Novel integrons and gene cassettes from a Cascadian submarine gas-hydrate-bearing core.

To determine whether integrons are present in a submarine gas hydrate community, metagenomic DNA was extracted from a gas-hydrate-bearing core, 150 m below the seafloor, from the Cascadian Margin. Integrons and gene cassettes were recovered by PCR from metagenomic DNA and sequenced. Thirty-seven integron integrase phylotypes were identified. The phylotypes were diverse and included members with homology to integrases from Methylomonas methanica, Desulfuromonas acetoxidans, Thermodesulfatator indicus, and marine uncultured bacteria. The gene cassette composition, 153 gene cassettes, was dominated by two types of encoded putative proteins. The first of these was predicted oxidoreductases, such as iron/sulfur cluster-binding proteins. A second type was alkyl transferases. Some cassette proteins showed homologies with those from methane-related archaea. These observations suggest that integrons may assist in the adaptation of microbial communities in this environment.

Integration of a laterally acquired gene into a cell network important for growth in a strain of Vibrio rotiferianus.

BACKGROUND: Lateral Gene Transfer (LGT) is a major contributor to bacterial evolution and up to 25% of a bacterium's genome may have been acquired by this process over evolutionary periods of time. Successful LGT requires both the physical transfer of DNA and its successful incorporation into the host cell. One system that contributes to this latter step by site-specific recombination is the integron. Integrons are found in many diverse bacterial Genera and is a genetic system ubiquitous in vibrios that captures mobile DNA at a dedicated site. The presence of integron-associated genes, contained within units of mobile DNA called gene cassettes makes up a substantial component of the vibrio genome (1-3%). Little is known about the role of this system since the vast majority of genes in vibrio arrays are highly novel and functions cannot be ascribed. It is generally regarded that strain-specific mobile genes cannot be readily integrated into the cellular machinery since any perturbation of core metabolism is likely to result in a loss of fitness. RESULTS: In this study, at least one mobile gene contained within the Vibrio rotiferianus strain DAT722, but lacking close relatives elsewhere, is shown to greatly reduce host fitness when deleted and tested in growth assays. The precise role of the mobile gene product is unknown but impacts on the regulation of outermembrane porins. This demonstrates that strain specific laterally acquired mobile DNA can be integrated rapidly into bacterial networks such that it becomes advantageous for survival and adaptation in changing environments. CONCLUSIONS: Mobile genes that are highly strain specific are generally believed to act in isolation. This is because perturbation of existing cell machinery by the acquisition of a new gene by LGT is highly likely to lower fitness. In contrast, we show here that at least one mobile gene, apparently unique to a strain, encodes a product that has integrated into central cellular metabolic processes such that it greatly lowers fitness when lost under those conditions likely to be commonly encountered for the free living cell. This has ramifications for our understanding of the role mobile gene encoded products play in the cell from a systems biology perspective.

Gene flow, mobile genetic elements and the recruitment of antibiotic resistance genes into Gram-negative pathogens.

Antibiotics were one of the great discoveries of the 20th century. However, resistance appeared even in the earliest years of the antibiotic era. Antibiotic resistance continues to become worse, despite the ever-increasing resources devoted to combat the problem. One of the most important factors in the development of resistance to antibiotics is the remarkable ability of bacteria to share genetic resources via Lateral Gene Transfer (LGT). LGT occurs on a global scale, such that in theory, any gene in any organism anywhere in the microbial biosphere might be mobilized and spread. With sufficiently strong selection, any gene may spread to a point where it establishes a global presence. From an antibiotic resistance perspective, this means that a resistance phenotype can appear in a diverse range of infections around the globe nearly simultaneously. We discuss the forces and agents that make this LGT possible and argue that the problem of resistance can ultimately only be managed by understanding the problem from a broad ecological and evolutionary perspective. We also argue that human activities are exacerbating the problem by increasing the tempo of LGT and bacterial evolution for many traits that are important to humans.

Marine integrons containing novel integrase genes, attachment sites, attI, and associated gene cassettes in polluted sediments from Suez and Tokyo Bays.

In order to understand the structure and biological significance of integrons and associated gene cassettes in marine polluted sediments, metagenomic DNAs were extracted from sites at Suez and Tokyo Bays. PCR amplicons containing new integrase genes, intI, linked with novel gene cassettes, were recovered and had sizes from 1.8 to 2.5 kb. This approach uncovered, for the first time, the structure and diversity of both marine integron attachment site, attI, and the first gene cassette, the most efficiently expressed integron-associated gene cassette. The recovered 13 and 20 intI phylotypes, from Suez and Tokyo Bay samples, respectively, showed a highly divergence, suggesting a difference in integron composition between the sampling sites. Some intI phylotypes showed similarity with that from Geobacter metallireducens, belonging to Deltaproteobacteria, the dominant class in both sampling sites, as determined by 16S rRNA gene analysis. Thirty distinct families of putative attI site, as determined by the presence of an attI-like simple site, were recovered. A total of 146 and 68 gene cassettes represented Suez and Tokyo Bay unsaturated cassette pools, respectively. Gene cassettes, including a first cassette, from both sampling sites encoded two novel families of glyoxalase/bleomycin antibiotic-resistance protein. Gene cassettes from Suez Bay encoded proteins similar to haloacid dehalogenases, protein disulfide isomerases and death-on-curing and plasmid maintenance system killer proteins. First gene cassettes from Tokyo Bay encoded a xenobiotic-degrading protein, cardiolipin synthetase, esterase and WD40-like ß propeller protein. Many of the first gene cassettes encoded proteins with no ascribable function but some of them were duplicated and possessed signal functional sites, suggesting efficient adaptive functions to their bacterial sources. Thus, each sampling site had a specific profile of integrons and cassette types consistent with the hypothesis that the environment shapes the genome.

Class 1 integrons in benthic bacterial communities: abundance, association with Tn402-like transposition modules and evidence for coselection with heavy-metal resistance.

The integron/gene cassette system contributes to lateral gene transfer of genetic information in bacterial communities, with gene cassette-encoded proteins potentially playing an important role in adaptation to stress. Class 1 integrons are a particularly important class as they themselves seem to be broadly disseminated among the Proteobacteria and have an established role in the spread of antibiotic resistance genes. The abundance and structure of class 1 integrons in freshwater sediment bacterial communities was assessed through sampling of 30 spatially distinct sites encompassing different substrate and catchment types from the Greater Melbourne Area of Victoria, Australia. Real-time PCR was used to demonstrate that the abundance of intI1 was increased as a result of ecosystem perturbation, indicated by classification of sample locations based on the catchment type and a strong positive correlation with the first principal component factor score, comprised primarily of the heavy metals zinc, mercury, lead and copper. Additionally, the abundance of intI1 at sites located downstream from treated sewage outputs was associated with the percentage contribution of the discharge to the basal flow rate. Characterization of class 1 integrons in bacteria cultured from selected sediment samples identified an association with complete Tn402-like transposition modules, and the potential for coselection of heavy-metal and antibiotic resistance mechanisms in benthic environments.

The integron/gene cassette system: an active player in bacterial adaptation.

The integron includes a site-specific recombination system capable of integrating and expressing genes contained in structures called mobile gene cassettes. Integrons were originally identified on mobile elements from pathogenic bacteria and were found to be a major reservoir of antibiotic-resistance genes. Integrons are now known to be ancient structures that are phylogenetically diverse and, to date, have been found in approximately 9% of sequenced bacterial genomes. Overall, gene diversity in cassettes is extraordinarily high, suggesting that the integron/gene cassette system has a broad role in adaptation rather than being confined to simply conferring resistance to antibiotics. In this chapter, we provide a review of the integron/gene cassette system highlighting characteristics associated with this system, diversity of elements contained within it, and their importance in driving bacterial evolution and consequently adaptation. Ideas on the evolution of gene cassettes and gene cassette arrays are discussed.

Structural genomics of the bacterial mobile metagenome: an overview.

Mobile gene cassettes collectively carry a highly diverse pool of novel genes, ostensibly for purposes of microbial adaptation. At the sequence level, putative functions can only be assigned to a minority of carried ORFs due to their inherent novelty. Having established these mobilized genes code for folded and functional proteins, the authors have recently adopted the procedures of structural genomics to efficiently sample their structures, thereby scoping their functional range. This chapter outlines protocols used to produce cassette-associated genes as recombinant proteins in Escherichia coli and crystallization procedures based on the dual screen/pH optimization approach of the SECSG (SouthEast Collaboratory for Structural Genomics). Crystal structures solved to date have defined unique members of enzyme fold classes associated with transport and nucleotide metabolism.

Influence of industrial contamination on mobile genetic elements: class 1 integron abundance and gene cassette structure in aquatic bacterial communities.

The acquisition of new genetic material via horizontal gene transfer allows bacteria to rapidly evolve. One key to estimating the contribution of horizontal gene transfer to bacterial evolution is to quantify the abundance of mobile genetic elements (MGEs) in bacterial communities under varying degrees of selective pressure. We quantified class 1 integrase (intI1) gene abundance in total community DNA extracted from contaminated and reference riverine and estuarine microhabitats, and in metal- or antibiotic-amended freshwater microcosms. The intI1 gene was more abundant in all contaminant-exposed communities indicating that relative gene transfer potential is higher in these communities. A second key to assessing the contributions of MGEs to bacterial evolution is to examine the structure and function of the MGE-associated gene pool. We determined that the gene cassette pool is a novel and diverse resource available for bacterial acquisition, but that contamination has no discernible effect on cassette richness. Gene cassette profiles were more similar within sites than among sites, yet bacterial community profiles were not, suggesting that selective pressures can shape the structure of the gene cassette pool. Of the 46 sequenced gene cassette products, 37 were novel sequences, while the 9 gene cassettes with similarity to database sequences were primarily to hypothetical proteins. That class 1 integrons are ubiquitous and abundant in environmental bacterial communities indicates that this group of MGEs can play a substantial role in the acquisition of a diverse array of gene cassettes beyond their demonstrated impact in mediating multidrug resistance in clinical bacteria.

Blood protein purification and simultaneous removal of nonenveloped viruses using tangential-flow preparative electrophoresis.

Gradiflow is new technology allowing purification of important blood proteins from viral contaminated plasma. Protein purification is based on unique scalable tangential-flow preparative electrophoresis, and is distinct from current technology because protein purification and virus removal are performed in the same step. This one-step removal and purification exploits both the size and charge of target proteins. The medically important blood proteins, immunoglobulin G (IgG) and alpha-1-antitrypsin, were chosen to demonstrate the ability of this process to purify proteins from contaminated plasma. Clearance factors achieved by infectivity assays and polymerase chain reaction (PCR) that meet regulatory requirements demonstrated removal of canine parvovirus (CPV). CPV is a model virus for pathogenic nonenveloped viruses, including parvovirus B19, not adequately removed or inactivated by most processes currently in practice. The recovery of proteins from plasma with high purity, recovery, and function, while simultaneously removing viruses, provides blood products with a level of purity compatible with clinical use more quickly and cheaply than available techniques.