Arsenic resistance genes in nocardioform bacteria

Abstract

Arsenic contamination is becoming a global dilemma making it imperative to study natural biomechanisms involved in arsenic detoxification in humans and other living organisms to gain insight in ways how arsenic poisoning can be treated. Arsenic resistance genes are being detected in ever increasing microorganisms with diverse gene organizations. Here, in this report the presence of ars genes in Rhodococcus rhodochrous, Arthrobacter oxydans, and Gordonia rubropertincta was investigated. Genomic libraries of these AsR actinomycete species were constructed and then screened for arsenic resistance and unique carbon source utilization genes. More than one hundred positive arsenate tolerant clones were obtained from a partially Pst I digested library of Gordonia rubropertincta of which ten random clones (pKL1-10) clearly showed the arsenic resistance phenotype in an AsS Gram-positive recipient strain. Nine of these ten clones demonstrated a conserved 3.5 kbp DNA fragment with only clone 1 containing a 5100 bp nucleotide segment. Restriction mapping and subcloning was performed on the designated pKL335 and pKL1 recombinant plasmid constructs after which the minimum DNA required for conferring arsenic resistance were sent for sequencing. Only the pKL316 and pKL120 subclones were successfully sequenced as putative secondary structures prevented the sequencing of the rest of pKL335 and pKL1. Four and three putative cistrons were predicted from the edited contiguous sequences of pKL335(ORF1-4) and pKL120(ORF5-7), respectively. Blastn,x,p and CD alignment searches revealed that ORF1 was a putative Acr3-type ArsB homolog, ORF2,3,4 and 7 showed significant homology with Trx-type ArsCs, and ORF5 and 6 are flavin-binding monooxygenase and thioredoxin reductase analogs, respectively. From the alignment data it can be deduced that pKL335 carries an arsRBCCC operon structure, such as C. efficiens YS-314, and that pKL1 probably comprises a unique arsRBCCCTO gene organization. This is only the third report of a thioredoxin reductase and a flavoprotein detected within an arsenic resistant gene cluster. Multiple ArsCs, an ArsT/TrxB and an ArsO/FMO-like homolog seems to be a unique feature amongst high GC-rich Gram(+) bacteria.