DNA on the move : investigation into two mobile genetic elements in Vibrio species

University of Technology, Sydney. Faculty of Science.Vibrios are a group of Gram negative rod-shaped bacteria that are ubiquitous in marine and estuarine environments. They exist as both free-living organisms and in association with a variety of hosts such as humans, coral, marine animals and plants. Vibrio cholerae is the most notorious of vibrios, being the causative agent of the devastating intestinal disease cholera in humans. Lateral gene transfer (LGT), a process that allows DNA transfer between bacterial cells, has largely driven the rapid evolution in V. cholerae and other Vibrio species. In some strains of Vibrio species at least 20% of genomic content has arisen from LGT events. With respect to V. cholerae, the two most important virulence factors: cholera toxin encoded by the ctxAB genes and intestinal adhesion encoded on the vibrio pathogenicity island (VPI-1) have been acquired via mobile genetic elements transferred by LGT. Thus, these two virulence factors convert toxigenic V. cholerae into a paradigm for the importance of LGT, demonstrating how seemingly avirulent strains of V. cholerae become capable of causing epidemic/pandemic outbreaks (Uma et al., 2003). Mobile genetic elements include but are not exclusive to: transposons, integrons, conjugative elements and genomic islands. Research performed in this thesis is focussed on the study of the integron and a genomic island and how phenotypes they confer contribute to the adaptation of two Vibrio species: V. rotiferianus and V. cholerae. Briefly, integrons are a two-component genetic recombination system present in the chromosome of almost all Vibrio species. The integron incorporates mobile genes termed gene cassettes into a reserved genetic site via site-specific recombination, named the integron/gene cassette system. The integron consists of three basic elements: an integrase gene (intI), an attachment site (attI) and a promoter (Pc). Gene cassettes generally contain a single open reading frame (ORF) and an IntI-identifiable recombination site called attC. Insertion (and excision) of gene cassettes is driven by an integrase-mediated recombination between attI and attC. Multiple insertion events lead to the accumulation of cassettes to form a cassette array. In vibrios, cassette arrays are uniquely large, sometimes containing hundreds of cassettes that make up a 1-3% of the entire genome. There is a consensus that these gene cassettes add to the adaptive potential of vibrios and have likely been an important driver in the evolution of vibrios into their respective niches. How this is achieved has been difficult to understand given that 80% of gene cassettes are novel and consequently of unknown physiological function. Using a number of chemical, proteomic and molecular techniques this thesis has shown that gene cassette(s) present in the chromosome of a model Vibrio organism (V. rotiferianus DAT722) are altering bacterial surface properties. Changes to bacterial surface properties can be important in bacterial-host interactions importantly; biofilm formation, protozoan grazing and pathogen-host association. This thesis also examines how another mobile genetic element; a novel genomic island, aids in the repair of damaged DNA in V. cholerae, giving the organism an advantage in both the environment and in the disease causing state within humans. Our knowledge of how LGT has and continues to drive bacterial adaptation and evolution has only uncovered the tip of the iceberg

The function of integron-associated gene cassettes in Vibrio species: The tip of the iceberg

The integron is a genetic element that incorporates mobile genes termed gene cassettes into a reserved genetic site via site-specific recombination. It is best known for its role in antibiotic resistance with one type of integron, the class 1 integron, a major player in the dissemination of antibiotic resistance genes across Gram negative pathogens and commensals. However, integrons are ancient structures with over 100 classes (including class 1) present in bacteria from the broader environment. While, the class 1 integron is only one example of an integron being mobilized into the clinical environment, it is by far the most successful. Unlike clinical class 1 integrons which are largely found on plasmids, other integron classes are found on the chromosomes of bacteria and carry diverse gene cassettes indicating a non-antibiotic resistance role(s). However, there is very limited knowledge on what these alternative roles are. This is particularly relevant to Vibrio species where gene cassettes make up approximately 1-3% of their entire genome. In this review, we discuss how emphasis on class 1 integron research has resulted in a limited understanding by the wider research community on the role of integrons in the broader environment. This has the capacity to be counterproductive in solving or improving the antibiotic resistance problem into the future. Furthermore, there is still a significant lack of knowledge on how gene cassettes in Vibrio species drive adaptation and evolution. From research in Vibrio rotiferianus DAT722, new insight into how gene cassettes affect cellular physiology offers new alternative roles for the gene cassette resource. At least a subset of gene cassettes are involved in host surface polysaccharide modification suggesting that gene cassettes may be important in processes such as bacteriophage resistance, adhesion/biofilm formation, protection from grazers and bacterial aggregation. © 2013 Rapa and Labbate

Deletion of Integron-Associated Gene Cassettes Impact on the Surface Properties of Vibrio rotiferianus DAT722

Background: The integron is a genetic recombination system that catalyses the acquisition of genes on mobilisable elements called gene cassettes. In Vibrio species, multiple acquired gene cassettes form a cassette array that can comprise 1-3% of the bacterial genome. Since 75% of these gene cassettes contain genes encoding proteins of uncharacterised function, how the integron has driven adaptation and evolution in Vibrio species remains largely unknown. A feature of cassette arrays is the presence of large indels. Using Vibrio rotiferianus DAT722 as a model organism, the aim of this study was to determine how large cassette deletions affect vibrio physiology with a view to improving understanding into how cassette arrays influence bacterial host adaptation and evolution. Methodology/Principal Findings: Biological assays and proteomic techniques were utilised to determine how artificially engineered deletions in the cassette array of V. rotiferianus DAT722 affected cell physiology. Multiple phenotypes were identified including changes to growth and expression of outer membrane porins/proteins and metabolic proteins. Furthermore, the deletions altered cell surface polysaccharide with Proton Nuclear Magnetic Resonance on whole cell polysaccharide identifying changes in the carbohydrate ring proton region indicating that gene cassette products may decorate host cell polysaccharide via the addition or removal of functional groups. Conclusions/Significance: From this study, it was concluded that deletion of gene cassettes had a subtle effect on bacterial metabolism but altered host surface polysaccharide. Deletion (and most likely rearrangement and acquisition) of gene cassettes may provide the bacterium with a mechanism to alter its surface properties, thus impacting on phenotypes such as biofilm formation. Biofilm formation was shown to be altered in one of the deletion mutants used in this study. Reworking surface properties may provide an advantage to the bacterium's interactions with organisms such as bacteriophage, protozoan grazers or crustaceans. © 2013 Rapa et al

A genomic island integrated into recA of Vibrio cholerae contains a divergent recA and provides multi-pathway protection from DNA damage

© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd. Lateral gene transfer (LGT) has been crucial in the evolution of the cholera pathogen, Vibrio cholerae. The two major virulence factors are present on two different mobile genetic elements, a bacteriophage containing the cholera toxin genes and a genomic island (GI) containing the intestinal adhesin genes. Non-toxigenic V.cholerae in the aquatic environment are a major source of novel DNA that allows the pathogen to morph via LGT. In this study, we report a novel GI from a non-toxigenic V.cholerae strain containing multiple genes involved in DNA repair including the recombination repair gene recA that is 23% divergent from the indigenous recA and genes involved in the translesion synthesis pathway. This is the first report of a GI containing the critical gene recA and the first report of a GI that targets insertion into a specific site within recA. We show that possession of the island in Escherichia coli is protective against DNA damage induced by UV-irradiation and DNA targeting antibiotics. This study highlights the importance of genetic elements such as GIs in the evolution of V.cholerae and emphasizes the importance of environmental strains as a source of novel DNA that can influence the pathogenicity of toxigenic strains