The prediction of the excitation and the emission maxima of green fluorescent protein (GFP) chromophores were investigated by a quantitative structure-property relationship study. A data set of 19 GFP color variants and an additional data set consisting of 29 synthetic GFP chromophores were collected from the literature. Artificial neural network implementing the back-propagation algorithm was employed. The proposed computational approach reliably predicted the excitation and the emission maxima of GFP chromophores with correlation coefficient exceeding 0.9. The usefulness of quantum chemical descriptors was revealed by a comparative study with other molecular descriptors. Assignment of appropriate protonation state of the chromophore for the GFP color variants data set was shown to be necessary for good predictive performance. Results suggest that the confinement of the GFP chromophore has no significant influence on the predictive performance of the data set used. A comparative investigation with the traditional modeling methods, particularly multiple linear regression and partial least squares, reveals that artificial neural network is the most suitable modeling approach for the GFP spectral properties. It is anticipated that this methodology has great potential in accelerating the design and engineering of novel GFP color variants of scientific or industrial interest.

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Cell to cell communication facilitated by chemical signals plays crucial roles in regulating various cellular functions in bacteria. Indole, one such signaling molecule has been demonstrated to control various bacterial phenotypes such as biofilm formation and virulence in diverse bacteria including Vibrio cholerae. The present study explores some key factors involved in indole production and the subsequent pathogenesis of V. cholerae. Indole production was higher at 37°C than at 30°C, although the growth at 37°C was slightly higher. A positive correlation was observed between indole production and biofilm formation in V. cholerae. Maximum indole production was detected at pH 7. There was no significant difference in indole production between clinical and environmental V. cholerae isolates, although indole production in one environmental isolate was significantly different. Both growth and indole production showed relevant changes with differences in salinity. An indole negative mutant strain was constructed using transposon mutagenesis and the direct effect of indole on the virulence of V. cholerae was evaluated using Galleria mellonella larvae model. Comparison to the wild type strain, the mutant significantly reduced the mortality of G. mellonella larvae which regained its virulence after complementation with exogenous indole. A gene involved in indole production and the virulence of V. cholerae was identified.

A novel solvent-exposed analyte channel, generated by F165G substitution, on the surface of green fluorescent protein (designated His6GFPuv/F165G) was successfully discovered by the aid of molecular modeling software (PyMOL) in conjunction with site-directed mutagenesis. Regarding the high predictive performance of PyMOL, two pore-containing mutants namely His6GFPuv/H148G and His6GFPuv/H148G/F165G were also revealed. The pore sizes of F165G, H148G, and the double mutant H148G/F165G were in the order of 4, 4.5 and 5.5 Å, respectively. These mutants were subjected to further investigation on the effect of small analytes (e.g. metal ions and hydrogen peroxide) as elucidated by fluorescence quenching experiments. Results revealed that the F165G mutant exhibited the highest metal sensitivity at physiological pH. Meanwhile, the other 2 mutants lacking histidine at position 148 had lower sensitivity against Zn2+ and Cu2+ than those of the template protein (His6GFPuv). Hence, a significant role of this histidine residue in mediating metal transfer toward the GFP chromophore was proposed and evidently demonstrated by testing in acidic condition. Results revealed that at pH 6.5 the order of metal sensitivity was found to be inverted whereby the H148G/F165G became the most sensitive mutant. The dissociation constants (Kd) to metal ions were in the order of 4.88×10-6 M, 16.67×10-6 M, 25×10-6 M, and 33.33×10-6 M for His6GFPuv/F165G, His6GFPuv, His6GFPuv/H148G/F165G and His6GFPuv/H148G, respectively. Sensitivity against hydrogen peroxide was in the order of H148G/F165G > H148G > F165G indicating the crucial role of pore diameters. However, it should be mentioned that H148G substitution caused a markedly decrease in pH- and thermo-stability. Taken together, our findings rendered the novel pore of GFP as formed by F165G substitution to be a high impact channel without adversely affecting the intrinsic fluorescent properties. This opens up a great potential of using F165G mutant in enhancing the sensitivity of GFP in future development of biosensors.

Bacterial communication system known as quorum-sensing (QS) is a pivotal system for bacterial survival, adaptation and pathogenesis. Members in the multicellular community may synthesize or acquire a signaling molecule in order to elicit downstream cellular processes. Roles of indole and derivatives, a new class of quorum-sensing signal molecules, in various bacterial physiologies and virulence have been reported recently. Indole is normally found in mammal gastrointestinal tract as a metabolite of tryptophan metabolism by microbiota. Therefore, interspecies connection via indole signaling among commensal bacteria and enteric pathogens could be anticipated. Effects of indole exposure on the virulence of Listeria monocytogenes were investigated by phenotypic and molecular approaches. Results demonstrated that synthetic indole and indole-rich conditioned medium significantly diminished biofilm formation and related virulence of L. monocytogenes including motility, cell aggregation and exopolysaccharide (EPS) production. Transcript levels of virulence-associated (pssE, dltA, flaA, fliI, motB, agrA and hly) and regulatory-genes (codY, sigB, prfA and gmaR) were substantially down-regulated in indole-treated cells. Only mogR gene encoding for a repressor of motility genes was up-regulated after indole exposure. Our findings raise the possibility that L. monocytogenes may acquire indole signaling from gut microbiota for resource-effective adaptation upon transition to new environment.

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Green fluorescent protein (GFP) is frequently utilized for metal ion detection and quantification. To improve the metal binding potential of GFP, three residues (N146, F165, and L201) were substituted to histidines. Each variant responded differently upon interaction with metal ions. More than 80% of N146H, having the most accessible surface area, could bind to immobilized metal ions. However, only F165H exhibited significant differences in quenching by soluble metal ions (22% fluorescence decrease) in comparison with the template protein (12%). These findings can be utilized for designing GFP variants for metal binding and sensor applications.

Background Acute hepatopancreatic necrosis syndrome (AHPND) caused by Vibrio parahaemolyticus strain (VPAHPND) impacts the shrimp industry worldwide. With the increasing problem of antibiotic abuse, studies on quorum sensing (QS) system and anti-QS compounds bring potential breakthroughs for disease prevention and treatment. Methods In this study, the cell-free culture supernatant (CFCS) and its extract of V. alginolyticus BC25 were investigated for anti-QS activity against a reporter bacteria, Chromobacterium violaceum DMST46846. The effects of CFCS and/ or extract on motility, biofilm formation and extracellular polymeric substances (EPSs) of VPAHPND PSU5591 were evaluated. Moreover, the effects of V. alginolyticus BC25 on virulence of VPAHPND PSU5591 were investigated by shrimp challenge test. The potentially active anti-QS compounds presented in the extract and effect on gene expression of VPAHPND PSU5591 were identified. Results The CFCS of V. alginolyticus BC25 and its extract showed a significant anti-QS activity against the reporter bacteria as well as swimming and swarming motilities, biofilms, and EPSs production by VPAHPND PSU5591. Transcriptome analysis revealed that V. alginolyticus BC25 extract significantly reduced the flagella genes involved in biofilm formation and iron-controlled virulence regulatory gene of VPAHPND PSU5591. Whereas, the LuxR family transcriptional regulator gene, c-factor, a cell-cell signaling gene, and capsular polysaccharide were up-regulated. The potentially active anti-QS compounds identified in extract were Cyclo-(L-Leu-L-Pro), and Cyclo-(L-Phe-L-Pro). Furthermore, V. alginolyticus BC25 enhanced disease resistance against VPAHPND PSU5591 in tested shrimp larvae. Conclusion These findings suggest that V. alginolyticus BC25 could provide natural anti-QS and anti-biofilms compounds and has great ability to be used as biocontrol agent against VPAHPND infection in shrimp aquaculture.