Genome mining and characterisation of a novel transaminase with remote stereoselectivity.

Microbial enzymes from pristine niches can potentially deliver disruptive opportunities in synthetic routes to Active Pharmaceutical Ingredients and intermediates in the Pharmaceutical Industry. Advances in green chemistry technologies and the importance of stereochemical control, further underscores the application of enzyme-based solutions in chemical synthesis. The rich tapestry of microbial diversity in the oceanic ecosystem encodes a capacity for novel biotransformations arising from the chemical complexity of this largely unexplored bioactive reservoir. Here we report a novel ω-transaminase discovered in a marine sponge Pseudovibrio sp. isolate. Remote stereoselection using a transaminase has been demonstrated for the first time using this novel protein. Application to the resolution of an intermediate in the synthesis of sertraline highlights the synthetic potential of this novel biocatalyst discovered through genomic mining. Integrated chemico-genomics revealed a unique substrate profile, while molecular modelling provided structural insights into this 'first in class' selectivity at a remote chiral centre.

Bile acids destabilise HIF-1α and promote anti-tumour phenotypes in cancer cell models.

BACKGROUND: The role of the microbiome has become synonymous with human health and disease. Bile acids, as essential components of the microbiome, have gained sustained credibility as potential modulators of cancer progression in several disease models. At physiological concentrations, bile acids appear to influence cancer phenotypes, although conflicting data surrounds their precise physiological mechanism of action. Previously, we demonstrated bile acids destabilised the HIF-1α subunit of the Hypoxic-Inducible Factor-1 (HIF-1) transcription factor. HIF-1 overexpression is an early biomarker of tumour metastasis and is associated with tumour resistance to conventional therapies, and poor prognosis in a range of different cancers. METHODS: Here we investigated the effects of bile acids on the cancer growth and migratory potential of cell lines where HIF-1α is known to be active under hypoxic conditions. HIF-1α status was investigated in A-549 lung, DU-145 prostate and MCF-7 breast cancer cell lines exposed to bile acids (CDCA and DCA). Cell adhesion, invasion, migration was assessed in DU-145 cells while clonogenic growth was assessed in all cell lines. RESULTS: Intracellular HIF-1α was destabilised in the presence of bile acids in all cell lines tested. Bile acids were not cytotoxic but exhibited greatly reduced clonogenic potential in two out of three cell lines. In the migratory prostate cancer cell line DU-145, bile acids impaired cell adhesion, migration and invasion. CDCA and DCA destabilised HIF-1α in all cells and significantly suppressed key cancer progression associated phenotypes; clonogenic growth, invasion and migration in DU-145 cells. CONCLUSIONS: These findings suggest previously unobserved roles for bile acids as physiologically relevant molecules targeting hypoxic tumour progression.

Aspirated bile: a major host trigger modulating respiratory pathogen colonisation in cystic fibrosis patients.

Chronic respiratory infections are a leading global cause of morbidity and mortality. However, the molecular triggers that cause respiratory pathogens to adopt persistent and often untreatable lifestyles during infection remain largely uncharacterised. Recently, bile aspiration caused by gastro-oesophageal reflux (GOR) has emerged as a significant complication associated with respiratory disease, and cystic fibrosis (CF) in particular. Based on our previous finding that the physiological concentrations of bile influence respiratory pathogens towards a chronic lifestyle in vitro, we investigated the impact of bile aspiration on the lung microbiome of respiratory patients. Sputum samples (n = 25) obtained from a cohort of paediatric CF patients were profiled for the presence of bile acids using high-resolution liquid chromatography-mass spectrometry (LC-MS). Pyrosequencing was performed on a set of ten DNA samples that were isolated from bile aspirating (n = 5) and non-bile aspirating (n = 5) patients. Both denaturing gradient gel electrophoresis (DGGE) and pyrosequencing revealed significantly reduced biodiversity and richness in the sputum samples from bile aspirating patients when compared with non-aspirating patients. Families and genera associated with the pervasive CF microbiome dominated aspirating patients, while bacteria associated with the healthy lung were most abundant in non-aspirating patients. Bile aspiration linked to GOR is emerging as a major host trigger of chronic bacterial infections. The markedly reduced biodiversity and increased colonisation by dominant proteobacterial CF-associated pathogens observed in the sputum of bile aspirating patients suggest that bile may play a major role in disease progression in CF and other respiratory diseases.

Genomic comparisons of Salmonella enterica serovar Dublin, Agona, and Typhimurium strains recently isolated from milk filters and bovine samples from Ireland, using a Salmonella microarray.

Salmonella-induced enterocolitis is the leading food-borne illness with a lethal outcome and causes millions of cases of gastroenteritis each year. We examined genomic variation among 12 environmental, veterinary, and clinical Salmonella enterica serovar Dublin, Agona, and Typhimurium strains isolated in Ireland between 2000 and 2003, as well as two clinical isolates from Canada and four archival isolates, which belonged to serovars Dublin and Agona. Using DNA-DNA hybridization to a microarray consisting of most of the predicted protein-encoding sequences of the S. enterica serovar Typhimurium LT2 genome, we identified a number of genomic regions that were absent in one or more serovars. The 34 genomic regions encoded proteins involved in sugar metabolism, transport, fimbrial and phage biogenesis, and transcriptional regulation, as well as inner and outer membrane-associated proteins. Two of the four prophages identified in strain LT2, prophages Gifsy-1 and Gifsy-2, were present in all six serovar Typhimurium strains examined. Prophage Fels-1 was absent from all 18 isolates examined, and Fels-2 was completely absent from the serovar Typhimurium isolates and the Salmonella Reference Collection B serovar Dublin strain Du2. All five Salmonella pathogenicity islands were present in all isolates. Plasmid pSLT was absent from all serovar Agona isolates, and only homologues of the spv genes were present in eight of the nine serovar Dublin strains. Only limited intraserovar diversity was found among the nine serovar Dublin, three serovar Agona, and six serovar Typhimurium isolates examined even though these isolates had extensive geographic, temporal, and source differences.

Molecular typing of epidemic and nonepidemic Vibrio cholerae isolates and differentiation of V. cholerae and V. mimicus isolates by PCR-single-strand conformation polymorphism analysis.

AIMS: To examine the utility of polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP) analysis to differentiate epidemic and nonepidemic Vibrio cholerae isolates as well as to differentiate V. cholerae and Vibrio mimicus isolates. METHODS AND RESULTS: By both PCR-restriction fragment length polymorphism (RFLP) and PCR-SSCP analysis of groEL-I on chromosome 1 and groEL-II on chromosome 2, V. cholerae isolates gave distinct profiles compared with V. mimicus isolates. In addition, PCR-SSCP analysis of groEL-I and groEL-II could differentiate between V. cholerae epidemic and nonepidemic isolates. Interestingly, the relationships among strains based on groEL-I from chromosome 1 and groEL-II from chromosome 2 were congruent with each other, highlighting the conserved evolutionary history of both chromosomes in this species. CONCLUSIONS: PCR-SSCP is a powerful typing technique, which has the ability to differentiate V. cholerae and V. mimicus isolates. The epidemic V. cholerae O1/O139 serogroup isolates represent a clonal complex distinct from non-O1/non-O139 isolates that can be identified by PCR-SSCP analysis. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlights the effectiveness of using reliable molecular typing methods and in particular PCR-SSCP, to identify genetic variation among V. cholerae and V. mimicus isolates.

Molecular characterisation and expression analysis of the first hemicellulase gene (bxl1) encoding beta-xylosidase from the thermophilic fungus Talaromyces emersonii.

The gene coding for beta-xylosidase, bxl1, has been cloned from the thermophilic filamentous fungus, Talaromyces emersonii. This is the first report of a hemicellulase gene from this novel source. At the genomic level, bxl1 consists of an open reading frame of 2388 nucleotides with no introns that encodes a putative protein of 796 amino acids. The bxl1 translation product contains a signal peptide of 21 amino acids that yields a mature protein of 775 amino acids, with a predicted molecular mass of 86.8 kDa. The deduced amino acid sequence of bxl1 exhibits considerable homology with the primary structures of the Aspergillus niger, Aspergillus nidulans, Aspergillus oryzae, and Trichoderma reesei beta-xylosidase gene products, and with some beta-glucosidases, all of which have been classified as Family 3 glycosyl hydrolases. Northern blot analysis of the bxl1 gene indicates that it is induced by xylan and methyl-beta-D-xylopyranoside. D-Xylose induced expression of bxl1 but was shown to repress induction of the gene at high concentrations. The presence of six CreA binding sites in the upstream regulatory sequence (URS) of the bxl1 gene indicates that the observed repression by D-glucose may be mediated, at least partly, by this catabolite repressor.