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Vibrio alginolyticus, an opportunistic pathogen that causes vibriosis in miscellaneous fish species, has brought about serious economic damage to the mariculture industry in South China. The mechanism of virulence regulation in V. alginolyticus is yet not known except a Vibrio harveyi-like quorum sensing (QS) system that is established to manipulate the expression of diverse genes including those encoding virulence determinants. In this study, a new TetR family QS regulator, luxT, was identified and characterized in V. alginolyticus. The transcription of luxT gene was cell density dependent and was positively regulated by LuxU, an established QS component relaying the signal from three paralleled QS regulatory systems in V. harveyi. In addition, luxT positively regulated both luxO at transcriptional level and luxR at post-transcriptional level, which is thoroughly different from the established QS regulation mode in V. harveyi and Vibrio vulnificus. The mutant of luxT deletion produced markedly decreased total extracellular proteases and reduced motility ability compared to the wild type and the complemented strain luxT (+). The fish infection results indicated that mutation of luxT led to marginal attenuation in the virulence of V. alginolyticus, suggesting that LuxT might play a role in the fine-tuning of the virulence via QS in V. alginolyticus.

The fish pathogen Vibrio alginolyticus virulence factors and regulation mechanism are barely known except the quorum sensing regulated extracellular products. In the present study, the cleavable isotope-coded affinity tags (cICATs) were used to analyze the differentially expressed extracellular proteins of V. alginolyticus wild-type and a ΔluxO mutant, and a putative peptidase Pep was identified. Quantitative real-time reverse transcription PCR confirmed that the transcription of pep was down-regulated by LuxO. Furthermore, western blotting demonstrated that the expression of Pep was cell-density dependent and quorum sensing regulated in V. alginolyticus. The pep in-frame mutant abolished the swimming and swarming motility, suggesting that it was involved in motility regulation in V. alginolyticus. In liquid medium, the polar flagellum was observed intact in ∆pep mutant while the chemotaxis related gene cheY, which is a response regulator causing change in direction of flagellar rotation, was down-regulated in ∆pep mutant. When ∆pep mutant was grown on hard agar plate, the lateral flagella disappeared and the transcriptions of flagellar biogenesis genes flaK, fliS, lafK, and lafA were down-regulated in ∆pep mutant. These results suggested that Pep was a luxO-regulated extracellular protein and involved in the motility of V. alginolyticus., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

All members of the Vibrionaceae harbour LuxO, a response regulator that integrates outputs from various signalling systems, ultimately controlling specific traits that are crucial to the distinct biology of each species. LuxO is phosphorylated in response to low cell density, activating the transcription of a family of small RNAs called Qrrs, which in turn, control the levels of a global regulatory protein conserved within the Vibrionaceae. Although the function of each Qrr is similar, the number of qrr genes varies among the different species. Using a bioinformatics approach, we have determined the number of qrr genes in fully sequenced Vibrionaceae members. Phylogenetic analysis suggests the most recent common ancestor of all Vibrionaceae shared a single, ancestral qrr gene, which duplicated and diverged into multiple qrr genes in some present-day vibrio lineages. To demonstrate that a single qrr gene is sufficient to mediate repression of LitR, the global regulator in Vibrio fischeri, we have performed a series of genetic and phenotypic analyses of the LuxO pathway and its output. Our studies contribute to a better understanding of the ancestral state of these pathways in vibrios, as well as to the evolution and divergence of other sRNAs within different bacterial lineages., (© 2010 Blackwell Publishing Ltd.)

Vibrio alginolyticus, a gram-negative marine bacterium, is one of the causative agents of fish vibriosis. Its virulence factors and pathogenesis mechanism are barely known except for some extracellular products (ECPs), which are implicated to be regulated by quorum sensing system. In the present study, microarray was used to analyze the transcription profiles of V. alginolyticus wild-type and a deletion mutant of luxO, the pivotal regulator in Vibrio quorum sensing systems, and a putative virulence factor MviN was identified. Quantitative real-time reverse transcription PCR confirmed that the transcription of mviN was up-regulated in the luxO mutant compared to wild-type and down-regulated in the luxO-con complemented strain. Furthermore, western blotting indicated that MviN was greatly induced in the late-exponential and stationary phases of growth, demonstrating the expression of MviN was cell-density dependent and quorum sensing regulated in V. alginolyticus. The mviN null mutant displayed a much slower growth rate than wild-type, suggesting its essential role in V. alginolyticus. Western blotting also revealed that MviN was present as an extracellular protein in V. alginolyticus. When the ECPs of the mviN mutant were subjected to treat EPC cells, no cytotoxicity was observed while pathological changes of EPC cells treated by the wild-type increased with the ECPs concentration and treating time. These data showed that MviN was a LuxO-regulated component in ECPs and involved in the pathogenicity of V. alginolyticus.

In Gram-negative bacteria, the TonB/ExbB/ExbD complex is required to energize the specific high-affinity receptors mediating iron uptake processes. In fish pathogen Vibrio alginolyticus MVP01, which exhibited capacities to scavenge iron sources with various specific iron uptake systems, we identified and characterized two sets of TonB systems. In V. alginolyticus, TonB1 and TonB2 systems were arrayed as TonB1-ExbB1-ExbD1 and ExbB2-ExbD2-TonB2, respectively. The transcription of the TonB systems was regulated by the iron concentration in culture medium. While TonB1 system specifically contributed to the hemin and hemoglobin utilization in V. alginolyticus MVP01, both of the TonB systems showed abilities to support the utilization of iron sources from ferrichrome and vibrioferrin, the endogenous siderophore of V. alginolyticus. In addition, when mutants of the TonB systems were inoculated intraperitoneally into the fish Brachydanio rerio, marked attenuation in virulence of V. alginolyticus was observed, indicating that both of the TonB systems are essential for the virulence of V. alginolyticus.

Vibrio alginolyticus, a marine bacterium, is an opportunistic pathogen capable of causing vibriosis with high mortality to fishes in the South China Sea. Stress resistance is very important for its survival in the natural environment and upon infection of the host. RpoS, an alternative sigma factor, is considered as an important regulator involved in stress response and virulence in many pathogens. In this study, the rpoS gene was cloned and characterized to evaluate the role of RpoS in V. alginolyticus. The predicted protein showed high identity with other reported rpoS gene products. The in-frame deleted mutation of rpoS in V. alginolyticus led to sensitivity of the strain to ethanol, hyperosmolarity, heat, and hydrogen peroxide challenges. Further studies showed that extracellular autoinducer 2 level, four of seven detected protease activities, and cytotoxicity of extracellular products were markedly decreased in the rpoS mutant compared with that in the wild-type strain. The results indicated that the global regulator RpoS was part of the regulatory networks of virulence and LuxS quorum sensing system.

The fish pathogen Vibrio alginolyticus contains two unique flagellar systems. The LuxS quorum sensing system is reported to regulate the expression of virulence factors in a wide variety of pathogenic bacteria. Our previous work demonstrated that inactive luxS led to decreased virulence in V. alginolyticus. In this study, LuxS-dependent regulation of motility and flagella biogenesis, the potential virulence factors in V. alginolyticus, were further investigated. A luxS-deleted mutant showed deficiency in motility and flagella formation, and an intact luxS complemented the defect. Since motility is flagella dependent, V. alginolyticus flagella biogenesis related genes, including the flagella regulator genes flaK and lafK and the sub-hierarchical flagellar genes fliS and lafA, were cloned and identified. Based on quantitative real-time reverse transcription-PCR, differential expression of these genes was confirmed in wild-type and luxS mutants. Our results indicate that LuxS plays an important role in the regulation of motility and flagella biogenesis in V. alginolyticus.