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357200.9988Comparative analysis of sequences flanking tet(W) resistance genes in multiple species of gut bacteria. tet(W) is one of the most abundant tetracycline resistance genes found in bacteria from the mammalian gut and was first identified in the rumen anaerobe Butyrivibrio fibrisolvens 1.230, where it is highly mobile and its transfer is associated with the transposable chromosomal element TnB1230. In order to compare the genetic basis for tet(W) carriage in different bacteria, we studied sequences flanking tet(W) in representatives of seven bacterial genera originating in diverse gut environments. The sequences 657 bp upstream and 43 bp downstream of tet(W) were 96 to 100% similar in all strains examined. A common open reading frame (ORF) was identified downstream of tet(W) in five different bacteria, while another conserved ORF that flanked tet(W) in B. fibrisolvens 1.230 was also present upstream of tet(W) in a human colonic Roseburia isolate and in another rumen B. fibrisolvens isolate. In one species, Bifidobacterium longum (strain F8), a novel transposase was located within the conserved 657-bp region upstream of tet(W) and was flanked by imperfect direct repeats. Additional direct repeats 6 bp long were identified on each end of a chromosomal ORF interrupted by the insertion of the putative transposase and the tet(W) gene. This tet(W) gene was transferable at low frequencies between Bifidobacterium strains. A putative minielement carrying a copy of tet(W) was identified in B. fibrisolvens transconjugants that had acquired the tet(W) gene on TnB1230. Several different mechanisms, including mechanisms involving plasmids and conjugative transposons, appear to be involved in the horizontal transfer of tet(W) genes, but small core regions that may function as minielements are conserved.200616870752
986610.9987Integrons in Xanthomonas: a source of species genome diversity. Integrons are best known for assembling antibiotic resistance genes in clinical bacteria. They capture genes by using integrase-mediated site-specific recombination of mobile gene cassettes. Integrons also occur in the chromosomes of many bacteria, notably beta- and gamma-Proteobacteria. In a survey of Xanthomonas, integrons were found in all 32 strains representing 12 pathovars of two species. Their chromosomal location was downstream from the acid dehydratase gene, ilvD, suggesting that an integron was present at this site in the ancestral xanthomonad. There was considerable sequence and structural diversity among the extant integrons. The majority of integrase genes were predicted to be inactivated by frameshifts, stop codons, or large deletions, suggesting that the associated gene cassettes can no longer be mobilized. In support, groups of strains with the same deletions or stop codons/frameshifts in their integrase gene usually contained identical arrays of gene cassettes. In general, strains within individual pathovars had identical cassettes, and these exhibited no similarity to cassettes detected in other pathovars. The variety and characteristics of contemporary gene cassettes suggests that the ancestral integron had access to a diverse pool of these mobile elements, and that their genes originated outside the Xanthomonas genome. Subsequent inactivation of the integrase gene in particular lineages has largely fixed the gene cassette arrays in particular pathovars during their differentiation and specialization into ecological niches. The acquisition of diverse gene cassettes by different lineages within Xanthomonas has contributed to the species-genome diversity of the genus. The role of gene cassettes in survival on plant surfaces is currently unknown.200515755815
45420.9987Nucleotide sequences and comparison of two large conjugative plasmids from different Campylobacter species. Two large tetracycline resistance (TcR) plasmids have been completely sequenced, the pTet plasmid (45.2 kb) from Campylobacter jejuni strain 81-176 and a plasmid pCC31 (44.7 kb) from Campylobacter coli strain CC31 that was isolated from a human case of severe gastroenteritis in the UK. Both plasmids are mosaic in structure, having homologues of genes found in a variety of different commensal and pathogenic bacteria, but nevertheless, showed striking similarities in DNA sequence and overall gene organization. Several predicted proteins encoded by genes involved in conjugation showed highest homology to proteins found in Actinobacillus actinomycetemcomitans, a periodontal pathogen. In addition to replication- and conjugation-associated genes, both plasmids carried a tet(O) gene encoding tetracycline resistance, a 6 kb ORF encoding a putative methylase and a number of genes of unknown function. The pTet plasmid co-exists in C. jejuni strain 81-176 with a smaller, previously characterized, non-conjugative plasmid pVir that also encodes a type IV secretion system (T4SS) that may affect virulence. In contrast, the T4SS encoded by pTet and pCC31 are shown to mediate bacterial conjugation between Campylobacter. The possible origin and evolution of pCC31 and pTet is discussed.200415470128
359230.9986A Functional Metagenomic Analysis of Tetracycline Resistance in Cheese Bacteria. Metagenomic techniques have been successfully used to monitor antibiotic resistance genes in environmental, animal and human ecosystems. However, despite the claim that the food chain plays a key role in the spread of antibiotic resistance, metagenomic analysis has scarcely been used to investigate food systems. The present work reports a functional metagenomic analysis of the prevalence and evolution of tetracycline resistance determinants in a raw-milk, blue-veined cheese during manufacturing and ripening. For this, the same cheese batch was sampled and analyzed on days 3 and 60 of manufacture. Samples were diluted and grown in the presence of tetracycline on plate count milk agar (PCMA) (non-selective) and de Man Rogosa and Sharpe (MRS) agar (selective for lactic acid bacteria, LAB). DNA from the cultured bacteria was then isolated and used to construct four fosmid libraries, named after the medium and sampling time: PCMA-3D, PCMA-60D, MRS-3D, and MRS-60D. Clones in the libraries were subjected to restriction enzyme analysis, PCR amplification, and sequencing. Among the 300 fosmid clones analyzed, 268 different EcoRI restriction profiles were encountered. Sequence homology of their extremes clustered the clones into 47 groups. Representative clones of all groups were then screened for the presence of tetracycline resistance genes by PCR, targeting well-recognized genes coding for ribosomal protection proteins and efflux pumps. A single tetracycline resistance gene was detected in each of the clones, with four such resistance genes identified in total: tet(A), tet(L), tet(M), and tet(S). tet(A) was the only gene identified in the PCMA-3D library, and tet(L) the only one identified in the PCMA-60D and MRS-60D libraries. tet(M) and tet(S) were both detected in the MRS-3D library and in similar numbers. Six representative clones of the libraries were sequenced and analyzed. Long segments of all clones but one showed extensive homology to plasmids from Gram-positive and Gram-negative bacteria. tet(A) was found within a sequence showing strong similarity to plasmids pMAK2 and pO26-Vir from Salmonella enterica and Escherichia coli, respectively. All other genes were embedded in, or near to, sequences homologous to those of LAB species. These findings strongly suggest an evolution of tetracycline resistance gene types during cheese ripening, which might reflect the succession of the microbial populations. The location of the tetracycline resistance genes in plasmids, surrounded or directly flanked by open reading frames encoding transposases, invertases or mobilization proteins, suggests they might have a strong capacity for transference. Raw-milk cheeses should therefore be considered reservoirs of tetracycline resistance genes that might be horizontally transferred.201728596758
357940.9986The Tetracycline Resistance Gene, tet(W) in Bifidobacterium animalis subsp. lactis Follows Phylogeny and Differs From tet(W) in Other Species. The tetracycline resistance gene tet(W) encodes a ribosomal protection protein that confers a low level of tetracycline resistance in the probiotic bacterium Bifidobacterium animalis subsp. lactis. With the aim of assessing its phylogenetic origin and potential mobility, we have performed phylogenetic and in silico genome analysis of tet(W) and its flanking genes. tet(W) was found in 41 out of 44 examined B. animalis subsp. lactis strains. In 38 strains, tet(W) was flanked by an IS5-like element and an open reading frame encoding a hypothetical protein, which exhibited a similar GC content (51-53%). These genes were positioned in the same genomic context within the examined genomes. Phylogenetically, the B. animalis subsp. lactis tet(W) cluster in a clade separate from tet(W) of other species and genera. This is not the case for tet(W) encoded by other bifidobacteria and other species where tet(W) is often found in association with transferable elements or in different genomic regions. An IS5-like element identical to the one flanking the B. animalis subsp. lactis tet(W) has been found in a human gut related bacterium, but it was not associated with any tet(W) genes. This suggests that the IS5-like element is not associated with genetic mobility. tet(W) and the IS5 element have previously been shown to be co-transcribed, indicating that co-localization may be associated with tet(W) expression. Here, we present a method where phylogenetic and in silico genome analysis can be used to determine whether antibiotic resistance genes should be considered innate (intrinsic) or acquired. We find that B. animalis subsp. lactis encoded tet(W) is part of the ancient resistome and thereby possess a negligible risk of transfer.202134335493
986750.9985Mosaic plasmids are abundant and unevenly distributed across prokaryotic taxa. Mosaic plasmids, plasmids composed of genetic elements from distinct sources, are associated with the spread of antibiotic resistance genes. Transposons are considered the primary mechanism for mosaic plasmid formation, though other mechanisms have been observed in specific instances. The frequency with which mosaic plasmids have been described suggests they may play an important role in plasmid population dynamics. Our survey of the confirmed plasmid sequences available from complete and draft genomes in the RefSeq database shows that 46% of them fit a strict definition of mosaic. Mosaic plasmids are also not evenly distributed over the taxa represented in the database. Plasmids from some genera, including Piscirickettsia and Yersinia, are almost all mosaic, while plasmids from other genera, including Borrelia, are rarely mosaic. While some mosaic plasmids share identical regions with hundreds of others, the median mosaic plasmid only shares with 8 other plasmids. When considering only plasmids from finished genomes (51.6% of the total), mosaic plasmids have significantly higher proportions of transposase and antibiotic resistance genes. Conversely, only 56.6% of mosaic fragments (DNA fragments shared between mosaic plasmids) contain a recognizable transposase gene, and only 1.2% of mosaic fragments are flanked by inverted repeats. Mosaic fragments associated with the IS26 transposase gene are 3.8-fold more abundant than any other sequence shared between mosaic plasmids in the database, though this is at least partly due to overrepresentation of Enterobacteriaceae plasmids. Mosaic plasmids are a complicated trait of some plasmid populations, only partly explained by transposition. Though antibiotic resistance genes led to the identification of many mosaic plasmids, mosaic plasmids are a broad phenomenon encompassing many more traits than just antibiotic resistance. Further research will be required to determine the influence of ecology, host repair mechanisms, conjugation, and plasmid host range on the formation and influence of mosaic plasmids. AUTHOR SUMMARY: Plasmids are extrachromosomal genetic entities that are found in many prokaryotes. They serve as flexible storage for genes, and individual cells can make substantial changes to their characteristics by acquiring, losing, or modifying a plasmid. In some pathogenic bacteria, such as Escherichia coli, antibiotic resistance genes are known to spread primarily on plasmids. By analyzing a database of 8592 plasmid sequences we determined that many of these plasmids have exchanged genes with each other, becoming mosaics of genes from different sources. We next separated these plasmids into groups based on the organism they were isolated from and found that different groups had different fractions of mosaic plasmids. This result was unexpected and suggests that the mechanisms and selective pressures causing mosaic plasmids do not occur evenly over all species. It also suggests that plasmids may provide different levels of potential variation to different species. This work uncovers a previously unrecognized pattern in plasmids across prokaryotes, that could lead to new insights into the evolutionary role that plasmids play.201930797764
357060.9985A newly discovered Bacteroides conjugative transposon, CTnGERM1, contains genes also found in gram-positive bacteria. Results of a recent study of antibiotic resistance genes in human colonic Bacteroides strains suggested that gene transfer events between members of this genus are fairly common. The identification of Bacteroides isolates that carried an erythromycin resistance gene, ermG, whose DNA sequence was 99% identical to that of an ermG gene found previously only in gram-positive bacteria raised the further possibility that conjugal elements were moving into Bacteroides species from other genera. Six of seven ermG-containing Bacteroides strains tested were able to transfer ermG by conjugation. One of these strains was chosen for further investigation. Results of pulsed-field gel electrophoresis experiments showed that the conjugal element carrying ermG in this strain is an integrated element about 75 kb in size. Thus, the element appears to be a conjugative transposon (CTn) and was designated CTnGERM1. CTnGERM1 proved to be unrelated to the predominant type of CTn found in Bacteroides isolates-CTns of the CTnERL/CTnDOT family-which sometimes carry another type of erm gene, ermF. A 19-kbp segment of DNA from CTnGERM1 was cloned and sequenced. A 10-kbp portion of this segment hybridized not only to DNA from all the ermG-containing strains but also to DNA from strains that did not carry ermG. Thus, CTnGERM1 seems to be part of a family of CTns, some of which have acquired ermG. The percentage of G+C content of the ermG region was significantly lower than that of the chromosome of Bacteroides species-an indication that CTnGERM1 may have entered Bacteroides strains from some other bacterial genus. A survey of strains isolated before 1970 and after 1990 suggests that the CTnGERM1 type of CTn entered Bacteroides species relatively recently. One of the genes located upstream of ermG encoded a protein that had 85% amino acid sequence identity with a macrolide efflux pump, MefA, from Streptococcus pyogenes. Our having found >90% sequence identity of two upstream genes, including mefA, and the remnants of two transposon-carried genes downstream of ermG with genes found previously only in gram-positive bacteria raises the possibility that gram-positive bacteria could have been the origin of CTnGERM1.200312902247
46770.9985Aerobic anoxygenic photosynthesis genes and operons in uncultured bacteria in the Delaware River. Photosynthesis genes and operons of aerobic anoxygenic photosynthetic (AAP) bacteria have been examined in a variety of marine habitats, but genomic information about freshwater AAP bacteria is lacking. The goal of this study was to examine photosynthesis genes of AAP bacteria in the Delaware River. In a fosmid library, we found two clones bearing photosynthesis gene clusters with unique gene content and organization. Both clones contained 37 open reading frames, with most of those genes encoding known AAP bacterial proteins. The genes in one fosmid were most closely related to those of AAP bacteria in the Rhodobacter genus. The genes of the other clone were related to those of freshwater beta-proteobacteria. Both clones contained the acsF gene, which is required for aerobic bacteriochlorophyll synthesis, suggesting that these bacteria are not anaerobes. The beta-proteobacterial fosmid has the puf operon B-A-L-M-C and is the first example of an uncultured bacterium with this operon structure. The alpha-3-proteobacterial fosmid has a rare gene order (Q-B-A-L-M-X), previously observed only in the Rhodobacter genus. Phylogenetic analyses of photosynthesis genes revealed a possible freshwater cluster of AAP beta-proteobacteria. The data from both Delaware River clones suggest there are groups of freshwater or estuarine AAP bacteria distinct from those found in marine environments.200516309388
453180.9985Various pAQU plasmids possibly contribute to disseminate tetracycline resistance gene tet(M) among marine bacterial community. Emergence of antibiotic-resistant bacteria in the aquaculture environment is a significant problem for disease control of cultured fish as well as in human public health. Conjugative mobile genetic elements (MGEs) are involved in dissemination of antibiotic resistance genes (ARGs) among marine bacteria. In the present study, we first designed a PCR targeting traI gene encoding essential relaxase for conjugation. By this new PCR, we demonstrated that five of 83 strains isolated from a coastal aquaculture site had traI-positive MGEs. While one of the five strains that belonged to Shewanella sp. was shown to have an integrative conjugative element of the SXT/R391 family (ICEVchMex-like), the MGEs of the other four strains of Vibrio spp. were shown to have the backbone structure similar to that of previously described in pAQU1. The backbone structure shared by the pAQU1-like plasmids in the four strains corresponded to a ~100-kbp highly conserved region required for replication, partition and conjugative transfer, suggesting that these plasmids constituted "pAQU group." The pAQU group plasmids were shown to be capable of conjugative transfer of tet(M) and other ARGs from the Vibrio strains to E. coli. The pAQU group plasmid in one of the examined strains was designated as pAQU2, and its complete nucleotide sequence was determined and compared with that of pAQU1. The results revealed that pAQU2 contained fewer ARGs than pAQU1 did, and most of the ARGs in both of these plasmids were located in the similar region where multiple transposases were found, suggesting that the ARGs were introduced by several events of DNA transposition into an ancestral plasmid followed by drug selection in the aquaculture site. The results of the present study indicate that the "pAQU group" plasmids may play an important role in dissemination of ARGs in the marine environment.201424860553
359690.9985Association of mercury resistance with antibiotic resistance in the gram-negative fecal bacteria of primates. Gram-negative fecal bacterial from three longitudinal Hg exposure experiments and from two independent survey collections were examined for their carriage of the mercury resistance (mer) locus. The occurrence of antibiotic resistance was also assessed in both mercury-resistant (Hgr) and mercury-susceptible (Hgs) isolates from the same collections. The longitudinal studies involved exposure of the intestinal flora to Hg released from amalgam "silver" dental restorations in six monkeys. Hgr strains were recovered before the installation of amalgams, and frequently these became the dominant strains while amalgams were installed. Such persistent Hgr strains always carried the same mer locus throughout the experiments. In both the longitudinal and survey collections, certain mer loci were preferentially associated with one genus, whereas other mer loci were recovered from many genera. In general, strains with any mer locus were more likely to be multiresistant than were strains without mer loci; this clustering tendency was also seen for antibiotic resistance genes. However, the association of antibiotic multiresistance with mer loci was not random; regardless of source, certain mer loci occurred in highly multiresistant strains (with as many as seven antibiotic resistances), whereas other mer loci were found in strains without any antibiotic resistance. The majority of highly multiresistant Hgr strains also carried genes characteristic of an integron, a novel genetic element which enables the formation of tandem arrays of antibiotic resistance genes. Hgr strains lacking antibiotic resistance showed no evidence of integron components.19979361435
3571100.9985Evidence for recent intergeneric transfer of a new tetracycline resistance gene, tet(W), isolated from Butyrivibrio fibrisolvens, and the occurrence of tet(O) in ruminal bacteria. We have previously reported high-frequency transfer of tetracycline resistance between strains of the rumen anaerobic bacterium Butyrivibrio fibrisolvens. Donor strains were postulated to carry two TcR genes, one of which is transferred on a novel chromosomal element. It is shown here that coding sequences within the non-transmissible gene in B. fibrisolvens 1.230 are identical to those of the Streptococcus pneumoniae tet(O) gene. This provides the first evidence for genetic exchange between facultatively anaerobic bacteria and rumen obligate anaerobes. In contrast, the product of the transmissible TcR gene shares only 68% amino acid sequence identity with the TetO and TetM proteins and represents a new class of ribosome protection tetracycline resistance determinant, designated Tet W. The tet(W) coding region shows a higher DNA G + C content (53%) than other B. fibrisolvens genes or other ribosome protection-type tet genes, suggesting recent acquisition from a high G + C content genome. Tet(W) genes with almost identical sequences are also shown to be present in TcR strains of B. fibrisolvens from Australian sheep and in TcR strains of two other genera of rumen obligate anaerobes, Selenomonas ruminantium and Mitsuokella multiacidus. This provides compelling evidence for recent intergeneric transfer of resistance genes between ruminal bacteria. Tet(W) is not restricted to ruminal bacteria, as it was also present in a porcine strain of M. multiacidus.199911207718
4524110.9985Functional genomics in Campylobacter coli identified a novel streptomycin resistance gene located in a hypervariable genomic region. Numerous aminoglycoside resistance genes have been reported in Campylobacter spp. often resembling those from Gram-positive bacterial species and located in transferable genetic elements with other resistance genes. We discovered a new streptomycin (STR) resistance gene in Campylobactercoli showing 27-34 % amino acid identity to aminoglycoside 6-nucleotidyl-transferases described previously in Campylobacter. STR resistance was verified by gene expression and insertional inactivation. This ant-like gene differs from the previously described aminoglycoside resistance genes in Campylobacter spp. in several aspects. It does not appear to originate from Gram-positive bacteria and is located in a region corresponding to a previously described hypervariable region 14 of C. jejuni with no other known resistance genes detected in close proximity. Finally, it does not belong to a multiple drug resistance plasmid or transposon. This novel ant-like gene appears widely spread among C. coli as it is found in strains originating both from Europe and the United States and from several, apparently unrelated, hosts and environmental sources. The closest homologue (60 % amino acid identity) was found in certain C. jejuni and C. coli strains in a similar genomic location, but an association with STR resistance was not detected. Based on the findings presented here, we hypothesize that Campylobacter ant-like gene A has originated from a common ancestral proto-resistance element in Campylobacter spp., possibly encoding a protein with a different function. In conclusion, whole genome sequencing allowed us to fill in a knowledge gap concerning STR resistance in C. coli by revealing a novel STR resistance gene possibly inherent to Campylobacter.201627154456
9963120.9985Hitherto-Unnoticed Self-Transmissible Plasmids Widely Distributed among Different Environments in Japan. Various conjugative plasmids were obtained by exogenous plasmid capture, biparental mating, and/or triparental mating methods from different environmental samples in Japan. Based on phylogenetic analyses of their whole-nucleotide sequences, new IncP/P-1 plasmids that could be classified into novel subgroups were obtained. Mini-replicons of the plasmids were constructed, and each of them was incompatible with at least one of the IncP/P-1 plasmids, although they showed diverse iteron sequences in their oriV regions. There were two large clades of IncP/P-1 plasmids, clade I and II. Plasmids in clade I and II included antibiotic resistance genes. Notably, nucleotide compositions of newly found plasmids exhibited different tendencies compared with those of the previously well-studied IncP/P-1 plasmids. Indeed, the host range of plasmids of clade II was different from that of clade I. Although few PromA plasmids have been reported, the number of plasmids belonging to PromAβ, and -γ subgroups detected in this study was close to that of IncP/P-1 plasmids. The host ranges of PromAγ and PromAδ plasmids were broad and transferred to different and distinct classes of Proteobacteria. Interestingly, PromA plasmids and many IncP/P-1 plasmids do not carry any accessory genes. These findings indicate the presence of "hitherto-unnoticed" conjugative plasmids, including IncP/P-1 or PromA derivative ones in nature. These plasmids would have important roles in the exchange of various genes, including antibiotic resistance genes, among different bacteria in nature. IMPORTANCE Plasmids are known to spread among different bacteria. However, which plasmids spread among environmental samples and in which environments they are present is still poorly understood. This study showed that unidentified conjugative plasmids were present in various environments. Different novel IncP/P-1 plasmids were found, whose host ranges were different from those of known plasmids, showing wide diversity of IncP/P-1 plasmids. PromA plasmids, exhibiting a broad host range, were diversified into several subgroups and widely distributed in varied environments. These findings are important for understanding how bacteria naturally exchange their genes, including antibiotic resistance genes, a growing threat to human health worldwide.202236069618
4662130.9984Characterization of a multiresistant mosaic plasmid from a fish farm Sediment Exiguobacterium sp. isolate reveals aggregation of functional clinic-associated antibiotic resistance genes. The genus Exiguobacterium can adapt readily to, and survive in, diverse environments. Our study demonstrated that Exiguobacterium sp. strain S3-2, isolated from marine sediment, is resistant to five antibiotics. The plasmid pMC1 in this strain carries seven putative resistance genes. We functionally characterized these resistance genes in Escherichia coli, and genes encoding dihydrofolate reductase and macrolide phosphotransferase were considered novel resistance genes based on their low similarities to known resistance genes. The plasmid G+C content distribution was highly heterogeneous. Only the G+C content of one block, which shared significant similarity with a plasmid from Exiguobacterium arabatum, fit well with the mean G+C content of the host. The remainder of the plasmid was composed of mobile elements with a markedly lower G+C ratio than the host. Interestingly, five mobile elements located on pMC1 showed significant similarities to sequences found in pathogens. Our data provided an example of the link between resistance genes in strains from the environment and the clinic and revealed the aggregation of antibiotic resistance genes in bacteria isolated from fish farms.201424362420
4500140.9984Mosaic tetracycline resistance genes encoding ribosomal protection proteins. First reported in 2003, mosaic tetracycline resistance genes are a subgroup of the genes encoding ribosomal protection proteins (RPPs). They are formed when two or more RPP-encoding genes recombine resulting in a functional chimera. To date, the majority of mosaic genes are derived from sections of three RPP genes, tet(O), tet(W) and tet(32), with others comprising tet(M) and tet(S). In this first review of mosaic genes, we report on their structure, diversity and prevalence, and suggest that these genes may be responsible for an under-reported contribution to tetracycline resistance in bacteria.201627494928
4530150.9984Novel conjugative transferable multiple drug resistance plasmid pAQU1 from Photobacterium damselae subsp. damselae isolated from marine aquaculture environment. The emergence of drug-resistant bacteria is a severe problem in aquaculture. The ability of drug resistance genes to transfer from a bacterial cell to another is thought to be responsible for the wide dissemination of these genes in the aquaculture environment; however, little is known about the gene transfer mechanisms in marine bacteria. In this study, we show that a tetracycline-resistant strain of Photobacterium damselae subsp. damselae, isolated from seawater at a coastal aquaculture site in Japan, harbors a novel multiple drug resistance plasmid. This plasmid named pAQU1 can be transferred to Escherichia coli by conjugation. Nucleotide sequencing showed that the plasmid was 204,052 base pairs and contained 235 predicted coding sequences. Annotation showed that pAQU1 did not have known repA, suggesting a new replicon, and contained seven drug resistance genes: bla(CARB-9)-like, floR, mph(A)-like, mef(A)-like, sul2, tet(M) and tet(B). The plasmid has a complete set of genes encoding the apparatus for the type IV secretion system with a unique duplication of traA. Phylogenetic analysis of the deduced amino acid sequence of relaxase encoded by traI in pAQU1 demonstrated that the conjugative transfer system of the plasmid belongs to MOB(H12), a sub-group of the MOB(H) plasmid family, closely related to the IncA/C type of plasmids and SXT/R391 widely distributed among species of Enterobacteriaceae and Vibrionaceae. Our data suggest that conjugative transfer is involved in horizontal gene transfer among marine bacteria and provide useful insights into the molecular basis for the dissemination of drug resistance genes among bacteria in the aquaculture environment.201222446310
493160.9984Mercury resistance transposons of gram-negative environmental bacteria and their classification. A total of 29 mercury resistance transposons were isolated from mercury-resistant environmental strains of proteobacteria collected in different parts of Eurasia and the USA and tested for hybridization with probes specific for transposase genes of known mercury resistance transposons. 9 were related to Tn21 in this test, 12 were related to Tn5053, 4 to Tn5041 and 1 to Tn5044; three transposons were negative in this test. Restriction mapping and DNA sequencing revealed that 12 transposons were identical or nearly identical to their corresponding relatives while the rest showed varying divergence from their closest relatives. Most of these previously unknown transposons apparently arose as a result of homologous or site-specific recombination. One of these, Tn5046, was completely sequenced, and shown to be a chimera with the mer operon and the transposition module derived from the transposons related to Tn5041 and to Tn5044, respectively. Transposon Tn5070, showing no hybridization with the specific probes used in this study, was also completely sequenced. The transposition module of Tn5070 was most closely related to that of Tn3 while the mer operon was most closely related to that of plasmid pMERPH. The merR of Tn5070 is transcribed in the same direction as the mer structural genes, which is typical for mer operons of gram-positive bacteria. Our data suggest that environmental bacteria may harbor many not yet recognized mercury resistance transposons and warrant their further inventory.200111763242
4501170.9984A Bacteroides tetracycline resistance gene represents a new class of ribosome protection tetracycline resistance. The ribosome protection type of tetracycline resistance (Tcr) has been found in a variety of bacterial species, but the only two classes described previously, Tet(M) and Tet(O), shared a high degree of amino acid sequence identity (greater than 75%). Thus, it appeared that this type of resistance emerged recently in evolution and spread among different species of bacteria by horizontal transmission. We obtained the DNA sequence of a Tcr gene from Bacteroides, a genus of gram-negative, obligately anaerobic bacteria that is phylogenetically distant from the diverse species in which tet(M) and tet(O) have been found. The Bacteroides Tcr gene defines a new class of ribosome protection resistance genes, Tet(Q), and has a deduced amino acid sequence that was only 40% identical to Tet(M) or Tet(O). Like tet(M) and tet(O), tet(Q) appears to have spread by horizontal transmission, but only within the Bacteroides group.19921339256
9965180.9984The 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.201222326849
477190.9984Novel nickel resistance genes from the rhizosphere metagenome of plants adapted to acid mine drainage. Metal resistance determinants have traditionally been found in cultivated bacteria. To search for genes involved in nickel resistance, we analyzed the bacterial community of the rhizosphere of Erica andevalensis, an endemic heather which grows at the banks of the Tinto River, a naturally metal-enriched and extremely acidic environment in southwestern Spain. 16S rRNA gene sequence analysis of rhizosphere DNA revealed the presence of members of five phylogenetic groups of Bacteria and the two main groups of Archaea mostly associated with sites impacted by acid mine drainage (AMD). The diversity observed and the presence of heavy metals in the rhizosphere led us to construct and screen five different metagenomic libraries hosted in Escherichia coli for searching novel nickel resistance determinants. A total of 13 positive clones were detected and analyzed. Insights about their possible mechanisms of resistance were obtained from cellular nickel content and sequence similarities. Two clones encoded putative ABC transporter components, and a novel mechanism of metal efflux is suggested. In addition, a nickel hyperaccumulation mechanism is proposed for a clone encoding a serine O-acetyltransferase. Five clones encoded proteins similar to well-characterized proteins but not previously reported to be related to nickel resistance, and the remaining six clones encoded hypothetical or conserved hypothetical proteins of uncertain functions. This is the first report documenting nickel resistance genes recovered from the metagenome of an AMD environment.200717675438