NaCl concentration in the medium modulates the secretion of active EmpA protease in Vibrio anguillarum at post-transcriptional level.

AIMS: This study focused on the influence of different amounts of NaCl in the medium in Vibrio anguillarum EmpA protease production at both the transcriptional and post-transcriptional levels. METHODS AND RESULTS: Vibrio anguillarum 975/I was cultivated in cM9 medium with varying concentrations of NaCl: 0·5, 1·5, 3·0%. EmpA protease was monitored in the supernatants by the skim milk test, azocasein assay and Western blot analysis. The empA gene expression was measured by real-time PCR. A mutant strain 975/I defective for the empA gene confirmed the specificity of the response for EmpA protease. Active protease production was induced by 0·5 and 1·5% NaCl-amended media; however, the strain cultivated in 3·0% NaCl was unable to secrete EmpA protease. The quantitative expression of the empA gene was very similar in all tested conditions. CONCLUSIONS: The NaCl concentration in the medium modulates the secretion of active EmpA protease in V. anguillarum at a post-transcriptional level. SIGNIFICANCE AND IMPACT OF THE STUDY: EmpA protease is one of the most important virulence factors in V. anguillarum. We demonstrated the influence of osmotic changes in the regulation of EmpA protease in the V. anguillarum 975/I strain. This finding has an important impact on the evaluation of factors determining the onset of disease in fish.

Application of relative real-time PCR to detect differential expression of virulence genes in Vibrio anguillarum under standard and stressed growth conditions.

In this study, we aimed to understand whether abiotic factors affect the expression of virulence genes in Vibrio anguillarum. We observed the in vitro responses of two Mediterranean strains of V. anguillarum to temperature, NaCl and iron concentration changes. We monitored growth performance and gene transcription levels by comparing the results obtained under stressed conditions (temperatures of 5 °C, 15 °C and 37 °C; NaCl concentrations of 3% and 5%; and iron depletion and excess) with those obtained under standard growth conditions (25 °C, 1.5% NaCl and 0.6 µm of iron). The results showed that the strains respond differently. The strain 975/I was most strongly affected by conditions of 15 °C and iron depletion; these conditions induced increased transcription levels of empA, angR and fatA. Growth of the strain 17/I was inhibited at 15 °C and in iron depletion conditions; this strain also showed dramatic changes in the transcription levels of toxR and tonB2 under increased NaCl concentrations. These results demonstrate that environmental stress affects the expression of virulence genes in V. anguillarum that have implications for the competitiveness, stress tolerance and the ability of V. anguillarum to cause infection.

Bacterial biofilms on medical masks disposed in the marine environment: a hotspot of biological and functional diversity.

The present paper was aimed at investigating the role of disposable medical masks as a substrate for microbial biofilm growth and for the selection of specific microbial traits in highly impacted marine environments. In this view, we have immerged masks in a coastal area affected by a continuous input of artisanal fishery wastes and hydrocarbons pollution caused by intense maritime traffic. Masks maintained one month in the field were colonized by a bacterial community significantly different from that detected in the natural matrices from the same areas (seawater and sediments). The masks served as a viable substrate for the growth and enrichment of phototrophic microorganisms (Oxyphotobacteria), as well as Ruminococcaceae, Gracilibacteria, and Holophageae. In a follow-up investigation, masks previously colonized in the field were transferred in lab-scale microcosms which were supplemented with hydrocarbons and which contained also a piece of a virgin mask. After one month, a shift in the community composition, likely triggered by hydrocarbons addition, was observed in the previously colonized mask, with signatures characteristic of hydrocarbon-degrading microbial groups. Such hydrocarbon-degrading bacteria were also found to colonize the virgin mask. Remarkably, SEM micrographs provided indications of the occurrence of morphological modifications of the surface components of the virgin masks colonized by hydrocarbonoclastic bacteria. Overall, for the first time, we have demonstrated the potential risk for human and animal health determined by the uncorrected disposal of masks which are suitable substrates for pathogens colonization, permanence and spreading. Moreover, we have herein strengthened the knowledge on the role of hydrocarbon-degrading bacteria in the colonization and modification of fossil-based plastics in marine environment.

Marine hydrocarbon-degrading bacteria breakdown poly(ethylene terephthalate) (PET).

Pollution of aquatic ecosystems by plastic wastes poses severe environmental and health problems and has prompted scientific investigations on the fate and factors contributing to the modification of plastics in the marine environment. Here, we investigated, by means of microcosm studies, the role of hydrocarbon-degrading bacteria in the degradation of poly(ethylene terephthalate) (PET), the main constituents of plastic bottles, in the marine environment. To this aim, different bacterial consortia, previously acclimated to representative hydrocarbons fractions namely, tetradecane (aliphatic fraction), diesel (mixture of hydrocarbons), and naphthalene/phenantrene (aromatic fraction), were used as inocula of microcosm experiments, in order to identify peculiar specialization in poly(ethylene terephthalate) degradation. Upon formation of a mature biofilm on the surface of poly(ethylene terephthalate) films, the bacterial biodiversity and degradation efficiency of each selected consortium was analyzed. Notably, significant differences on biofilm biodiversity were observed with distinctive hydrocarbons-degraders being enriched on poly(ethylene terephthalate) surface, such as Alcanivorax, Hyphomonas, and Cycloclasticus species. Interestingly, ATR-FTIR analyses, supported by SEM and water contact angle measurements, revealed major alterations of the surface chemistry and morphology of PET films, mainly driven by the bacterial consortia enriched on tetradecane and diesel. Distinctive signatures of microbial activity were the alteration of the FTIR spectra as a consequence of PET chain scission through the hydrolysis of the ester bond, the increased sample hydrophobicity as well as the formation of small cracks and cavities on the surface of the film. In conclusion, our study demonstrates for the first time that hydrocarbons-degrading marine bacteria have the potential to degrade poly(ethylene terephthalate), although their degradative activity could potentially trigger the formation of harmful microplastics in the marine environment.

Bioremediation technologies for polluted seawater sampled after an oil-spill in Taranto Gulf (Italy): A comparison of biostimulation, bioaugmentation and use of a washing agent in microcosm studies.

One of the main challenges of bioremediation is to define efficient protocols having a low environmental impact. We have investigated the effect of three treatments in oily-seawater after a real oil-spill occurred in the Gulf of Taranto (Italy). Biostimulation with inorganic nutrients allowed the biodegradation of the 73±2.4% of hydrocarbons, bioaugmentation with a selected hydrocarbonoclastic consortium consisting of Alcanivorax borkumensis, Alcanivorax dieselolei, Marinobacter hydrocarbonoclasticus, Cycloclasticus sp. 78-ME and Thalassolituus oleivorans degraded 79±3.2%, while the addition of nutrients and a washing agent has allowed the degradation of the 69±2.6%. On the other hand, microbial community was severely affected by the addition of the washing agent and the same product seemed to inhibit the growth of the majority of strains composing the selected consortium at the tested concentration. The use of dispersant should be accurately evaluated also considering its effect on the principal actors of biodegradation.

Alcanivorax borkumensis produces an extracellular siderophore in iron-limitation condition maintaining the hydrocarbon-degradation efficiency.

Obligate marine hydrocarbonoclastic bacteria possess genetic and physiological features to use hydrocarbons as sole source of carbon and to compete for the uptake of nutrients in usually nutrient-depleted marine habitats. In the present work we have studied the siderophore-based iron uptake systems in Alcanivorax borkumensis SK2 and their functioning during biodegradation of an aliphatic hydrocarbon, tetradecane, under iron limitation conditions. The antiSMASH analysis of SK2 genome revealed the presence of two different putative operons of siderophore synthetases. Search for the predicted core structures indicated that one siderophore is clearly affiliated to the family of complex oligopeptidic siderophores possessing an Orn-Ser-Orn carboxyl motif whereas the second one is likely to belong to the family of SA (salicylic acid)-based siderophores. Analyzing the supernatant of SK2 culture, an extracellular siderophore was identified and its structure was resolved. Thus, along with the recently described membrane-associated amphiphilic tetrapeptidic siderophore amphibactin, strain SK2 additionally produces an extracellular type of iron-chelating molecule with structural similarity to pseudomonins. Comparative Q-PCR analysis of siderophore synthetases demonstrated their significant up-regulation in iron-depleted medium. Different expression patterns were recorded for two operons during the early and late exponential phases of growth, suggesting a different function of these two siderophores under iron-depleted conditions.