Recovery and evolutionary analysis of complete integron gene cassette arrays from Vibrio

BACKGROUND: Integrons are genetic elements capable of the acquisition, rearrangement and expression of genes contained in gene cassettes. Gene cassettes generally consist of a promoterless gene associated with a recombination site known as a 59-base element (59-be). Multiple insertion events can lead to the assembly of large integron-associated cassette arrays. The most striking examples are found in Vibrio, where such cassette arrays are widespread and can range from 30 kb to 150 kb. Besides those found in completely sequenced genomes, no such array has yet been recovered in its entirety. We describe an approach to systematically isolate, sequence and annotate large integron gene cassette arrays from bacterial strains. RESULTS: The complete Vibrio sp. DAT722 integron cassette array was determined through the streamlined approach described here. To place it in an evolutionary context, we compare the DAT722 array to known vibrio arrays and performed phylogenetic analyses for all of its components (integrase, 59-be sites, gene cassette encoded genes). It differs extensively in terms of genomic context as well as gene cassette content and organization. The phylogenetic tree of the 59-be sites collectively found in the Vibrio gene cassette pool suggests frequent transfer of cassettes within and between Vibrio species, with slower transfer rates between more phylogenetically distant relatives. We also identify multiple cases where non-integron chromosomal genes seem to have been assembled into gene cassettes and others where cassettes have been inserted into chromosomal locations outside integrons. CONCLUSION: Our systematic approach greatly facilitates the isolation and annotation of large integrons gene cassette arrays. Comparative analysis of the Vibrio sp. DAT722 integron obtained through this approach to those found in other vibrios confirms the role of this genetic element in promoting lateral gene transfer and suggests a high rate of gene gain/loss relative to most other loci on vibrio chromosomes. We identify a relationship between the phylogenetic distance separating two species and the rate at which they exchange gene cassettes, interactions between the non-mobile portion of bacterial genomes and the vibrio gene cassette pool as well as intragenomic translocation events of integrons in vibrios

Slektskap og kolonisering hos ørekyt

Målet med dette prosjektet var å studere populasjonsgenetikken til ørekyt i Norge med sikte på å få økt kunnskap om artens spredningsmekanismer. Ørekyten utvider stadig sitt leveområde, og det er uklart hvordan spredningen skjer. Vi har kunnet fastslå at noen av de nyetablerte bestandene på Hardangervidda har en genetisk historie (en unik mitokondrie-DNA haplotype) som er svært forskjellig fra hva den naturlig utbredte ørekyten har. Innen det naturlige utbredelsesområdet finner vi bare en haplotype. Dette betyr at noe av spredningen trolig skyldes langtransport av levende fisk, dette kan skyldes bruk av levende agn (importert), eller utsetting av ikke kontrollert settefisk importert fra utlandet (med ørekyt som blindpassasjer). En del av den nyetablerte ørekyten har samme genetiske historie som naturlig utbredt ørekyt. I to vann (Legereidvann og Sandvatn) fant vi ørekyt med begge haplotypene; dette tyder på at ørekyten i disse vannene har blitt etablert som følge av to ulike spredningshendelser. Analysene av DNA-fingerprint viste at det var stor genetisk variasjon innen de ”naturlige” ørekytpopulasjonene. Det var som ventet mindre variasjon hos de nyetablerte populasjonene; noen av populasjonene oppviste imidlertid relativt høy genetisk variasjon. Den høye variasjonen i DNA-fingerprintdata hos ørekyt fra Sandvatn og Legereidvatn kan skyldes at det har forekommet minst to spredningshendelser (vi fant to ulike mitokondrie-DNA haplotyper). Også i de andre populasjonene hvor vi fant stor variasjon (Vinstri, Lesjaskogsvatn, Vålåsjøen) antar vi at årsaken er multiple spredningshendelser. Alle disse lokalitetene ligger i populære fiskeområder. Vi kan ikke fastslå i større detalj de ulike ørekytpopulasjonens opphav, til det trengs mer omfattende analyser

Suppressive Subtractive Hybridization Detects Extensive Genomic Diversity in Thermotoga maritima

Comparisons between genomes of closely related bacteria often show large variations in gene content, even between strains of the same species. Such studies have focused mainly on pathogens; here, we examined Thermotoga maritima, a free-living hyperthermophilic bacterium, by using suppressive subtractive hybridization. The genome sequence of T. maritima MSB8 is available, and DNA from this strain served as a reference to obtain strain-specific sequences from Thermotoga sp. strain RQ2, a very close relative (∼96% identity for orthologous protein-coding genes, 99.7% identity in the small-subunit rRNA sequence). Four hundred twenty-six RQ2 subtractive clones were sequenced. One hundred sixty-six had no DNA match in the MSB8 genome. These differential clones comprise, in sum, 48 kb of RQ2-specific DNA and match 72 genes in the GenBank database. From the number of identical clones, we estimated that RQ2 contains 350 to 400 genes not found in MSB8. Assuming a similar genome size, this corresponds to 20% of the RQ2 genome. A large proportion of the RQ2-specific genes were predicted to be involved in sugar transport and polysaccharide degradation, suggesting that polysaccharides are more important as nutrients for this strain than for MSB8. Several clones encode proteins involved in the production of surface polysaccharides. RQ2 encodes multiple subunits of a V-type ATPase, while MSB8 possesses only an F-type ATPase. Moreover, an RQ2-specific MutS homolog was found among the subtractive clones and appears to belong to a third novel archaeal type MutS lineage. Southern blot analyses showed that some of the RQ2 differential sequences are found in some other members of the order Thermotogales, but the distribution of these variable genes is patchy, suggesting frequent lateral gene transfer within the group

Recombination in Thermotoga: Implications for Species Concepts and Biogeography

Here we characterize regions of the genomes of eight members of the hyperthermophilic genus Thermotoga. These bacteria differ from each other physiologically and by 3–20% in gene content and occupy physically distinct environments in widely disparate regions of the globe. Among the four different lineages (represented by nine different strains) that we compare, no two are closer than 96% in the average sequences of their genes. By most accepted recent definitions these are different “ecotypes” and different “species.” And yet we find compelling evidence for recombination between them. We suggest that no single prokaryotic species concept can accommodate such uncoupling of ecotypic and genetic aspects of cohesion and diversity, and that without a single concept, the question of whether or not prokaryotic species might in general be cosmopolitan cannot be sensibly addressed. We can, however, recast biogeographical questions in terms of the distribution of genes and their alleles

Searching for Mesophilic Thermotogales Bacteria: “Mesotogas” in the Wild▿ †

All cultivated Thermotogales are thermophiles or hyperthermophiles. However, optimized 16S rRNA primers successfully amplified Thermotogales sequences from temperate hydrocarbon-impacted sites, mesothermic oil reservoirs, and enrichment cultures incubated at <46°C. We conclude that distinct Thermotogales lineages commonly inhabit low-temperature environments but may be underreported, likely due to “universal” 16S rRNA gene primer bias

Class 1 Integrons Potentially Predating the Association with Tn402-Like Transposition Genes Are Present in a Sediment Microbial Community

Integrons are genetic elements that contribute to lateral gene transfer in bacteria as a consequence of possessing a site-specific recombination system. This system facilitates the spread of genes when they are part of mobile cassettes. Most integrons are contained within chromosomes and are confined to specific bacterial lineages. However, this is not the case for class 1 integrons, which were the first to be identified and are one of the single biggest contributors to multidrug-resistant nosocomial infections, carrying resistance to many antibiotics in diverse pathogens on a global scale. The rapid spread of class 1 integrons in the last 60 years is partly a result of their association with a specific suite of transposition functions, which has facilitated their recruitment by plasmids and other transposons. The widespread use of antibiotics has acted as a positive selection pressure for bacteria, especially pathogens, which harbor class 1 integrons and their associated antibiotic resistance genes. Here, we have isolated bacteria from soil and sediment in the absence of antibiotic selection. Class 1 integrons were recovered from four different bacterial species not known to be human pathogens or commensals. All four integrons lacked the transposition genes previously considered to be a characteristic of this class. At least two of these integrons were located on a chromosome, and none of them possessed antibiotic resistance genes. We conclude that novel class 1 integrons are present in a sediment environment in various bacteria of the β-proteobacterial class. These data suggest that the dispersal of this class may have begun before the “antibiotic era.