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Bacteria employ secondary metabolism to combat competitors, and xenobiotic metabolism to survive their chemical environment. This project has aimed to introduce a bacterial collection enabling comprehensive comparative investigations of those functions. The collection comprises 120 strains (Proteobacteria, Actinobacteria and Firmicutes), and was compiled on the basis of the broad taxonomic range of isolates and their postulated biosynthetic and/or xenobiotic detoxification capabilities. The utility of the collection was demonstrated in two ways: first, by performing 5144 co-cultures, recording inhibition between isolates and employing bioinformatics to predict biosynthetic gene clusters in sequenced genomes of species; second, by screening for xenobiotic sensitivity of isolates against 2-benzoxazolinone and 2-aminophenol. The co-culture medium of Bacillus siamensis D9 and Lysinibacillus sphaericus DSM 28T was further analysed for possible antimicrobial compounds, using liquid chromatography-mass spectrometry (LC-MS), and guided by computational predictions and the literature. Finally, LC-MS analysis demonstrated N-acetylation of 3,4-dichloroaniline (a toxic pesticide residue of concern) by the actinobacterium Tsukamurella paurometabola DSM 20162T which is highly tolerant of the xenobiotic. Microbial collections enable "pipeline" comparative screening of strains: on the one hand, bacterial co-culture is a promising approach for antibiotic discovery; on the other hand, bioremediation is effective in combating pollution, but requires knowledge of microbial xenobiotic metabolism. The presented outcomes are anticipated to pave the way for studies that may identify bacterial strains and/or metabolites of merit in biotechnological applications.

Human NAT1 gene for N-acetyltransferase 1 modulates xenobiotic metabolism of arylamine drugs and mutagens. Beyond pharmacogenetics, NAT1 is also relevant to breast cancer. The population history of human NAT1 suggests evolution through purifying selection, but it is unclear whether this pattern is evident in other primate lineages where population studies are scarce. We report NAT1 polymorphism in 25 rhesus macaques (Macaca mulatta) and describe the haplotypic and functional characteristics of 12 variants. Seven non-synonymous single nucleotide variations (SNVs) were identified and experimentally demonstrated to compromise enzyme function, mainly through destabilization of NAT1 protein and consequent activity loss. One non-synonymous SNV (c.560G > A, p.Arg187Gln) has also been characterized for human NAT1 with similar effects. Population haplotypic and functional variability of rhesus NAT1 was considerably higher than previously reported for its human orthologue, suggesting different environmental pressures in the two lineages. Known functional elements downstream of human NAT1 were also differentiated in rhesus macaque and other primates. Xenobiotic metabolizing enzymes play roles beyond mere protection from exogenous chemicals. Therefore, any link to disease, particularly carcinogenesis, may be via modulation of xenobiotic mutagenicity or more subtle interference with cell physiology. Comparative analyses add the evolutionary dimension to such investigations, assessing functional conservation/diversification among primates.

Actinobacteria in the Tsukamurella genus are aerobic, high-GC, Gram-positive mycolata, considered as opportunistic pathogens and isolated from various environmental sources, including sites contaminated with oil, urban or industrial waste and pesticides. Although studies look into xenobiotic biotransformation by Tsukamurella isolates, the relevant enzymes remain uncharacterized. We investigated the arylamine N-acetyltransferase (NAT) enzyme family, known for its role in the xenobiotic metabolism of prokaryotes and eukaryotes. Xenobiotic sensitivity of Tsukamurella paurometabola type strain DSM 20162T was assessed, followed by cloning, recombinant expression and functional characterization of its single NAT homolog (TSUPD)NAT1. The bacterium appeared quite robust against chloroanilines, but more sensitive to 4-anisidine and 2-aminophenol. However, metabolic activity was not evident towards those compounds, presumably due to mechanisms protecting cells from xenobiotic entry. Of the pharmaceutical arylhydrazines tested, hydralazine was toxic, but the bacterium was less sensitive to isoniazid, a drug targeting mycolic acid biosynthesis in mycobacteria. Although (TSUPD)NAT1 protein has an atypical Cys-His-Glu (instead of the expected Cys-His-Asp) catalytic triad, it is enzymatically active, suggesting that this deviation is likely due to evolutionary adaptation potentially serving a different function. The protein was indeed found to use malonyl-CoA, instead of the archetypal acetyl-CoA, as its preferred donor substrate. Malonyl-CoA is important for microbial biosynthesis of fatty acids (including mycolic acids) and polyketide chains, and the corresponding enzymatic systems have common evolutionary histories, also linked to xenobiotic metabolism. This study adds to accummulating evidence suggesting broad phylogenetic and functional divergence of microbial NAT enzymes that goes beyond xenobiotic metabolism and merits investigation.

Plant-pathogenic fungi and their hosts engage in chemical warfare, attacking each other with toxic products of secondary metabolism and defending themselves via an arsenal of xenobiotic metabolizing enzymes. One such enzyme is homologous to arylamine N-acetyltransferase (NAT) and has been identified in Fusarium infecting cereal plants as responsible for detoxification of host defence compound 2-benzoxazolinone. Here we investigate functional diversification of NAT enzymes in crop-compromising species of Fusarium and Aspergillus, identifying three groups of homologues: Isoenzymes of the first group are found in all species and catalyse reactions with acetyl-CoA or propionyl-CoA. The second group is restricted to the plant pathogens and is active with malonyl-CoA in Fusarium species infecting cereals. The third group generates minimal activity with acyl-CoA compounds that bind non-selectively to the proteins. We propose that fungal NAT isoenzymes may have evolved to perform diverse functions, potentially relevant to pathogen fitness, acetyl-CoA/propionyl-CoA intracellular balance and secondary metabolism.