RESUMEN
BACKGROUND: Aeromonas salmonicida subsp. salmonicida is a ubiquitous psychrophilic waterborne bacterium and a fish pathogen. The numerous mobile elements, especially insertion sequences (IS), in its genome promote rearrangements that impact its phenotype. One of the main virulence factors of this bacterium, its type three secretion system (TTSS), is affected by these rearrangements. In Aeromonas salmonicida subsp. salmonicida most of the TTSS genes are encoded in a single locus on a large plasmid called pAsa5, and may be lost when the bacterium is cultivated at a higher temperature (25 °C), producing non-virulent mutants. In a previous study, pAsa5-rearranged strains that lacked the TTSS locus on pAsa5 were produced using parental strains, including 01-B526. Some of the generated deletions were explained by homologous recombination between ISs found on pAsa5, whereas the others remained unresolved. To investigate those rearrangements, short- and long-read high-throughput sequencing technologies were used on the A. salmonicida subsp. salmonicida 01-B526 whole genome. RESULTS: Whole genome sequencing of the 01-B526 strain revealed that its pAsa5 has an additional IS copy, an ISAS5, compared to the reference strain (A449) sequence, which allowed for a previously unknown rearrangement to occur. It also appeared that 01-B526 bears a second large plasmid, named pAsa9, which shares 40 kbp of highly similar sequences with pAsa5. Following these discoveries, previously unexplained deletions were elucidated by genotyping. Furthermore, in one of the derived strains a fusion of pAsa5 and pAsa9, involving the newly discovered ISAS5 copy, was observed. CONCLUSION: The loss of TTSS and hence virulence is explained by one consistent mechanism: IS-driven homologous recombination. The similarities between pAsa9 and pAsa5 also provide another example of genetic diversity driven by ISs.
Asunto(s)
Aeromonas salmonicida/genética , Plásmidos/genética , Sistemas de Secreción Tipo III/genética , Técnicas de Genotipaje , Especificidad de la EspecieRESUMEN
The draft genome of multidrug-resistant Aeromonas sp. ARM81 isolated from a wastewater treatment plant in Warsaw (Poland) was obtained. Sequence analysis revealed multiple genes conferring resistance to aminoglycosides, ß-lactams or tetracycline. Three different ß-lactamase genes were identified, including an extended-spectrum ß-lactamase gene bla PER-1. The antibiotic susceptibility was experimentally tested. Genome sequencing also allowed us to investigate the plasmidome and transposable mobilome of ARM81. Four plasmids, of which two carry phenotypic modules (i.e., genes encoding a zinc transporter ZitB and a putative glucosyltransferase), and 28 putative transposase genes were identified. The mobility of three insertion sequences (isoforms of previously identified elements ISAs12, ISKpn9 and ISAs26) was confirmed using trap plasmids.
Asunto(s)
Aeromonas/efectos de los fármacos , Aeromonas/aislamiento & purificación , Antibacterianos/farmacología , Farmacorresistencia Bacteriana , Aguas Residuales/microbiología , Aeromonas/clasificación , Aeromonas/genética , Elementos Transponibles de ADN , Genoma Bacteriano , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Plásmidos/genética , Plásmidos/metabolismo , Polonia , Análisis de Secuencia de ADN , beta-Lactamasas/genéticaRESUMEN
Pseudomonas strains isolated from the heavily contaminated Lubin copper mine and Zelazny Most post-flotation waste reservoir in Poland were screened for the presence of integrons. This analysis revealed that two strains carried homologous DNA regions composed of a gene encoding a DNA_BRE_C domain-containing tyrosine recombinase (with no significant sequence similarity to other integrases of integrons) plus a three-component array of putative integron gene cassettes. The predicted gene cassettes encode three putative polypeptides with homology to (i) transmembrane proteins, (ii) GCN5 family acetyltransferases, and (iii) hypothetical proteins of unknown function (homologous proteins are encoded by the gene cassettes of several class 1 integrons). Comparative sequence analyses identified three structural variants of these novel integron-like elements within the sequenced bacterial genomes. Analysis of their distribution revealed that they are found exclusively in strains of the genus Pseudomonas.
Asunto(s)
Integrones , Metales Pesados/metabolismo , Pseudomonas/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Minería , Datos de Secuencia Molecular , Filogenia , Polonia , Pseudomonas/enzimología , Pseudomonas/aislamiento & purificación , Pseudomonas/metabolismo , Recombinasas/genética , Recombinasas/metabolismo , Aguas Residuales/microbiologíaRESUMEN
Antibiotic resistance determinants are frequently associated with plasmids and other mobile genetic elements, which simplifies their horizontal transmission. Several groups of plasmids (including replicons of the IncL/M incompatibility group) were found to play an important role in the dissemination of resistance genes encoding ß-lactamases. The IncL/M plasmids are large, broad host range, and self-transmissible replicons. We have identified and characterized two novel members of this group: pARM26 (isolated from bacteria inhabiting activated sludge from a wastewater treatment plant) and pIGT15 (originating from a clinical strain of Escherichia coli). This instigated a detailed comparative analysis of all available sequences of IncL/M plasmids encoding ß-lactamases. The core genome of these plasmids is comprised of 20 genes with conserved synteny. Phylogenetic analyses of these core genes allowed clustering of the plasmids into four separate groups, which reflect their antibiotic resistance profiles. Examination of the biogeography of the IncL/M plasmids revealed that they are most frequently found in bacteria of the family Enterobacteriaceae originating from the Mediterranean region and Western Europe and that they are able to persist in various ecological niches even in the absence of direct antibiotic selection pressure.
Asunto(s)
Enterobacteriaceae/genética , Transferencia de Gen Horizontal/genética , Genes Bacterianos , Variación Genética , Plásmidos/genética , Resistencia betalactámica/genética , ADN Circular/genética , Genómica , Filogeografía , beta-Lactamasas/genéticaRESUMEN
Several trap plasmids (enabling positive selection of transposition events) were used to identify a pool of functional transposable elements (TEs) residing in bacteria of the genus Paracoccus (Alphaproteobacteria). Complex analysis of 25 strains representing 20 species of this genus led to the capture and characterization of (i) 37 insertion sequences (ISs) representing 9 IS families (IS3, IS5, IS6, IS21, IS66, IS256, IS1182, IS1380 and IS1634), (ii) a composite transposon Tn6097 generated by two copies of the ISPfe2 (IS1634 family) containing two predicted genetic modules, involved in the arginine deiminase pathway and daunorubicin/doxorubicin resistance, (iii) 3 non-composite transposons of the Tn3 family, including Tn5393 carrying streptomycin resistance and (iv) a transposable genomic island TnPpa1 (45 kb). Some of the elements (e.g. Tn5393, Tn6097 and ISs of the IS903 group of the IS5 family) were shown to contain strong promoters able to drive transcription of genes placed downstream of the target site of transposition. Through the application of trap plasmid pCM132TC, containing a promoterless tetracycline resistance reporter gene, we identified five ways in which transposition can supply promoters to transcriptionally silent genes. Besides highlighting the diversity and specific features of several TEs, the analyses performed in this study have provided novel and interesting information on (i) the dynamics of the process of transposition (e.g. the unusually high frequency of transposition of TnPpa1) and (ii) structural changes in DNA mediated by transposition (e.g. the generation of large deletions in the recipient molecule upon transposition of ISPve1 of the IS21 family). We also demonstrated the great potential of TEs and transposition in the generation of diverse phenotypes as well as in the natural amplification and dissemination of genetic information (of adaptative value) by horizontal gene transfer, which is considered the driving force of bacterial evolution.
Asunto(s)
Alphaproteobacteria/genética , Elementos Transponibles de ADN/fisiología , Paracoccus/genética , Evolución Biológica , Transferencia de Gen Horizontal , Genes Bacterianos , Plásmidos/genética , Regiones Promotoras GenéticasRESUMEN
Plasmid pAMI7 of the methylotrophic bacterium Paracoccus aminophilus JCM 7686 (Alphaproteobacteria) encodes a functional type II restriction-modification (R-M) system designated PamI. Homologous systems were identified in the genomes of distinct taxonomic groups of Bacteria and Archaea, which provides evidence that horizontal gene transfer has contributed to the wide dissemination of R-M modules - even between domains. Analysis of the cleavage specificity of the R.PamI endonuclease revealed that this protein is an isoschizomer of restriction enzyme NcoI. Interestingly, bioinformatic analyses suggest that R.PamI and NcoI are accompanied by methyltransferases of different methylation specificities (C5-methylcytosine and N4-methylcytosine methyltransferases, respectively), which possibly exemplifies recombinational shuffling of genes coding for individual components of R-M systems. The PamI system can stabilize plasmid pAMI7 in a bacterial population, most probably at the postsegregational level. Therefore, it functions in an analogous manner to plasmid-encoded toxin-antitoxin (TA) systems. Since the TA system of pAMI7 is nonfunctional, it is highly probable that this lack is compensated by the stabilizing activity of PamI. This indicates the crucial role of the analyzed R-M system in the stable maintenance of pAMI7, which is, to our knowledge, the first report of 'symbiosis' between a R-M system and a plasmid in the Alphaproteobacteria.
Asunto(s)
Enzimas de Restricción-Modificación del ADN/genética , Enzimas de Restricción-Modificación del ADN/metabolismo , Desoxirribonucleasas de Localización Especificada Tipo II/genética , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Paracoccus/enzimología , Paracoccus/genética , Plásmidos , Secuencia de Aminoácidos , ADN/metabolismo , Transferencia de Gen Horizontal , Datos de Secuencia Molecular , Homología de Secuencia , Especificidad por SustratoRESUMEN
We have developed a DIY (Do It Yourself) series of genetic cassettes, which facilitate construction of novel versatile vectors for Alphaproteobacteria. All the cassettes are based on defined genetic modules derived from three natural plasmids of Paracoccus aminophilus JCM 7686. We have constructed over 50 DIY cassettes, which differ in structure and specific features. All of them are functional in eight strains representing three orders of Alphaproteobacteria: Rhodobacterales, Rhizobiales and Caulobacterales. Besides various replication and stabilization systems, many of the cassettes also contain selective markers appropriate for Alphaproteobacteria (40 cassettes) and genetic modules responsible for mobilization for conjugal transfer (24 cassettes). All the DIY cassettes are bordered by different types of polylinkers, which facilitate vector construction. Using these DIY cassettes, we have created a set of compatible Escherichia coli-Alphaproteobacteria mobilizable shuttle vectors (high or low copy number in E. coli), which will greatly assist the genetic manipulation of Alphaproteobacteria.