Mining of two novel aldehyde dehydrogenases (DHY-SC-VUT5 and DHY-G-VUT7) from metagenome of hydrocarbon contaminated soils

Background Aldehyde dehydrogenases are vital for aerobic hydrocarbon degradation and is involved in the last step of catalysing the oxidation of aldehydes to carboxylic acids. With the global increase in hydrocarbon pollution of different environments, these enzymes have the potential to be used in enzymatic bioremediation applications. Results Fifteen fosmid clones with hydrocarbon degrading potential were functionally screened to identify dehydrogenase enzymes. Accordingly, the fosmid insert of the positive clones were sequenced using PacBio next generation sequencing platform and de novo assembled using CLC Genomic Work Bench. The 1233 bp long open reading frame (ORF) for DHY-SC-VUT5 was found to share a protein sequence similarity of 97.7% to short-chain dehydrogenase from E. coli. The 1470 bp long ORF for DHY-G-VUT7 was found to share a protein sequence similarity of 23.9% to glycine dehydrogenase (decarboxylating) (EC 1.4.4.2) from Caulobacter vibrioides (strain NA1000 / CB15N) (Caulobacter crescentus). The in silico analyses and blast against UNIPROT protein database with the stated similarity show that the two dehydrogenases are novel. Biochemical characterization revealed, that the highest relative activity was observed at substrate concentrations of 150 mM and 50 mM for DHY-SC-VUT5 and DHY-G-VUT7, respectively. The Km values were found to be 13.77 mM with a Vmax of 0.009135 μmol.min− 1 and 2.832 mM with a Vmax of 0.005886 μmol.min− 1 for DHY-SC-VUT5 and DHY-G-VUT7, respectively. Thus, a potent and efficient enzyme for alkyl aldehyde conversion to carboxylic acid. Conclusion The microorganisms overexpressing the novel aldehyde dehydrogenases could be used to make up microbial cocktails for biodegradation of alkanes. Moreover, since the discovered enzymes are novel it would be interesting to solve their structures by crystallography and explore the downstream applications.

Reevaluating the Fusobacterium Virulence Factor Landscape

ABSTRACTFusobacteríum are Gram-negative, anaerobic, opportunistic pathogens involved in multiple diseases, including the oral pathogen Fusobacterium nucleatum being linked to the progression and severity of colorectal cancer. The global identification of virulence factors in Fusobacterium has been greatly hindered by a lack of properly assembled and annotated genomes. Using newly completed genomes from nine strains and seven species of Fusobacterium, we report the identification and correction of virulence factors from the Type 5 secreted autotransporter and FadA protein families, with a focus on the genetically tractable strain F. nucleatum subsp. nucleatum ATCC 23726 and the classic typed strain F. nucleatum subsp. nucleatum ATCC 25586. Within the autotransporters, we employed protein sequence similarity networks to identify subsets of virulence factors, and show a clear differentiation between the prediction of outer membrane adhesins, serine proteases, and proteins with unknown function. These data have defined protein subsets within the Type 5a effectors that are present in predicted invasive strains but are broadly lacking in passively invading strains; a key phenotype associated with Fusobacterium virulence. However, our data shows that prior bioinformatic analysis that predicted species of Fusobacterium to be non-¡nvasive can indeed invade human cells, and that pure phylogenetic analysis to determine the virulence within this bacterial genus should be used cautiously and subsequently paired with experiments to validate these hypotheses. In addition, we provide data that show a complex interplay between autotransporters, MORN2 domain containing proteins, and FadA adhesins that we hypothesize synergistically contribute to host cell interactions and invasion. In summary, we report that accurate open reading frame annotations using complete Fusobacterium genomes, in combination with experimental validation of invasion, redefines the repertoire of virulence factors that could be contributing to the species specific pathology of multiple Fusobacterium induced infections and diseases.IMPORTANCEFusobacterium are emerging pathogens that contribute to the progression and severity of multiple mammalian and human infectious diseases, including colorectal cancer. Despite a validated connection with disease, a limited number of proteins have been characterized that define a direct molecular mechanism for pathogenesis in a diverse range of host tissue infections. We report a comprehensive examination of virulence associated protein families in multiple Fusobacterium species, and show that complete genomes facilitate the correction and identification of multiple, large Type 5a secreted autotransporter genes in previously misannotated or fragmented genomes. In addition, we use protein sequence similarity networks and human cell invasion experiments to show that previously predicted non-invasive strains can indeed enter human cells, and that this is likely due to the expansion of specific virulence proteins that drive F. nucleatum infections and disease.