Detection of different quorum-sensing signal molecules in a virulent Edwardsiella tarda strain LTB-4

Aims: The aim of this study was to elucidate the potential quorum-sensing (QS) signal molecules of an emerging pathogen (Edwardsiella tarda strain LTB-4) of cultured turbot (Scophthalmus maximus). Methods and Results: A sensitive and rapid double-layer plate method using biosensor strain Agrobacterium tumefaciens KYC55 was developed to detect the N-acylhomoserine lactone (AHL)-related compounds in bacteria. LTB-4 was found to have two QS systems, one was based on the AHLs and the other was based on the autoinducer-2 (AI-2). The AI-2 activity produced by LTB-4 was growth phase dependent and topped at OD600 of 1 center dot 0. The protocol to detect cholerae autoinducer 1 (CAI-1) activity in bacteria was modified, lowering the background luminescence of biosensor strain Vibrio harveyi JAF375. CAI-1 activity could not be detected in LTB-4. Conclusion: Edwardsiella tarda LTB-4 produced at least four kinds of AHLs during its whole growth phase. In comparison with the AHL-inducing QS, AI-2 may be the first predominant signal, functioning at early exponential phase. LTB-4 did not produce any CAI-1 activity. Significance and Impact of the Study: Different QS signal molecules of Edw. tarda LTB-4 were clarified by improved bioassays. In contrast to earlier studies detecting two types of AHLs, strain LTB-4 produced at least four kinds of AHLs, which seemed to be C-4-HSL, C-6-HSL, 3-oxo-C-6-HSL and an uncharacterized AHL molecule

The use of selected bacteria and yeasts to control vibrio spp. in live food

Vibrio species are a significant causative of mass mortality in mariculture worldwide, which can quickly accumulate in live food and transmit into the larval gut. With restrictions on the use of antibiotics in aquaculture, finding a proper solution to reduce the risk of Vibriosis is vital. This study aimed to evaluate the susceptibility of Vibrio harveyi, V. campbellii, V. anguillarum, and V. parahaemolyticus to twenty-six bacterial and yeast strains and use the beneficial ones to enrich live food (Branchiopod, Artemia franciscana, rotifer, Brachionus plicatilis and copepod, Tigriopus japonicus). Thus, a modified disk diffusion method was applied. After a susceptibility assay, the bacteria and yeast beneficial in suppressing the Vibrio species were labeled by fluorescent stain and used to measure the accumulation potential in different live foods. Also, the beneficial bacteria and yeast were used to enrich live foods, and then the count of loaded Vibrio was estimated after 5, 10, 15, and 20 h by the serial dilution method. From the total bacteria and yeast strains that were used, Candida parapsilosis, Pseudoalteromonas flavipulchra, Lactobacillus sakei, Bacillus natto, and B. amyloliquefaciens inhibited all four Vibrio species. The results of microbial labeling showed that L. sakei in Artemia, C. parapsilosis in rotifers, and V. harveyi in copepods had the highest accumulation rate. The results of the estimation of loaded Vibrio in different live foods also showed that the use of beneficial bacteria and yeast each significantly reduced the count of Vibrio. Application of bacteria and yeast to suppress pathogenic Vibrio maybe a sustainable method for preventing this pathogen from harmfully invading aquaculture and may also aid in reducing the chances of antibiotic resistance in pathogenic Vibrio

Progress in strategies for sequence diversity library creation for directed evolution

Protein engineering has been the most attractive strategy for biologists to redesign enzymes. As the simplest technique of protein engineering, directed evolution has been applied to many fields, such as industry, agriculture and medicine. An experiment of directed evolution comprises mutant libraries creation and screening or selection for enzyme variants with desired properties. Therefore, a successful application of directed evolution depends on whether or not one can generate a quality library and perform effective screening to find the desired properties. Directed evolution is already increasingly used in many laboratories to improve protein stability and activity, alter enzyme substrate specificity, or design new activities. Meanwhile, many more effective novel strategies of mutant library generation and screening or selection have emerged in recent years, and will continue to be developed. Combining computational/rational design with directed evolution has been developed as more available means to redesign enzymes.Keywords: Protein engineering, directed evolution, sequence diversity creation, novel strategy, computational design, rational desig

Validation of reference genes for gene expression studies in tartary buckwheat (Fagopyrum tataricum Gaertn.) using quantitative real-time PCR

Quantitative real-time reverse transcriptase polymerase chain reaction is a sensitive technique for quantifying gene expression levels. By implementing three distinct algorithms (geNorm, normFinder and BestKeeper), we have validated the stability of the expression of seven candidate reference genes in tartary buckwheat, including FtSAND, FtCACS, FtExpressed1, FtGAPDH, FtActin, FtEF-1a and FtH3. In this study, the results indicated that FtCACS and FtSAND were the best reference genes for ‘abiotic cotyledons’, FtExpressed1 and FtEF-1α were the best reference genes for aluminium treatment, FtCACS and FtExpressed1 performed the best for the immature seed stage, FtCACS was best for the abiotic treatment, and FtH3 appeared to be the most suitable reference gene for the abiotic treatment in hypocotyls and all samples in this study. In contrast, FtActin and FtGAPDH are unsuitable genes. Our findings offer additional stable reference genes for gene expression research on tartary buckwheat at the immature seed stage and under abiotic treatment

Distribution of five vibrio virulence-related genes among Vibrio harveyi isolates

Short Communicatio

Effects of feeding regime and probionts on the diverting microbial communities in rotifer Brachionus culture

Rotifer growth performance and microbial community changes associated with rotifer cultures were monitored while different feed types (Nannochloropsis oculata paste and the commercial yeast based feed CS-3000), different regimes (daily changes, changes per batch and no changes) and mixtures of three probionts (Phenylobacterium sp.; Gluconobacter sp. and Paracoccus denitrificans) were provided. It was shown that the dominant bacterial species in the cultures receiving either N. oculata or CS-3000 were different. However, in cultures receiving both feeds (either switching between feeds on a daily basis or on a batch basis), a high similarity in microbial community fingerprint was found. The presence of probionts was detected by the end of four batch culture cycles in spite of strong shifts of the bacterial community. By group discriminant analysis, it was found that Phenylobacterium sp. and Paracoccus sp. contributed positively to the CS-3000-fed group, while Gluconobacter sp. contributed positively to the N. oculata-fed group, although they did not appear as very dominant species