yeast

A third gene encoding glutathione S-transferase (GSTIII) was cloned from the fission yeast Schizosaccharomyces pombe. The nucleotide sequence determined was found to contain 2110 base pairs including an open reading frame of 242 amino acids that would encode a protein of a molecular mass of 26,620 Da. The cloned GSTIII gene could be expressed in S. pombe, S. cerevisiae and Escherichia coli cells which gave 1.4-, 2.1-, and 3.0-fold higher GST activity in an assay using 1-chloro-2,4-dinitrobenzene as a substrate, respectively. The cloned GSTIII gene caused higher survivals of S. pombe cells on solid media with cadmium chloride or mercuric chloride. The GSTIII protein has 16% and 18% homologies with the GSTI and GSTII proteins, respectively. To independently monitor the regulation of the GSTIII gene, its 1168 bp upstream region and N-terminal 33 amino acid-coding region was fused into the promoterless beta-galactosidase gene of the shuttle vector YEp357. The synthesis of beta-galactosidase from the fusion plasmid pGY357 was greatly enhanced by cadmium chloride (50 microM), cupric chloride (10 microM), aluminum chloride (5 mM, 10 mM), mercuric chloride (1 microM), and zinc chloride (10 mM). However, the synthesis of beta-galactosidase from the fusion plasmid pGY357 was not affected by superoxide-generating menadione, and o-dinitrobenzene, whereas they could significantly induce the expression of the GSTI and GSTII genes of S. pombe. The overproduced Pap1 inhibited the induction of beta-galactosidase synthesis from the fusion plasmid pGY357 by cadmium chloride, which is opposite to the previously known role of Pap1 in the response to oxidative stress. Our results collectively indicate that the three GST genes of S. pombe are subjected to different regulatory mechanisms. The major role of the GSTIII protein in S. pombe may be the detoxification of various metals.     

Method for the transfer of large cryptic, non-self-transmissible plasmids: ex planta transfer of the virulence plasmid of Agrobacterium rhizogenes

A method is presented for the (ex planta) transfer of large, cryptic plasmids that are (phenotypically) non-self-transmissible. The procedure consists of introducing a mobilizing R plasmid into the strain carrying the cryptic plasmid followed by random transposon mutagenesis (e.g., by conjugation with a bacterium carrying a suicide plasmid). Tn-carrying derivatives with a copy of the Tn in the cryptic plasmid are subsequently identified by their ability to transfer the Tn-encoded markers via R plasmid mobilization. The method was applied for the labeling of a large cryptic plasmid present in Agrobacterium rhizogenes 1855 and for its subsequent ex planta transfer to a Ti plasmidless A. tumefaciens strain. The plasmid gave the new host the capacity to induce the hairy root disease in plants, and thus turned out to be an Ri plasmid. From the labeled Ri plasmid derepressed mutants were isolated that were transmissible both in planta and ex planta.     

A new insertion sequence from Sinorhizobium meliloti with homology to IS1357 from Methylobacterium sp. and IS1452 from Acetobacter pasteurianus

The insertion sequence ISRm8 was identified by sequence analysis of the cryptic plasmid pRmeGR4b of Sinorhizobium meliloti GR4. ISRm8 is 1451 bp in length and carries 22/24-bp terminal imperfect inverted repeats with seven mismatches and a direct target site duplication of 3 bp. ISRm8 carries a unique open reading frame whose putative protein showed significant similarity to the insertion sequences IS1357 and IS1452, isolated from Methylobacterium sp. and Acetobacter pasteurianus, respectively. Two copies of this IS element were found in strain GR4; one of them is linked to plasmid pRmeGR4b, whereas the other is localized out of the non-pSym plasmids. In S. meliloti field populations ISRm8 shows a limited distribution (50% of the strains tested carry the IS element), with a copy number ranging from 1 to 6.     

Plasmidome-Analysis of ESBL-Producing Escherichia coli Using Conventional Typing and High-Throughput Sequencing

Infections caused by Extended spectrum β-lactamase (ESBL)-producing E. coli are an emerging global problem, threatening the effectiveness of the extensively used β-lactam antibiotics. ESBL dissemination is facilitated by plasmids, transposons, and other mobile elements. We have characterized the plasmid content of ESBL-producing E. coli from human urinary tract infections. Ten diverse isolates were selected; they had unrelated pulsed-field gel electrophoresis (PFGE) types (<90% similarity), were from geographically dispersed locations and had diverging antibiotic resistance profiles. Three isolates belonged to the globally disseminated sequence type ST131. ESBL-genes of the CTX-M-1 and CTX-M-9 phylogroups were identified in all ten isolates. The plasmid content (plasmidome) of each strain was analyzed using a combination of molecular methods and high-throughput sequencing. Hidden Markov Model-based analysis of unassembled sequencing reads was used to analyze the genetic diversity of the plasmid samples and to detect resistance genes. Each isolate contained between two and eight distinct plasmids, and at least 22 large plasmids were identified overall. The plasmids were variants of pUTI89, pKF3-70, pEK499, pKF3-140, pKF3-70, p1ESCUM, pEK204, pHK17a, p083CORR, R64, pLF82, pSFO157, and R721. In addition, small cryptic high copy-number plasmids were frequent, containing one to seven open reading frames per plasmid. Three clustered groups of such small cryptic plasmids could be distinguished based on sequence similarity. Extrachromosomal prophages were found in three isolates. Two of them resembled the E. coli P1 phage and one was previously unknown. The present study confirms plasmid multiplicity in multi-resistant E. coli. We conclude that high-throughput sequencing successfully provides information on the extrachromosomal gene content and can be used to generate a genetic fingerprint of possible use in epidemiology. This could be a valuable tool for tracing plasmids in outbreaks.     

Isolation and Characterization of a Rolling-Circle-Type Plasmid from Rhodococcus erythropolis and Application of the Plasmid to Multiple-Recombinant-Protein Expression

We isolated, sequenced, and characterized the cryptic plasmid pRE8424 from Rhodococcus erythropolis DSM8424. Plasmid pRE8424 is a 5,987-bp circular plasmid; it carries six open reading frames and also contains cis-acting elements, specifically a single-stranded origin and a double-stranded origin, which are characteristic of rolling-circle-replication plasmids. Experiments with pRE8424 derivatives carrying a mutated single-stranded origin sequence showed that single-stranded DNA intermediates accumulated in the cells because of inefficient conversion from single-stranded DNA to double-stranded DNA. This result indicates that pRE8424 belongs to the pIJ101/pJV1 family of rolling-circle-replication plasmids. Expression vectors that are functional in several Rhodococcus species were constructed by use of the replication origin from pRE8424. We previously reported a cryptic plasmid, pRE2895, from R. erythropolis, which may replicate by a θ-type mechanism, like ColE2 plasmids. The new expression vectors originating from pRE8424 were compatible with those derived from pRE2895. Coexpression experiments with these compatible expression vectors indicated that the plasmids are suitable for the simultaneous expression of multiple recombinant proteins.     

Design and Application of a New Cryptic-Plasmid-Based Shuttle Vector for Magnetospirillum magneticum

A 3.7-kb cryptic plasmid designated pMGT was found in Magnetospirillum magneticum MGT-1. It was characterized and used for the development of an improved expression system in strain AMB-1 through the construction of a shuttle vector, pUMG. An electroporation method for magnetic bacteria that uses the cryptic plasmid was also developed.     

Sequence Analysis of the Cryptic Plasmid pMG101 from Rhodopseudomonas palustris and Construction of Stable Cloning Vectors

A 15-kb cryptic plasmid was obtained from a natural isolate of Rhodopseudomonas palustris. The plasmid, designated pMG101, was able to replicate in R. palustris and in closely related strains of Bradyrhizobium japonicum and phototrophic Bradyrhizobium species. However, it was unable to replicate in the purple nonsulfur bacterium Rhodobacter sphaeroides and in Rhizobium species. The replication region of pMG101 was localized to a 3.0-kb SalI-XhoI fragment, and this fragment was stably maintained in R. palustris for over 100 generations in the absence of selection. The complete nucleotide sequence of this fragment revealed two open reading frames (ORFs), ORF1 and ORF2. The deduced amino acid sequence of ORF1 is similar to sequences of Par proteins, which mediate plasmid stability from certain plasmids, while ORF2 was identified as a putative rep gene, coding for an initiator of plasmid replication, based on homology with the Rep proteins of several other plasmids. The function of these sequences was studied by deletion mapping and gene disruptions of ORF1 and ORF2. pMG101-based Escherichia coli-R. palustris shuttle cloning vectors pMG103 and pMG105 were constructed and were stably maintained in R. palustris growing under nonselective conditions. The ability of plasmid pMG101 to replicate in R. palustris and its close phylogenetic relatives should enable broad application of these vectors within this group of α-proteobacteria.     

Genetic map of the virulence plasmid of Salmonella enteritidis and nucleotide sequence of its replicons

Partial sequencing of a genomic library of the virulence plasmid of Salmonella enteritidis has been used to localize in the restriction map of this plasmid the genetic loci already described in other Salmonella plasmids. The comparison of the vestigial tra region with the corresponding genes in the F plasmid allowed us to define the extent of the deletions that the S. enteritidis plasmid should have suffered. The putative replicons of the plasmid, repB and repC, were isolated and both proved to be functional in Escherichia coli, but repC was segregationally unstable. The nucleotide sequence of repB showed the typical organization of RepFIIA replicons, although the similarity was lower than usual in this group of replicons. The highest homology was found with the replicon of the virulence plasmid pYVe439-80 from Yersinia enterocolitica (72.5%). Replicon repC also showed a maximum identity of 72.6% with known replicons, namely the RepFIB of pColV-K30 and P307, both virulence plasmids isolated from E. coli. We conclude that the S. enteritidis plasmid could arise from the S. typhimurium plasmid through deletions, and that they are evolutionary distant from other IncFI and IncFII plasmids.     

Characterization,sequence and mode of replication of plasmid pNB2 from the thermophilic bacterium Clostridium thermosaccharolyticum

The 1882-bp nucleotide sequence of the cryptic plasmid pNB2 isolated from the thermophilic bacterium Clostridium thermosaccharolyticum was determined. pNB2 DNA has very low GC content (27%) and may serve as a model for studying the modes of maintenance and replication of AT-rich DNA under conditions of thermophilic growth. The plasmid sequence revealed three open reading frames (ORFs) which would encode polypeptides of 289, 68 and 59 amino acids, respectively, and these proteins were synthesized in E. coli extracts primed with the plasmid. We found that the product of ORF289 may be initiated at the non-ATG start codon, TTG, and has similarities with the conserved motifs of Rep proteins encoded by rolling circle (RC) plasmids of the pC194/pUB110 family. Southern hybridization analysis of lysates of C. thermosaccharolyticum cells harboring pNB2 revealed single-stranded intermediates, suggesting that this plasmid is able to replicate in clostridial cells via the RC mechanism. The most significant similarities are found between pNB2 Rep protein and the Rep proteins of three RC plasmids of the pC194 family (pTB913, pBC1 and pST1) isolated from thermophilic bacteria. Comparative analysis of these Rep proteins showed that despite the significant level of divergence, these Rep proteins share a high degree of similarity in the regions of five well-known conserved domains of RC Rep proteins and fall into two groups in accordance with the similarities found in their active sites.     

Involvement of umuDC ST genes in nitropyrene-induced -CG frameshift mutagenesis at the repetitive CG sequence in the hisD3052 allele of Salmonella typhimurium

Expression of the umuDC operon is required for UV and most chemical mutagenesis in Escherichia coli. The closely related species Salmonella typhimurium has two sets of umuDC-like operons, umuDC _ST on the chromosome and samAB on a 60-MDa cryptic plasmid. The roles of theumuDC-like operons in chemically induced frameshift mutagenesis of the hisD3052 allele of S. typhimurium were investigated. Introduction of a pBR322-derived plasmid carrying umuDCST increased the rate of reversion of hisD3052, following treatment with 1-nitropyrene (1-NP) or 1,8-dinitropyrene (1,-8DNP) tenfold and fivefold, respectively, whereas it did not substantially increase the rate of reversion induced by other frameshift mutagens, i.e. 2-nitrofluorene (2NF) and 2-amino- 3-methyldipyrido[1,2-a:3 ′,2′-d]imi-dazole (Glu-P-1). Introduction of a pBR322-derived plasmid carrying samAB did not increase the incidence of reversion of hisD3052 observed with any of the mutagens examined. Deletion of umuDC _STSubstantially lowered the reversion rate induced by l-NP or 1,8-DNP, but it did not affect reversion induced by 2-NF, Glu-P-1 or N-hydroxyacetylaminofluorene (N-OH-AAF). Deletion of samAB had little impact on reversion incidence induced by any of the five frameshift mutagens. DNA amplification using the polymerase chain reaction technique followed by restriction enzyme analysis using BssHII, suggested that the mutations induced by the five frameshift mutagens were all CG deletions at the CGCGCGCG sequence in hisD3052. These results suggest that umuDCST, but not samAB, is involved in the -2 frameshift mutagenesis induced by l-NP and 1,8-DNP at the repetitive CG sequence, whereas neither operon participates in induction of the same type of mutations by 2-NF, Glu-P-1 or N-OH-AAF.     

Identification of Frankia Strains by Direct DNA Hybridization of Crushed Nodules

A hybridization procedure was developed to identify Frankia strains inside actinorhizae by direct probing of crushed root nodules. The probe consisted of an indigenous cryptic plasmid. This well-conserved, 8-kilobase plasmid was detected in Frankia isolates that were very close taxonomically (they possessed a very high DNA sequence homology). The probe did not hybridize to the DNA of Frankia isolates which did not carry the plasmid. Endophyte DNA was extracted by a modification of a technique originally developed for the detection of plasmids in Frankia isolates. The hybridization procedure applied to nodules collected in a stand of alder permitted determination of a distribution map of the plasmid-bearing Frankia strains.     

Sequence analysis of a 1296-nucleotide plasmid from Xylella fastidiosa

A cryptic plasmid from Xylella fastidiosa strain ATCC 35868 was cloned, sequenced, and the sequence entered into GenBank (U71220). The plasmid is 1296 nucleotides in length with 55% GC content and three open reading frames. A plasmid with sequence homology was found in only one other strain of X. fastidiosa , ATCC 35878. Searches of the GenBank reveal nucleotide sequence homology with plasmid pNKH43 from Stenotrophomonas maltophilia , and amino acid sequence homology with phage Pf3 from Pseudomonas aeruginosa , plasmid pAP12875 from Acetobacter pasteurianus , and plasmid pVT736-1 from Actinobacillus actinomycetemcomitans .