Interplasmid Recombination Research Articles

High Prevalence of Plasmid-Mediated Quinolone Resistance among ESBL/AmpC-Producing Enterobacterales from Free-Living Birds in Poland.

In this study, we investigated the occurrence of plasmid-mediated quinolone resistance (PMQR) in extended-spectrum β-lactamase- (ESBL) and/or AmpC-type β-lactamase-producing Enterobacterales isolates from free-living birds in Poland. The prevalence of the qnrB19 gene was 63%, and the distribution of isolates in terms of bacterial species was as follows: 67% (22/33) corresponded to Escherichia coli, 83% (5/6) to Rahnella aquatilis, 44% (4/9) to Enterobacter cloacae and 33% (1/3) to Klebsiella pneumoniae. The qnrB19 gene was also found in a single isolate of Citrobacter freundii. The molecular characteristics of qnrB19-positive isolates pointed to extended-spectrum beta lactamase CTX-M as the most prevalent one (89%) followed by TEM (47%), AmpC (37%) and SHV (16%). This study demonstrates the widespread occurrence of PMQR-positive and ESBL/AmpC-producing Enterobacterales isolates in fecal samples from wild birds. In this work, plasmid pAM1 isolated from Escherichia coli strain SN25556 was completely sequenced. This plasmid is 3191 nucleotides long and carries the qnrB19 gene, which mediates decreased susceptibility to quinolones. It shares extensive homology with other previously described small qnrB19-harboring plasmids. The nucleotide sequence of pAM1 showed a variable region flanked by an oriT locus and a Xer recombination site. The presence of a putative recombination site was detected, suggesting that interplasmid recombination events might have played a role in the development of pAM1. Our results highlight the broad geographical spread of ColE-type Qnr resistance plasmids in clinical and environmental isolates of Enterobacterales. As expected from the results of phenotypic susceptibility testing, no resistance genes other than qnrB19 were identified.

Giardia duodenalis carries out canonical homologous recombination and single-strand annealing

In the past decades, the ability of Giardia duodenalis to perform homologous recombination has been suggested, supported by the observations of genomic integration of foreign plasmids and the disruption of genes using CRISPR technology. Unfortunately, the direct study of a HR mechanism has not been addressed, which would be pertinent in a minimalist organism lacking fundamental DNA-repair elements and even complete pathways. In addition, the constant ploidy changes through the life cycle of this parasite highlight the conservation and relevance of homologous recombination in maintaining genomic stability. In this research, we analyzed different recombinable plasmid systems and their outcomes after G. duodenalis transfection, using this approach we determined genomic, intra-plasmid and inter-plasmid recombination, moreover, we examined the presence of the non-conservative single-strand annealing pathway. With the intention of corroborating that the observed processes were done by homologous recombination, we used a chemical inhibitor named Mirin, which specifically inhibits Mre11 3′- 5′ exonuclease activity, one of the first steps involved in homologous recombination and fundamental to success in repairing. Overall, these results describe the multiple recombinational substrates used by G. duodenalis to achieve HR and demonstrate the presence and use of single-strand annealing recombination.

Transfer of a mobile Staphylococcus saprophyticus plasmid isolated from fermented seafood that confers tetracycline resistance.

The complete nucleotide sequence of a tetracycline-resistance gene (tetK)-carrying plasmid from a Staphylococcus saprophyticus isolate from jeotgal, a Korean high-salt-fermented seafood, was determined. The plasmid, designated pSSTET1, was 4439 bp in length and encoded typical elements found in plasmids that replicate via a rolling-circle mechanism, including the replication protein gene (rep), a double-stranded origin of replication, a single-stranded origin of replication, and a counter-transcribed RNA sequence. Additionally, the plasmid recombination enzyme gene (pre), which may be involved in inter-plasmid recombination and conjugation, was found. Each gene exhibited >94% sequence identity with those harbored in other Staphylococcus species. pSSTET1 was conditionally transferred to Staphylococcus species in a host-dependent manner and transferred to an Enterococcus faecalis strain in vitro. Antibiotic susceptibility of the transconjugants was host-dependent and transconjugants maintained a tetracycline-resistant phenotype in the absence of selective pressure over 100 generations.

Detection of a Novel qnrB19-Carrying Plasmid Variant Mediating Decreased Fluoroquinolone Susceptibility in Salmonella enterica Serovar Hadar.

Thirty Salmonella enterica subsp. enterica serovar Hadar isolates of avian origin collected between 2007 and 2010 from chicken carcasses in five geographically spread abattoirs in Germany were investigated for plasmid-mediated quinolone resistance determinants. Four isolates were identified by PCR analysis and hybridization experiments to carry qnrB genes. The isolates were indistinguishable by their XbaI macrorestriction patterns and did not exhibit a mutation in the quinolone resistance-determining regions of the DNA gyrase and topoisomerase IV genes. The qnrB genes were found to be located on small plasmids of ∼2.6 kb, which mediated decreased susceptibility only to quinolones. The plasmids were assigned to the same type, pHAD28, and transformation studies into an Escherichia coli recipient strain confirmed their transferability. Sequence analysis of the complete plasmid pHAD28 revealed the presence of a qnrB19 gene. The gene was found on a novel variant of qnrB19-harboring plasmids with high similarity to plasmids pPAB19-3 from E. coli and pPAB19-4 from Salmonella sp. M9397. A presumptive recombination side was detected, suggesting that interplasmid recombination events might have played a role in the development of this plasmid variant.

Characterization of Mobile Staphylococcus equorum Plasmids Isolated from Fermented Seafood That Confer Lincomycin Resistance.

The complete nucleotide sequences of lincomycin-resistance gene (lnuA)-containing plasmids in Staphylococcus equorum strains isolated from the high-salt-fermented seafood jeotgal were determined. These plasmids, designated pSELNU1–3, are 2638-bp long, have two polymorphic sites, and encode typical elements found in plasmids that replicate via a rolling-circle mechanism including the replication protein gene (rep), a double-stranded origin of replication, a single-stranded origin of replication, and counter-transcribed RNA sequence, as well as lnuA. Plasmid sequences exhibit over 83% identity to other Staphylococcus plasmids that harbor rep and lnuA genes. Further, three pairs of identified direct repeats may be involved in inter-plasmid recombination. One plasmid, pSELNU1, was successfully transferred to other Staphylococcus species, Enterococcus faecalis, and Tetragenococcus halophilus in vitro. Antibiotic susceptibility of the transconjugants was host-dependent, and transconjugants maintained a lincomycin resistance phenotype in the absence of selective pressure over 60 generations.

Novel plasmid-borne multidrug resistance gene cluster including lsa(E) from a linezolid-resistant Enterococcus faecium isolate of swine origin.

A novel nonconjugative plasmid of 28,489 bp from a porcine linezolid-resistant Enterococcus faecium isolate was completely sequenced. This plasmid harbored a novel type of multiresistance gene cluster that comprised the resistance genes lnu(B), lsa(E), spw, aadE, aphA3, and two copies of erm(B), which account for resistance to macrolides, lincosamides, streptogramins, pleuromutilins, streptomycin, spectinomycin, and kanamycin/neomycin. Structural comparisons suggested that this plasmid might have developed from other enterococcal plasmids by insertion element (IS)-mediated interplasmid recombination processes.

Complete sequence of a multiresistance plasmid from a methicillin-resistant Staphylococcus epidermidis ST5 isolated in a small animal clinic

Sir, In a previous study, we identified a methicillin-resistant Staphylococcus epidermidis (MRSE) clone being present in nine different samples obtained from two cats hospitalized in a small animal clinic, an employee and the clinic environment. Members of this MRSE clone exhibited SCCmec type IV, dru type dt10a, multilocus sequence type ST5 and very similar or indistinguishable SmaI macrorestriction patterns. These MRSE isolates were resistant to aminoglycosides/ aminocyclitols, b-lactams, fluoroquinolones, lincosamides, macrolides, chloramphenicol/florfenicol, pleuromutilins, sulphonamides, tetracyclines and trimethoprim, and shared the same plasmid profile including one plasmid of 28 kb in addition to several smaller plasmids. The aim of this study was to investigate this 28 kbplasmidfor itsstructureandthepresenceofresistancegenes. Plasmids from all nine isolates were transferred into Staphylococcus aureus RN4220 by protoplast transformation with selection on regeneration plates supplemented with tiamulin (10 mg/L), trimethoprim (30 mg/L), tetracycline (20 mg/L), erythromycin (10 mg/L) or florfenicol (20 mg/L). The resistance phenotype of the transformants was determined by broth microand macrodilution according to documents VET01-A4 and VET01-S2 (formerly M31-A3) of the CLSI. The antimicrobial agents tested are listed in Table S1 (available as Supplementary data at JAC Online). Plasmid DNA from one representative transformant, namely that from the feline MRSE isolate 47, was cut separately with HindIII and BglII. The resulting fragments were cloned into pBluescript II SK+ and sequenced starting with the M13 universal and reverse primers. Sequence determination was completed by primer walking on both strands. Sequence analysis was performed with the basic local alignment tools BLASTN and BLASTP (http://www.ncbi. nlm.nih.gov/BLAST), the ORF Finder program (http://www.ncbi. nlm.nih.gov/gorf/gorf.html) and the IS finder program (https:// www-is.biotoul.fr//) (all URLs last accessed in July 2013). From each of the nine isolates, the 28 kb plasmid was successfully transferred into S. aureus RN4220 after selection on plates supplemented with tetracycline, tiamulin or trimethoprim. All the transformed plasmids showed indistinguishable restriction patterns with BglII, EcoRI and HindIII. All nine transformants exhibited increased MICs compared with the empty recipient S. aureus RN4220 (Table S1) for clindamycin, tobramycin, neomycin, minocycline, tetracycline, tiamulin, trimethoprim/sulfamethoxazole and trimethoprim. Sequence analysis of one representative plasmid, designated pSWS47, showed that it had a total size of 28743 bp and revealed 27 open reading frames, 9 of which were truncated (Figure 1). The mosaic structure of plasmid pSWS47 strongly suggested that this plasmid has been developed from parts of plasmids and transposons previously described in staphylococci, but also other Gram-positive bacteria, including pUR4128 from a methicillinresistant S. aureus (MRSA), pSERP from human MRSE, pERGB from human MRSA, pPECL-3 and pPECL-4 from Pediococcus claussenii, pMD5057 from Lactobacillus plantarum and pSAP016A from S. epidermidis (Figure 1). Similarity to segments of the aforementioned plasmids was often terminated by insertion sequences, such as IS431, IS257 or IS1310. This might point towards the ability of insertion sequences to mediate the integration of small plasmids into larger plasmids. During such interplasmid recombination processes, insertion sequences may also be truncated, as seen in the present case. Moreover, closer inspection of the 8 bp sequences usually found immediately upand downstream of complete IS431 and IS257 revealed that none of these 8 bp sequences represented direct repeats (Figure 1). This observation suggested that multiple recombination processes, in which these insertion sequences had been involved, might have taken place. This scenario may also be supported by the detection of two segments of the same repU gene disrupted by IS257 and another three intact plasmid replication genes, a plasmid recombination gene, a relaxase gene and two plasmid mobilization genes, originating at least in part from different ancestral plasmids (Figure 1). Plasmid pSWS47 carried five functionally active resistance genes, accounting for the resistance phenotype observed: vga(A) (clindamycin and tiamulin resistance), dfrK (trimethoprim resistance), aadD (neomycin and tobramycin resistance), tet(L) and tet(M) (tetracycline and tetracycline/minocycline resistance) (Figure 1). When comparing plasmid pSWS47 with other genetic elements carrying resistance genes, three interesting observations were made. First, plasmid pSWS47 also harboured an inactive blaZ b-lactamase gene. Sequence analysis revealed an additional adenine residue at position 21227, which resulted in a frameshift mutationintheblaZgene.Second,thestartcodonforthereadingframeof the 20 amino acid peptide in the tet(L) translational attenuator was changed from ATG to AGG in pSWS47. However, MIC testing (32 mg/ L) of the4079 bpHindIIIclonethatcarried the tet(L)geneand its regulatory region confirmed that the tet(L) gene is functionally active despite this mutation. This observation is in agreement with thedetection of functionally active tet(L) genes in Pasteurella multocida and Mannheimia spp. that lacked the entire translational attenuator. Third, a segment that corresponded to the plasmid pMD5057 from

The Asd + -DadB + Dual-Plasmid System Offers a Novel Means To Deliver Multiple Protective Antigens by a Recombinant Attenuated Salmonella Vaccine

We developed means to deliver multiple heterologous antigens on dual plasmids with non-antibiotic-resistance markers in a single recombinant attenuated vaccine strain of Salmonella enterica serotype Typhimurium. The first component of this delivery system is a strain of S. Typhimurium carrying genomic deletions in alr, dadB, and asd, resulting in obligate requirements for diaminopimelic acid (DAP) and d-alanine for growth. The second component is the Asd(+)-DadB(+) plasmid pair carrying wild-type copies of asdA and dadB, respectively, to complement the mutations. To evaluate the protection efficacy of the dual-plasmid vaccine, S. Typhimurium strain χ9760 (a strain with multiple attenuating mutations: Δasd Δalr ΔdadB ΔrecF) was transformed with Asd(+) and DadB(+) plasmids specifying pneumococcal antigens PspA and PspC, respectively. Both plasmids were stable in χ9760 for 50 generations when grown in nonselective medium. This was significantly (P < 0.05) greater than the stability seen in its recF(+) counterpart χ9590 and could be attributed to reduced interplasmid recombination in χ9760. Oral immunization of BALB/c mice with 1 × 10(9) CFU of χ9760 (carrying Asd(+)-PspA and DadB(+)-PspC plasmids) elicited a dominant Th1-type serum IgG response against both antigens and protected mice against intraperitoneal challenge with 200 50% lethal doses (LD(50)s) of virulent Streptococcus pneumoniae strain WU2 or intravenous challenge with 100 LD(50)s of virulent S. pneumoniae strain L81905 or intranasal challenge with a lethal dose of S. pneumoniae A66.1 in a pneumonia model. Protection offered by χ9760 was superior to that offered by the mixture of two strains, χ9828 (Asd(+)-PspA) and χ11026 (DadB(+)-PspC). This novel dual-plasmid system marks a remarkable improvement in the development of live bacterial vaccines.

Analysis of bla CTX-M -Carrying Plasmids from Escherichia coli Isolates Collected in the BfT-GermVet Study

In this study, 417 Escherichia coli isolates from defined disease conditions of companion and farm animals collected in the BfT-GermVet study were investigated for the presence of extended-spectrum β-lactamase (ESBL) genes. Three ESBL-producing E. coli isolates were identified among the 100 ampicillin-resistant isolates. The E. coli isolates 168 and 246, of canine and porcine origins, respectively, harbored bla(CTX-M-1), and the canine isolate 913 harbored bla(CTX-M-15), as confirmed by PCR and sequence analysis. The isolates 168 and 246 belonged to the novel multilocus sequence typing (MLST) types ST1576 and ST1153, respectively, while isolate 913 had the MLST type ST410. The ESBL genes were located on structurally related IncN plasmids in isolates 168 and 246 and on an IncF plasmid in isolate 913. The bla(CTX-M-1) upstream regions of plasmids pCTX168 and pCTX246 were similar, whereas the downstream regions showed structural differences. The genetic environment of the bla(CTX-M-15) gene on plasmid pCTX913 differed distinctly from that of both bla(CTX-M-1) genes. Detailed sequence analysis showed that the integration of insertion sequences, as well as interplasmid recombination events, accounted for the structural variability in the bla(CTX-M) gene regions.

Improving Salmonella vector with recmutation to stabilize the DNA cargoes

BackgroundSalmonella has been employed to deliver therapeutic molecules against cancer and infectious diseases. As the carrier for target gene(s), the cargo plasmid should be stable in the bacterial vector. Plasmid recombination has been reduced in E. coli by mutating several genes including the recA, recE, recF and recJ. However, to our knowledge, there have been no published studies of the effect of these or any other genes that play a role in plasmid recombination in Salmonella enterica.ResultsThe effect of recA, recF and recJ deletions on DNA recombination was examined in three serotypes of Salmonella enterica. We found that (1) intraplasmid recombination between direct duplications was RecF-independent in Typhimurium and Paratyphi A, but could be significantly reduced in Typhi by a ΔrecA or ΔrecF mutation; (2) in all three Salmonella serotypes, both ΔrecA and ΔrecF mutations reduced intraplasmid recombination when a 1041 bp intervening sequence was present between the duplications; (3) ΔrecA and ΔrecF mutations resulted in lower frequencies of interplasmid recombination in Typhimurium and Paratyphi A, but not in Typhi; (4) in some cases, a ΔrecJ mutation could reduce plasmid recombination but was less effective than ΔrecA and ΔrecF mutations. We also examined chromosome-related recombination. The frequencies of intrachromosomal recombination and plasmid integration into the chromosome were 2 and 3 logs lower than plasmid recombination frequencies in Rec+ strains. A ΔrecA mutation reduced both intrachromosomal recombination and plasmid integration frequencies.ConclusionsThe ΔrecA and ΔrecF mutations can reduce plasmid recombination frequencies in Salmonella enterica, but the effect can vary between serovars. This information will be useful for developing Salmonella delivery vectors able to stably maintain plasmid cargoes for vaccine development and gene therapy.

Analysis of an Actinobacillus pleuropneumoniae multi-resistance plasmid, pHB0503

A plasmid containing multidrug resistance genes has been discovered from a clinical Actinobacillus pleuropneumoniae strain isolated in China. The complete 15079 kb sequence of this plasmid, designated pHB0503, was analyzed with regard to the organization and evolution of multidrug resistance genes. The deduced amino acid sequences from seven open reading frames ( sul2 catA3, aacC2, strA, truncated strB ( strB′), bla ROB-1 and aph( 3′) -I) identified in pHB0503 were entirely or nearly identical to resistance genes of plasmids both within and outside of the family Pasteurellaceaea, indicating that pHB0503 arose through inter-plasmid recombination processes among them. In addition, co-transcription of the cluster of resistance genes from the promoter upstream of sul2 and bla ROB-1 was confirmed by RT-PCR. This is the first report of a complete sequence of the plasmid containing seven resistance genes from A. pleuropneumoniae.

Mutational Analysis of the Ribosomal Protein Rpl10 from Yeast

Yeast Rpl10 belongs to the L10e family of ribosomal proteins. In the large (60 S) subunit, Rpl10 is positioned in a cleft between the central protuberance and the GTPase-activating center. It is loaded into the 60 S subunit at a late step in maturation. We have shown previously that Rpl10 is required for the release of the Crm1-dependent nuclear export adapter Nmd3, an event that also requires the cytoplasmic GTPase Lsg1. Here we have carried out an extensive mutational analysis of Rpl10 to identify mutations that would allow us to map activities to distinct domains of the protein to begin to understand the molecular interaction between Rpl10 and Nmd3. We found that mutations in a central loop (amino acids 102-112) had a significant impact on the release of Nmd3. This loop is unstructured in the crystal and solution structures of prokaryotic Rpl10 orthologs. Thus, the loop is not necessary for stable interaction of Rpl10 with the ribosome, suggesting that it plays a dynamic role in ribosome function or regulating the association of other factors. We also found that mutant Rpl10 proteins were engineered to be unable to bind to the ribosome accumulated in the nucleus. This was unexpected and may suggest a nuclear role for Rpl10.

Identification of the plasmid-borne quinolone resistance gene qnrS in Salmonella enterica serovar Infantis

A Salmonella enterica serovar Infantis isolate of avian origin was investigated for the presence of the gene qnrS, its transferability and its association with other resistance genes. The Salmonella Infantis isolate was investigated for its susceptibility to antimicrobial agents and its plasmid content. Hybridization experiments and PCR assays were performed to identify the resistance genes while transformation and conjugation studies were conducted to show their transferability. The quinolone resistance-determining regions of the genes gyrA, gyrB, parC and parE were sequenced. Moreover, extended sequence analysis was performed to gain insight into the structure and organization of the qnrS gene area. The Salmonella Infantis isolate exhibited the Asp87-->Tyr87 mutation in gyrA, but no resistance-mediating mutations in the other target genes. It carried a conjugative plasmid, pINF5, on which a qnrS gene was detected in close proximity to a Tn3-like, blaTEM-1-carrying transposon. Homology to the qnrS-carrying plasmid pAH0376 of Shigella flexneri was limited to the Tn3-qnrS region. Sequence analysis of an approximately 13.4 kb region of pINF5 identified truncated insertion sequences of types IS26 and IS2 as well as an internal segment of the CS12 fimbrial gene cluster of Escherichia coli up- and downstream of the qnrS gene. This is to the best of our knowledge the first report of a qnrS gene in a Salmonella isolate. The analysis of the regions flanking the qnrS gene suggested that this region developed in multiple steps that included not only the integration of insertion sequences and a Tn3-like transposon, but also interplasmid recombination events.

In Vivo Evidence for a recA -Independent Recombination Process in Escherichia coli That Permits Completion of Replication of DNA Containing UV Damage in Both Strands

We have investigated recombination mechanisms promoting the completion of replication in the face of unrepaired DNA damage by transforming an isogenic set of uvrA6 excision-defective Escherichia coli strains with pUC-based plasmids in which each strand carried, at staggered positions, a single thymine-thymine pyrimidine (6-4) pyrimidinone lesion. The distance between the lesions was 28 or 8 bp in one orientation relative to the unidirectional ColE1 origin of replication or, in the other orientation, 30 or 10 bp. C-C mismatches placed opposite each of the T-T photoproducts permit unambiguous detection of the three events that can lead to the completion of replication: sister-strand recombination, translesion replication (TR) on the leading strand, and TR on the lagging strand. We find that E. coli possesses a largely constitutive, recA-independent sister-strand recombination mechanism that allows 9% or more of these severely compromised plasmids to be fully replicated. In one orientation, such recombination depends partly on recG and priA but not on ruvA, ruvB, ruvC, or mutS and is largely independent of recF. In the other orientation, recombination is dependent on none of the genes. The strains used did not contain the cryptic phage encoding recET, which encodes enzymes that promote interplasmid recombination. The nature of the recA-independent recombination mechanism is not known but could perhaps result from a template-strand-switching, or copy choice, process.

Nucleotide sequence and organization of the multiresistance plasmid pSCFS1 from Staphylococcus sciuri.

The multiresistance plasmid pSCFS1 from Staphylococcus sciuri was sequenced completely and analysed with regard to its gene organization and the putative role of a novel ABC transporter in antimicrobial resistance. Plasmid pSCFS1 was transformed into Staphylococcus aureus RN4220, overlapping restriction fragments were cloned into Escherichia coli plasmid vectors and sequenced. For further analysis of the ABC transporter, a approximately 3 kb EcoRV-HpaI fragment was cloned into the staphylococcal plasmid pT181MCS and the respective S. aureus RN4220 transformants were subjected to MIC determination. A total of 14 ORFs coding for proteins of >100 amino acids were detected within the 17 108 bp sequence of pSCFS1. Five of them showed similarity to recombination/mobilization genes while another two were similar to plasmid replication genes. In addition to the previously described genes cfr for chloramphenicol/florfenicol resistance and erm(33) for inducible resistance to macrolide-lincosamide-streptogramin B resistance, a Tn554-like spectinomycin resistance gene and Tn554-related transposase genes were identified. Moreover, a novel ABC transporter was detected and shown to mediate low-level lincosamide resistance. Plasmid pSCFS1 is composed of various parts which show similarity to sequences known to occur on plasmids or transposons of Gram-positive, but also Gram-negative bacteria. It is likely that pSCFS1 represents the result of inter-plasmid recombination events also involving the truncation of a Tn554-like transposon.

Organization of the antiseptic resistance gene qacA and Tn552-related beta-lactamase genes in multidrug- resistant Staphylococcus haemolyticus strains of animal and human origins.

A part (12 kb) of a plasmid containing the beta-lactamase genes of Tn552, the disinfectant resistance gene qacA, and flanking DNA has been cloned from a Staphylococcus haemolyticus isolate and sequenced. This region was used to map the corresponding regions in six other multiresistant S. haemolyticus isolates of human and animal origin. The organizations of the genetic structures were almost identical in all isolates studied. The beta-lactamase and qacA genes from S. haemolyticus have >99.9% identities at the nucleotide level with the same genes from S. aureus, demonstrating that various staphylococcal species able to colonize animal and human hosts can exchange the genetic elements involved in resistance to antibiotics and disinfectants. The use of antibiotics and disinfectants in veterinary practice and animal husbandry may also contribute to the selection and maintenance of resistance factors among the staphylococcal species. Different parts of the 12-kb section analyzed had high degrees of nucleotide identity with regions from several other different Staphylococcus aureus plasmids. This suggests the contribution of interplasmid recombination in the evolutionary makeup of this 12-kb section involving plasmids that can intermingle between various staphylococcal species. The lateral spread of resistance genes between various staphylococcal species is probably facilitated by the generation of large multiresistance plasmids and the subsequent interspecies exchange of them.

Evidence that extrachromosomal double-strand break repair can be coupled to the repair of chromosomal double-strand breaks in mammalian cells.

Transfected linear DNA molecules are substrates for double-strand break (DSB) repair in mammalian cells. The DSB repair process can involve recombination between the transfected DNA molecules, between the transfected molecules and chromosomal DNA, or both. In order to determine whether these different types of repair events are linked, we devised assays enabling us to follow the fate of linear extrachromosomal DNA molecules involved in both interplasmid and chromosome-plasmid recombination, in the presence or absence of a pre-defined chromosomal DSB. Plasmid-based vectors were designed that could either recombine via interplasmid recombination or chromosome-plasmid recombination to produce a functional beta-galactosidase (betagal) fusion gene. By measuring the frequency of betagal+ cells at 36 h post-transfection versus the frequency of betagal+ clones after 14 days, we found that the number of cells containing extrachromosomal recombinant DNA molecules at 36 h (i.e., betagal+), either through interplasmid or chromosome-plasmid recombination, was nearly the same as the number of cells integrating these recombinant molecules. Furthermore, when a predefined DSB was created at a chromosomal site, the extrachromosomal recombinant DNA molecules were shown to integrate preferentially at that site by Southern and fiber-FISH (fluorescence in situ hybridization) analysis. Together these data indicate that the initial recombination event can potentiate or commit extrachromosomal DNA to integration in the genome at the site of a chromosomal DSB. The efficiency at which extrachromosomal recombinant molecules are used as substrates in chromosomal DSB repair suggests extrachromosomal DSB repair can be coupled to the repair of chromosomal DSBs in mammalian cells.

The REC41 gene of Saccharomyces cerevisiae: isolation and genetic analysis

Recombination-deficient strains have been proven useful for the understanding of the genetic control of homologous recombination. As the genetic screens used to isolate recombination-deficient (rec−) yeast mutants have not been saturated, we sought to develop a simple colony color assay to identify mutants with low or elevated rates of recombination. Using this system we isolated a collection of rec− mutants. We report the characterization of the REC41 gene identified in this way. REC41 is required for normal levels of interplasmid recombination and γ-ray induced mitotic interchromosomal recombination. The rec41-1 mutant failed to grow at 37°C. Microscopic analysis of plated cells showed that 45–50% of them did not form visible colonies at permissive temperature. Haploid cells of the rec41 mutant show the same γ-ray sensitivity as wild type ones. However, the diploid rec41 mutant shows γ-ray sensitivity which is comparable with heterozygous REC41/rec41-1 diploid cells. This fact indicates semidominance of the rec41-1 mutation. Diploid strains homozygous for the rec41 rad52 mutations had the same γ-ray sensitivity as single rad52 diploids and exhibited dramatically decreased growth rate. The expression of the HO gene does not lead to inviability of rec41 cells. The rec41 mutation has an effect on meiosis, likely meiotic recombination, even in the heterozygous state. We cloned the REC41 gene. Sequence analysis revealed that the REC41 gene is encoded by ORF YDR245w. Earlier, this ORF was attributed to MNN10, BED1, SLC2, CAX5 genes. Two multicopy plasmids with suppressers of the rec41-1 mutation (pm21 and pm32) were isolated. The deletion analysis showed that only DNA fragments with the CDC43 and HAC1 genes can partially complement the rec41-1 mutation.

A small cryptic plasmid from Ruminobacter amylophilus NIAH-3 possesses functional mobilization properties

The complete nucleotide sequence of a small cryptic plasmid designated pRAO1, from the Gram-negative ruminal bacterium Ruminobacter amylophilus NIAH-3, was determined. The plasmid is a circular DNA molecule, 2140 bp in size, with a GC content of 40%. Computer-assisted analysis identified three open reading frames (ORFs), one of which, ORF3 (347 amino acids), displayed a high degree of amino acid identity with the Mob proteins involved in conjugative mobilization and interplasmid recombination of plasmids from Gram-positive bacteria. We proved the mobilization properties of pRAO1 in the Escherichia coli system using the coresident IncW broad-host-range conjugative plasmid R388. These data demonstrated, for the first time, the mobilization properties of small cryptic plasmids from Gram-negative inhabitants of the rumen.

A small cryptic plasmid from Ruminobacter amylophilus NIAH-3 possesses functional mobilization properties.

The complete nucleotide sequence of a small cryptic plasmid designated pRAO1, from the Gram-negative ruminal bacterium Ruminobacter amylophilus NIAH-3, was determined. The plasmid is a circular DNA molecule, 2140 bp in size, with a GC content of 40%. Computer-assisted analysis identified three open reading frames (ORFs), one of which, ORF3 (347 amino acids), displayed a high degree of amino acid identity with the Mob proteins involved in conjugative mobilization and interplasmid recombination of plasmids from Gram-positive bacteria. We proved the mobilization properties of pRAO1 in the Escherichia coli system using the coresident IncW broad-host-range conjugative plasmid R388. These data demonstrated, for the first time, the mobilization properties of small cryptic plasmids from Gram-negative inhabitants of the rumen.