Integrative metagenomic and biochemical studies on rifamycin ADP-ribosyltransferases discovered in the sediment microbiome

Abstract

Antibiotic resistance is a serious and growing threat to human health. The environmental microbiome is a rich reservoir of resistomes, offering opportunities to discover new antibiotic resistance genes. Here we demonstrate an integrative approach of utilizing gene sequence and protein structural information to characterize unidentified genes that are responsible for the resistance to the action of rifamycin antibiotic rifampin, a first-line antimicrobial agent to treat tuberculosis. Biochemical characterization of four environmental metagenomic proteins indicates that they are adenosine diphosphate (ADP)-ribosyltransferases and effective in the development of resistance to FDA-approved rifamycins. Our analysis suggests that even a single residue with low sequence conservation plays an important role in regulating the degrees of antibiotic resistance. In addition to advancing our understanding of antibiotic resistomes, this work demonstrates the importance of an integrative approach to discover new metagenomic genes and decipher their biochemical functions.

Genomic epidemiology of rifampicin ADP-ribosyltransferase (Arr) in the Bacteria domain

“…Here, we observed that this H-Y-[QED] motif is highly conserved in almost all hundreds of Arr sequences analyzed, indicating its importance in Arr activity, and suggesting that these Arr sequences could be functional. Indeed, the Arr enzymes already functionally characterized (Arr-ms, Arr-2 to Arr-8, Arr-cb, Arr-sc, Arr_Mab, Arr-wd1-4) 2 , 4 , 6 – 8 and those of the present study were observed along the phylogenetic tree, showing that despite the sequence diversity, even in the RIF binding sites, their activities against rifamycins are maintained. These patterns were observed in sequences carried by bacteria belonging to high and low antibiotic-impacted environments.…”

“…Highly conserved residues (≥ 90%), considering all Arr sequences analyzed in the phylogeny, are represented below the alignment by the fuchsia blocks. References: Arr-2, Arr-sc, and Arr-ms 1 ; Arr-4 and Arr-5 6 ; Arr-8 8 ; Arr-w1, Arr-w2, Arr-w3, and Arr-w4 4 ; ArrMab 7 ; Arr-cb 2 . …”

“…Interestingly, one of the Arr Proteobacteria clades, mainly formed by Arr encoded in clinical pathogenic Proteobacteria plasmids, has a phylogenetic relationship with a clade that encompasses only chromosomal Arr of environmental Actinobacteria , which suggests, in this case, the environment as a source of Arr currently impacting the clinic. This scenario substantially enlarged the occurrence and distribution of Arr within the Bacteria domain, since the few previous studies on this issue considered a restricted set of bacteria carrying Arr 1 , 4 .…”

“…Arr is a small enzyme (16 kDa) with low sequence similarity to other known ARTs, however, shares structural homology with them. The arr gene was firstly described in Mycolicibacterium smegmatis ( arr -ms or arr -1) 3 , and since then other alleles have been identified in some organisms of Actinobacteria , Proteobacteria , and Firmicutes phyla, in addition to marine and soil microbiomes 1 , 2 , 4 , 5 . However, Arr current distribution and prevalence in the Bacteria domain is unknown.…”

“…The genomic context of the arr gene might vary by organism; for instance, while Arr-ms is chromosomally encoded in the saprophyte organism M. smegmatis , Arr-2 has been associated with mobilizable and mobile elements (transposons and integrons) of Gram-negative pathogenic bacteria (e.g., Pseudomonas aeruginosa and Klebsiella pneumoniae ) 1 . Besides these Arr enzymes, few others (Arr-3 to Arr-8, Arr-cb, Arr-sc, Arr_Mab) have already been characterized 2 , 4 , 6 – 8 . Although Arr has been associated primarily with resistance to clinically important rifamycin antibiotics, some authors suggested that it may be able to act on other substrates and metabolic pathways 1 , 5 , 9 .…”