Comparative genomic survey of microbial arylamine N-acetyltransferases

Authors: GAREFALAKI, V - Democritus University Of Thrace; BOURAKI, G - Democritus University Of Thrace; OLBASALIS, I - Democritus University Of Thrace; SAVVIDOU, O - Democritus University Of Thrace; KONTOMINA, E - Democritus University Of Thrace; KOTSELI, V - Democritus University Of Thrace; DAVIS, B - University Of Georgia; FELFOLDI, T - Eotvos Lorand University; MARIALIGETI, K - Eotvos Lorand University; Glenn, Anthony - Tony; BOUKOUVALA, S - Democritus University Of Thrace

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 4/15/2016
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: Introduction: Microorganisms are constantly exposed to exogenous chemical influences. Our previous genomic surveys have identified putative NAT genes across a phylogenetic spectrum of prokaryotic and eukaryotic microorganisms. We are currently pursuing two lines of investigation: The first looks into the distribution and roles of NATs in bacteria isolated from natural or polluted environments. The second focuses on NATs of plant-associated ascomycetes, demonstrating considerable functional divergence. Methods: We have surveyed >30,000 sequenced prokaryotic genomes, performing arduous reconstruction of NAT open reading frames. We perform phylogenetic analyses and experiment with tools to predict horizontal gene transfer or possible localization within gene clusters. Laboratory investigation involves 94 representative bacterial isolates, classified by 16S rRNA gene sequencing, for which we assess arylamine tolerance. Cloning of NAT genes is attempted for isolates with sequenced relatives, followed by protein expression and enzymatic investigation. Similar experiments have been performed for 13 recombinant NAT homologues of ascomycetes. Endogenous expression of those homologues is investigated using enzymatic activity assays and quantitative RT-PCR, particularly in xenobiotic challenged fungi. Results: In bacteria, our survey identified 3009 NAT genes from 141 genera of Proteobacteria, Actinobacteria, Firmicutes, Chlamidiae, Verrucomicrobia, Chloroflexi, Cyanobacteria, Nitrospinae, Planctomycetes and Spirochaetes. In archaea, only 5 NAT genes were annotated in Halobacteria. Variable tolerance of arylamine was recorded for 60 representative isolates tested. Fifteen of the cloned NAT genes generated recombinant protein that was active with acetyl- and propionyl-CoA. One NAT homologue (of the actinobacterium Tsukamurella paurometabola) additionally utilized malonyl- and succinyl-CoA as donor substrates. In fungi, functional diversification of NAT enzymes in species of Fusarium and Aspergillus was evident, demonstrating three groups of homologues. Group I homologues were found in all species and utilized acetyl-CoA and propionyl-CoA. Group II homologues were restricted to plant pathogens and were active with malonyl-CoA in Fusarium species infecting cereals; those homologues were inducible by arylamine and their function is linked to Fusarium survival of host antimicrobials. Group III homologues generated minimal activity with acyl-CoA compounds that bound non-selectively to the proteins. Conclusion(s): We speculate that microbial NATs may have evolved to perform diverse functions, potentially relevant to pathogen fitness, acetyl-CoA/propionyl-CoA intracellular balance and secondary metabolism.