Computational approach for identification, characterization, three-dimensional structure modelling and machine learning-based thermostability prediction of xylanases from the genome of Aspergillus fumigatus.

Identification of thermostable and alkaline xylanases from different fungal and bacterial species have gained an interest for the researchers because of its biotechnological relevance in many industries, such as pulp, paper, and bioethanol. In this study, we have identified and characterized xylanases from the genome of the thermophilic fungus of Aspergillus fumigatus by in silico analysis. Genome data mining revealed that the A fumigatus genome has six xylanase genes that belong to GH10, GH11, GH43 glycoside hydrolase families. In general, most of the bacterial and fungal GH11 xylanases are alkaline, and GH10 xylanases are acidic; however, we found that one identified xylanase from A fumigatus that belongs to the GH10 family is alkaline while the rest are acidic. Moreover, physicochemical properties also stated that most of the xylanases identified have lower molecular weight except one that belongs to the GH43 family. Structure prediction by homology modelling gave optimized structures of the xylanases. It suggests that GH10 family structure models adapt (ß∕α) 8 barrel type, GH11 homology models adapt ß-jelly type, and the GH43 family has a fivefold ß-propeller type structure. Molecular docking of identified xylanases with xylan revealed that GH11 xylanases have strong interaction (-9.6 kcal/mol) with xylan than the GH10 (-8.5 and -9.3 kcal/mol) and GH43 (-8.8 kcal/mol). We used the machine learning approach based TAXyl server to predict the thermostability of the xylanases. It revealed that two GH10 xylanases and one GH11 xylanase are thermo-active up to 75ᵒC. We have explored the physiochemical properties responsible for maintaining thermostability for bacterial and fungal GH10 and GH11 xylanases by comparing crystal structures. All the analyzed parameters specified that GH10 xylanases from both the fungi and bacteria are more thermostable due to higher hydrogen bonds, salt bridges, and helical content.

Functional Insights Into the Role of gppA in (p)ppGpp Metabolism of Vibrio cholerae.

The stringent response, an adaptive response to nutrient limitation and exposure to xenobiotics in bacteria, is mediated by two intracellular signaling molecules, pppGpp and ppGpp, together called (p)ppGpp. The cellular level of (p)ppGpp in bacterial cells is controlled by the Rel/Spo family of proteins. In the cholera pathogen, Vibrio cholerae, (p)ppGpp metabolism is regulated by the products of at least three genes relA, spoT, and relV. In this study, we identify and characterize the function of the guanosine-5'-triphosphate 3'-diphosphate pyrophosphatase A (GppA) encoding gene gppA of V. cholerae. Genomic analysis indicates that the gppA locus is conserved in vibrios and organized as a bicistronic operon along with the rhlB gene. We engineered the genome of V. cholerae to develop different mutants devoid of GppA and/or other phosphate metabolic enzymes. Our findings indicate that in V. cholerae, GppA plays an important role in the conversion of pppGpp to ppGpp during amino acid deprivation but not during glucose starvation. Quantitative analyses of the gppA transcript level reveal its differential expression pattern at different growth phases and starvation conditions. It has been observed that the GppA deficiency during amino acid starvation condition could be complemented by overexpressing the exopolyphosphatase coding gene ppx of V. cholerae. By deletion analysis, we further demonstrate that the amino and carboxy terminal sequences flanking the Ppx-GppA motif of the GppA protein of V. cholerae are also important for its enzymatic function.

Small alarmones (p)ppGpp regulate virulence associated traits and pathogenesis of Salmonella enterica serovar Typhi.

How Salmonella enterica serovar Typhi (S. Typhi), an important human pathogen, survives the stressful microenvironments inside the gastrointestinal tract and within macrophages remains poorly understood. We report here that S. Typhi has a bonafide stringent response (SR) system, which is mediated by (p)ppGpp and regulates multiple virulence-associated traits and the pathogenicity of the S. Typhi Ty2 strain. In an iron overload mouse model of S. Typhi infection, the (p)ppGpp0 (Ty2ΔRelAΔSpoT) strain showed minimal systemic spread and no mortality, as opposed to 100% death of the mice challenged with the isogenic wild-type strain. Ty2ΔRelAΔSpoT had markedly elongated morphology with incomplete septa formation and demonstrated severely attenuated motility and chemotaxis due to the loss of flagella. Absence of the Vi-polysaccharide capsule rendered the mutant strain highly susceptible to complement-mediated lysis. The phenotypes of Ty2ΔRelAΔSpoT was contributed by transcriptional repression of several genes, including fliC, tviA, and ftsZ, as found by reverse transcriptase quantitative polymerase chain reaction and gene complementation studies. Finally, Ty2ΔRelAΔSpoT had markedly reduced invasion into intestinal epithelial cells and significantly attenuated survival within macrophages. To the best of our knowledge, this was the first study that addressed SR in S. Typhi and showed that (p)ppGpp was essential for optimal pathogenic fitness of the organism.

DksA-HapR-RpoS axis regulates haemagglutinin protease production in Vibrio cholerae.

DksA acts as a co-factor for the intracellular small signalling molecule ppGpp during the stringent response. We recently reported that the expression of the haemagglutinin protease (HAP), which is needed for shedding of the cholera pathogen Vibrio cholerae during the late phase of infection, is significantly downregulated in V. cholerae ∆dksA mutant (∆dksAVc) cells. So far, it has been shown that HAP production by V. cholerae cells is critically regulated by HapR and also by RpoS. Here, we provide evidence that V. cholerae DksA (DksAVc) positively regulates HapR at both the transcriptional and post-transcriptional levels. We show that in ∆dksAVc cells the CsrB/C/D sRNAs, required for the maintenance of intracellular levels of hapR transcripts during the stationary growth, are distinctly downregulated. Moreover, the expression of exponential phase regulatory protein Fis, a known negative regulator of HapR, was found to continue even during the stationary phase in ∆dksAVc cells compared to that of wild-type strain, suggesting another layer of complex regulation of HapR by DksAVc. Extensive reporter construct-based and quantitative reverse-transcriptase PCR (qRT-PCR) analyses supported that RpoS is distinctly downregulated at the post-transcriptional/translational levels in stationary phase-grown ∆dksAVc cells. Since HAP expression through HapR and RpoS is stationary phase-specific in V. cholerae, it appears that DksAVc is also a critical stationary phase regulator for fine tuning of the expression of HAP. Moreover, experimental evidence provided in this study clearly supports that DksAVc is sitting at the top of the hierarchy of regulation of expression of HAP in V. cholerae.

Genetic and mutational characterization of the small alarmone synthetase gene relV of Vibrio cholerae.

In Vibrio cholerae, the causative agent of cholera, products of three genes, relA, spoT and relV, govern nutritional stress related stringent response (SR). SR in bacteria is critically regulated by two intracellular small molecules, guanosine 3'-diphosphate 5'-triphosphate (pppGpp) and guanosine 3',5'-bis(diphosphate) (ppGpp), collectively called (p)ppGpp or alarmone. Evolution of relV is unique in V. cholerae because other Gram-negative bacteria carry only relA and spoT genes. Recent reports suggest that RelV is needed for pathogenesis. RelV carries a single (p)ppGpp synthetase or RelA-SpoT domain (SYNTH/RSD) and belongs to the small alarmone synthetase (SAS) family of proteins. Here, we report extensive functional characterizations of the relV gene by constructing several deletion and site-directed mutants followed by their controlled expression in (p)ppGpp(0) cells of Escherichia coli or V. cholerae. Substitution analysis indicated that the amino acid residues K107, D129, R132, L150 and E188 of the RSD region of RelV are essential for its activity. While K107, D129 and E188 are highly conserved in RelA and SAS proteins, L150 appears to be conserved in the latter group of enzymes, and the R132 residue was found to be unique in RelV. Extensive progressive deletion analysis indicated that the amino acid residues at positions 59 and 248 of the RelV protein are the functional N- and C-terminal boundaries, respectively. Since the minimal functional length of RelV was found to be 189 aa, which includes the 94 aa long RSD region, it seems that the flanking residues of the RSD are also important for maintaining the (p)ppGpp synthetase activity.

Mutational analysis of the (p)ppGpp synthetase activity of the Rel enzyme of Mycobacterium tuberculosis.

Rel(Mtb), a GTP pyrophosphokinase encoded by the Mycobacterium tuberculosis (Mtb) genome, catalyzes synthesis of (p)ppGpp from ATP and GDP(GTP) and its hydrolysis to GDP(GTP) and pyrophosphate to mediate stringent response, which helps bacteria to survive during nutrient limitation. Like other members of Rel_Spo homologs, Rel(Mtb) has four distinct domains: HD, Rel_Spo (RSD), TGS and ACT. The N-terminal HD and RSD are responsible for (p)ppGpp hydrolysis and synthesis, respectively. In this study, we have dissected the rel(Mtb) gene function and determined the minimal region essential for (p)ppGpp synthetic activity. The Rel(Mtb) and its truncated derivatives were expressed from an arabinose inducible promoter (P(BAD)), and in vivo functional analyses were done in a (p)ppGpp null Escherichia coli strain. Our results indicate that only 243 amino acids (188-430 residues) containing fragment are sufficient for Rel(Mtb) (p)ppGpp synthetic activity. The results were further confirmed by in vitro assays using purified proteins. We further characterized the RSD of Rel(Mtb) by substituting several conserved amino acids with structurally related residues and identified six such residues, which appeared to be critical for maintaining its catalytic activity. Furthermore, we have also extended our analysis to an RSD encoding gene rv1366 of Mtb, and experimental results indicated that the encoded protein Rv1366 is unable to synthesize (p)ppGpp.

Functional characterization of the stringent response regulatory gene dksA of Vibrio cholerae and its role in modulation of virulence phenotypes.

In bacteria, nutrient deprivation evokes the stringent response, which is mediated by the small intracellular signaling molecule ppGpp. In Gram negatives, the RelA enzyme synthesizes and SpoT hydrolyzes ppGpp, although the latter protein also has weak synthetase activity. DksA, a recently identified RNA polymerase binding transcription factor, acts as a coregulator along with ppGpp for controlling the stringent response. Recently, we have shown that three genes, relA, spoT, and relV, govern cellular levels of ppGpp during various starvation stresses in the Gram-negative cholera pathogen Vibrio cholerae. Here we report functional characterization of the dksA gene of V. cholerae (dksA(Vc)), coding for the protein DksA(Vc). Extensive genetic analyses of the ΔdksA(Vc) mutants suggest that DksA(Vc) is an important component involved in the stringent response in V. cholerae. Further analysis of mutants revealed that DksA(Vc) positively regulates various virulence-related processes, namely, motility, expression of the major secretory protease, called hemagglutinin protease (HAP), and production of cholera toxin (CT), under in vitro conditions. We found that DksA(Vc) upregulates expression of the sigma factor FliA (σ(28)), a critical regulator of motility in V. cholerae. Altogether, it appears that apart from stringent-response regulation, DksA(Vc) also has important roles in fine regulation of virulence-related phenotypes of V. cholerae.

Genetic components of stringent response in Vibrio cholerae.

Nutritional stress elicits stringent response in bacteria involving modulation of expression of several genes. This is mainly triggered by the intracellular accumulation of two small molecules, namely, guanosine 3'-diphosphate 5'-triphosphate and guanosine 3',5'-bis(diphosphate), collectively called (p)ppGpp. Like in other Gram-negative bacteria, the cellular level of (p)ppGpp is maintained in Vibrio cholerae, the causative bacterial pathogen of the disease cholera, by the products of two genes relA and spoT. However, apart from relA and spoT, a novel gene relV has recently been identified in V. cholerae, the product of which has been shown to be involved in (p)ppGpp synthesis under glucose or fatty acid starvation in a ∆relA ∆spoT mutant background. Furthermore, the GTP binding essential protein CgtA and a non-DNA binding transcription factor DksA also seem to play several important roles in modulating stringent response and regulation of other genes in this pathogen. The present review briefly discusses about the role of all these genes mainly in the management of stringent response in V. cholerae.

Isolation of an unusual metabolite 2-allyloxyphenol from a marine actinobacterium, its biological activities and applications.

A marine actinobacterium isolated from the Bay of Bengal, India and previously found to be producing an antimicrobial and cytotoxic terpenoid was further investigated for antimicrobial metabolites. The bacterium was preliminarily identified as a new species of the genus Streptomyces (strain MS1/7). The cell-free culture broth was extracted with n-butanol and purified using silica gel column chromatography and high-performance liquid chromatography. Molecular characterization was done using ESI mass, IR and 1H and 13C NMR spectrometry. 2-Allyloxyphenol (MW 150; C9H10O2), a synthetic drug and chemical intermediate, was obtained as a natural product for the first time. Serendipitous natural occurrence provided new insights into the synthetic molecule. 2-Allyloxyphenol was found to be inhibitory to 21 bacteria and three fungi in the minimum range 0.2-1.75 mg mL(-1) determined by agar dilution method. 2-Allyoxyphenol possesses strong antioxidant property (IC(50) 22 microg mL(-1), measured by 1, 1-diphenyl-2-picryl hydrazyl scavenging activity). Hydroxyl and allyloxy groups in 2-allyloxyphenol were responsible for antimicrobial and antioxidant activities. 2-Allyloxyphenol has marked resemblance to smoky aroma and is two to three times more active as an antimicrobial than some commercial smoke-flavour compounds. Absence of hemolytic toxicity, potential carcinogenicity, cytotoxicity and reports of toxic reactions in literature suggest possible application of 2-allyloxyphenol as a food preservative and an oral disinfectant.