Identification of genes required for gold and silver tolerance in Burkholderia cenocepacia H111 by transposon sequencing.

Members of the genus Burkholderia show remarkable abilities to adapt to a wide range of environmental conditions and is frequently isolated from soils contaminated with heavy metals. In this study, we used a transposon sequencing approach to identify 138 and 164 genes that provide a benefit for growth of the opportunistic pathogen Burkholderia cenocepacia H111 in the presence of silver and gold ions respectively. The data suggest that arginine metabolism and citrate biosynthesis are important for silver tolerance, while components of an ABC transporter (BCAL0307-BCAL0308) and de novo cysteine biosynthesis are required for tolerance to gold ions. We show that determinants that affect tolerance to both metal ions include the two-component systems BCAL0497/99 and BCAL2830/31 and genes that are involved in maintaining the integrity of the cell envelope, suggesting that membrane proteins represent important targets of silver and gold ions. Furthermore, we show that that the P-type ATPase CadA (BCAL0055), which confers tolerance to cadmium contributes to silver but not gold tolerance. Our results may be useful for improving the antibacterial effect of silver and gold ions to combat drug-resistant pathogens.

Copper resistance genes of Burkholderia cenocepacia H111 identified by transposon sequencing.

Copper is an essential element but in excess is highly toxic and therefore cytoplasmic levels must be tightly controlled. Member of the genus Burkholderia are highly resistant to various heavy metals and are often isolated from acidic soils where copper bioavailability is high. In this study, we employed transposon sequencing (Tn-Seq) to identify copper resistance genes in Burkholderia cenocepacia H111. We identified a copper efflux system that shares similarities with the plasmid-based copper detoxification systems found in Escherichia coli and Pseudomonas syringae. We also found that several of the identified resistance determinants are involved in maintaining the integrity of the cell envelope, suggesting that proteins located in the outer membrane and periplasmic space are particularly sensitive to copper stress. Given that several of the resistance genes are required for the repair and turnover of misfolded proteins, we suggest that copper toxicity is caused by protein damage rather than by oxidative stress.

Dynamics of the Pacific oyster pathobiota during mortality episodes in Europe assessed by 16S rRNA gene profiling and a new target enrichment next-generation sequencing strategy.

Infectious agents such as the bacteria Vibrio aestuarianus or Ostreid herpesvirus 1 have been repeatedly associated with dramatic disease outbreaks of Crassostrea gigas beds in Europe. Beside roles played by these pathogens, microbial infections in C. gigas may derive from the contribution of a larger number of microorganisms than previously thought, according to an emerging view supporting the polymicrobial nature of bivalve diseases. In this study, the microbial communities associated with a large number of C. gigas samples collected during recurrent mortality episodes at different European sites were investigated by real-time PCR and 16SrRNA gene-based microbial profiling. A new target enrichment next-generation sequencing protocol for selective capturing of 884 phylogenetic and virulence markers of the potential microbial pathogenic community in oyster tissue was developed allowing high taxonomic resolution analysis of the bivalve pathobiota. Comparative analysis of contrasting C. gigas samples conducted using these methods revealed that oyster experiencing mortality outbreaks displayed signs of microbiota disruption associated with the presence of previously undetected potential pathogenic microbial species mostly belonging to genus Vibrio and Arcobacter. The role of these species and their consortia should be targeted by future studies aiming to shed light on mechanisms underlying polymicrobial infections in C. gigas.

Exposure to TiO2 nanoparticles induces shifts in the microbiota composition of Mytilus galloprovincialis hemolymph.

It is now recognized that host microbiome, the community of microorganisms that colonize the animal body (e.g. microbiota) and their genomes, play an important role in the health status of all organisms, from nutrient processing to protection from disease. In particular, the complex, bilateral interactions between the host innate immune system and the microbiota are crucial in maintaining whole body homeostasis. The development of nanotechnology is raising concern on the potential impact of nanoparticles-NPs on human and environmental health. Titanium dioxide-nTiO2, one of the most widely NP in use, has been shown to affect the gut microbiota of mammals and fish, as well as to potentially alter microbial communities. In the marine bivalve Mytilus galloprovincialis, nTiO2 has been previously shown to interact with hemolymph components, thus resulting in immunomodulation. However, no information is available on the possible impact of NPs on the microbiome of marine organisms. Bivalves host high microbial abundance and diversity, and alteration of their microbiota, in both tissues and hemolymph, in response to stressful conditions has been linked to a compromised health status and susceptibility to diseases. In this work, the effects of nTiO2 exposure (100 µg/L, 4 days) on Mytilus hemolymph microbiota were investigated by 16S rRNA gene-based profiling. Immune parameters were also evaluated. Although hemolymph microbiota of control and nTiO2-treated mussels revealed a similar core composition, nTiO2 exposure affected the abundance of different genera, with decreases in some (e.g. Shewanella, Kistimonas, Vibrio) and increases in others (e.g. Stenotrophomonas). The immunomodulatory effects of nTiO2 were confirmed by the increase in the bactericidal activity of whole hemolymph. These represent the first data on the effects of NPs on the microbiome of marine invertebrates, and suggest that the shift in hemolymph microbiome composition induced by nTiO2 may result from the interplay between the microbiota and the immune system.

The Essential Genome of Burkholderia cenocepacia H111.

The study of the minimum set of genes required to sustain life is a fundamental question in biological research. Recent studies on bacterial essential genes suggested that between 350 and 700 genes are essential to support autonomous bacterial cell growth. Essential genes are of interest as potential new antimicrobial drug targets; hence, our aim was to identify the essential genome of the cystic fibrosis (CF) isolate Burkholderia cenocepacia H111. Using a transposon sequencing (Tn-Seq) approach, we identified essential genes required for growth in rich medium under aerobic and microoxic conditions as well as in a defined minimal medium with citrate as a sole carbon source. Our analysis suggests that 398 genes are required for autonomous growth in rich medium, a number that represents only around 5% of the predicted genes of this bacterium. Five hundred twenty-six genes were required to support growth in minimal medium, and 434 genes were essential under microoxic conditions (0.5% O2). A comparison of these data sets identified 339 genes that represent the minimal set of essential genes required for growth under all conditions tested and can be considered the core essential genome of B. cenocepacia H111. The majority of essential genes were found to be located on chromosome 1, and few such genes were located on chromosome 2, where most of them were clustered in one region. This gene cluster is fully conserved in all Burkholderia species but is present on chromosome 1 in members of the closely related genus Ralstonia, suggesting that the transfer of these essential genes to chromosome 2 in a common ancestor contributed toward the separation of the two genera.IMPORTANCE Transposon sequencing (Tn-Seq) is a powerful method used to identify genes that are essential for autonomous growth under various conditions. In this study, we have identified a set of "core essential genes" that are required for growth under multiple conditions, and these genes represent potential antimicrobial targets. We also identified genes specifically required for growth under low-oxygen and nutrient-limited environments. We generated conditional mutants to verify the results of our Tn-Seq analysis and demonstrate that one of the identified genes was not essential per se but was an artifact of the construction of the mutant library. We also present verified examples of genes that were not truly essential but, when inactivated, showed a growth defect. These examples have identified so-far-underestimated shortcomings of this powerful method.