Does virulence assessment of Vibrio anguillarum using sea bass (Dicentrarchus labrax) larvae correspond with genotypic and phenotypic characterization?

Background: Vibriosis is one of the most ubiquitous fish diseases caused by bacteria belonging to the genus Vibrio such as Vibrio (Listonella) anguillarum. Despite a lot of research efforts, the virulence factors and mechanism of V. anguillarum are still insufficiently known, in part because of the lack of standardized virulence assays. Methodology/Principal Findings: We investigated and compared the virulence of 15 V. anguillarum strains obtained from different hosts or non-host niches using a standardized gnotobiotic bioassay with European sea bass (Dicentrarchus labrax L.) larvae as model hosts. In addition, to assess potential relationships between virulence and genotypic and phenotypic characteristics, the strains were characterized by random amplified polymorphic DNA (RAPD) and repetitive extragenic palindromic PCR (rep-PCR) analyses, as well as by phenotypic analyses using Biolog's Phenotype MicroArray (TM) technology and some virulence factor assays. Conclusions/Significance: Virulence testing revealed ten virulent and five avirulent strains. While some relation could be established between serotype, genotype and phenotype, no relation was found between virulence and genotypic or phenotypic characteristics, illustrating the complexity of V. anguillarum virulence. Moreover, the standardized gnotobiotic system used in this study has proven its strength as a model to assess and compare the virulence of different V. anguillarum strains in vivo. In this way, the bioassay contributes to the study of mechanisms underlying virulence in V. anguillarum

Identification and application of bacterial volatiles to attract a generalist aphid parasitoid from laboratory to greenhouse assays

BACKGROUND: Recent studies have shown that microorganisms emit volatile compounds that affect insect behaviour. However, it remains largely unclear whether microbes can be exploited as a source of attractants to improve biological control of insect pests. In this study, we used a combination of coupled gas chromatography electroantennography (GC-EAG) and Y-tube olfactometer bioassays to identify attractive compounds in the volatile extracts of three bacterial strains that are associated with the habitat of the generalist aphid parasitoid Aphidius colemani, and to create mixtures of synthetic compounds to find attractive blends for A. colemani. Subsequently, the most promising blend was evaluated in two-choice cage experiments under greenhouse conditions. RESULTS: GC-EAG analysis revealed 20 compounds that were linked to behaviourally attractive bacterial strains. A mixture of two EAG-active compounds, styrene and benzaldehyde applied at a respective dose of 1 μg and 10 ng, was more attractive than the single compounds or the culture medium of the bacteria in Y-tube olfactometer bioassays. Application of this synthetic mixture under greenhouse conditions resulted in significant attraction of the parasitoids, and outperformed application of the bacterial culture medium. CONCLUSION: Compounds isolated from bacterial blends were capable of attracting parasitoids both in laboratory and greenhouse assays, indicating that microbial culture are an effective source of insect attractants. This opens new opportunities to attract and retain natural enemies of pest species and to enhance biological pest control

Bacterial phylogeny predicts volatile organic compound composition and olfactory response of an aphid parasitoid

There is increasing evidence that microorganisms emit a wide range of volatile compounds (mVOCs, microbial volatile organic compounds) that act as insect semiochemicals, and therefore play an important role in insect behaviour. Although it is generally believed that phylogenetically closely related microbes tend to have similar phenotypic characteristics and therefore may elicit similar responses in insects, currently little is known about whether the evolutionary history and phylogenetic relationships among microorganisms have an impact on insect‐microbe interactions. In this study, we tested the hypothesis that phylogenetic relationships among 40 Bacillus strains isolated from diverse environmental sources predicted mVOC composition and the olfactory response of the generalist aphid parasitoid Aphidius colemani . Results revealed that phylogenetically closely related Bacillus strains emitted similar blends of mVOCs and elicited a comparable olfactory response of A. colemani in Y‐tube olfactometer bioassays, varying between attraction and repellence. Analysis of the chemical composition of the mVOC blends showed that all Bacillus strains produced a highly similar set of volatiles, but often in different concentrations and ratios. Benzaldehyde was produced in relatively high concentrations by strains that repel A. colemani , while attractive mVOC blends contained relatively higher amounts of acetoin, 2,3‐butanediol, 2,3‐butanedione, eucalyptol and isoamylamine. Overall, these results indicate that bacterial phylogeny had a strong impact on mVOC compositions and as a result on the olfactory responses of insects

Identification and application of bacterial volatiles to attract a generalist aphid parasitoid: from laboratory to greenhouse assays

BACKGROUND Recent studies have shown that microorganisms emit volatile compounds that affect insect behaviour. However, it remains largely unclear whether microbes can be exploited as a source of attractants to improve biological control of insect pests. In this study, we used a combination of coupled gas chromatography‐electroantennography (GC–EAG) and Y‐tube olfactometer bioassays to identify attractive compounds in the volatile extracts of three bacterial strains that are associated with the habitat of the generalist aphid parasitoid Aphidius colemani, and to create mixtures of synthetic compounds to find attractive blends for A. colemani. Subsequently, the most attractive blend was evaluated in two‐choice cage experiments under greenhouse conditions. RESULTS GC–EAG analysis revealed 20 compounds that were linked to behaviourally attractive bacterial strains. A mixture of two EAG‐active compounds, styrene and benzaldehyde applied at a respective dose of 1 μg and 10 ng, was more attractive than the single compounds or the culture medium of the bacteria in Y‐tube olfactometer bioassays. Application of this synthetic mixture under greenhouse conditions resulted in significant attraction of the parasitoids, and outperformed application of the bacterial culture medium. CONCLUSION Compounds isolated from bacterial blends were capable of attracting parasitoids both in laboratory and greenhouse assays, indicating that microbial cultures are an effective source of insect attractants. This opens new opportunities to attract and retain natural enemies of pest species and to enhance biological pest control

Novel Genetic Tools for Diaminopimelic Acid Selection in Virulence Studies of Yersinia pestis

Molecular studies of bacterial virulence are enhanced by expression of recombinant DNA during infection to allow complementation of mutants and expression of reporter proteins in vivo. For highly pathogenic bacteria, such as Yersinia pestis, these studies are currently limited because deliberate introduction of antibiotic resistance is restricted to those few which are not human treatment options. In this work, we report the development of alternatives to antibiotics as tools for host-pathogen research during Yersinia pestis infections focusing on the diaminopimelic acid (DAP) pathway, a requirement for cell wall synthesis in eubacteria. We generated a mutation in the dapA-nlpB(dapX) operon of Yersinia pestis KIM D27 and CO92 which eliminated the expression of both genes. The resulting strains were auxotrophic for diaminopimelic acid and this phenotype was complemented in trans by expressing dapA in single and multi-copy. In vivo, we found that plasmids derived from the p15a replicon were cured without selection, while selection for DAP enhanced stability without detectable loss of any of the three resident virulence plasmids. The dapAX mutation rendered Y. pestis avirulent in mouse models of bubonic and septicemic plague which could be complemented when dapAX was inserted in single or multi-copy, restoring development of disease that was indistinguishable from the wild type parent strain. We further identified a high level, constitutive promoter in Y. pestis that could be used to drive expression of fluorescent reporters in dapAX strains that had minimal impact to virulence in mouse models while enabling sensitive detection of bacteria during infection. Thus, diaminopimelic acid selection for single or multi-copy genetic systems in Yersinia pestis offers an improved alternative to antibiotics for in vivo studies that causes minimal disruption to virulence

Defining the Pseudomonas Genus: Where Do We Draw the Line with Azotobacter?

The genus Pseudomonas has gone through many taxonomic revisions over the past 100 years, going from a very large and diverse group of bacteria to a smaller, more refined and ordered list having specific properties. The relationship of the Pseudomonas genus to Azotobacter vinelandii is examined using three genomic sequence-based methods. First, using 16S rRNA trees, it is shown that A. vinelandii groups within the Pseudomonas close to Pseudomonas aeruginosa. Genomes from other related organisms (Acinetobacter, Psychrobacter, and Cellvibrio) are outside the Pseudomonas cluster. Second, pan genome family trees based on conserved gene families also show A. vinelandii to be more closely related to Pseudomonas than other related organisms. Third, exhaustive BLAST comparisons demonstrate that the fraction of shared genes between A. vinelandii and Pseudomonas genomes is similar to that of Pseudomonas species with each other. The results of these different methods point to a high similarity between A. vinelandii and the Pseudomonas genus, suggesting that Azotobacter might actually be a Pseudomonas

Azotobacter genomes: the genome of Azotobacter chroococcum NCIMB 8003 (ATCC 4412)

The genome of the soil-dwelling heterotrophic N2-fixing Gram-negative bacterium Azotobacter chroococcum NCIMB 8003 (ATCC 4412) (Ac-8003) has been determined. It consists of 7 circular replicons totalling 5,192,291 bp comprising a circular chromosome of 4,591,803 bp and six plasmids pAcX50a, b, c, d, e, f of 10,435 bp, 13,852, 62,783, 69,713, 132,724, and 311,724 bp respectively. The chromosome has a G+C content of 66.27% and the six plasmids have G+C contents of 58.1, 55.3, 56.7, 59.2, 61.9, and 62.6% respectively. The methylome has also been determined and 5 methylation motifs have been identified. The genome also contains a very high number of transposase/inactivated transposase genes from at least 12 of the 17 recognised insertion sequence families. The Ac-8003 genome has been compared with that of Azotobacter vinelandii ATCC BAA-1303 (Av-DJ), a derivative of strain O, the only other member of the Azotobacteraceae determined so far which has a single chromosome of 5,365,318 bp and no plasmids. The chromosomes show significant stretches of synteny throughout but also reveal a history of many deletion/insertion events. The Ac-8003 genome encodes 4628 predicted protein-encoding genes of which 568 (12.2%) are plasmid borne. 3048 (65%) of these show > 85% identity to the 5050 protein-encoding genes identified in Av-DJ, and of these 99 are plasmid-borne. The core biosynthetic and metabolic pathways and macromolecular architectures and machineries of these organisms appear largely conserved including genes for CO-dehydrogenase, formate dehydrogenase and a soluble NiFe-hydrogenase. The genetic bases for many of the detailed phenotypic differences reported for these organisms have also been identified. Also many other potential phenotypic differences have been uncovered. Properties endowed by the plasmids are described including the presence of an entire aerobic corrin synthesis pathway in pAcX50f and the presence of genes for retro-conjugation in pAcX50c. All these findings are related to the potentially different environmental niches from which these organisms were isolated and to emerging theories about how microbes contribute to their communities