Arsenic Resistance inHalobacteriumsp. Strain NRC-1 Examined by Using an Improved Gene Knockout System

Abstract

The genome sequence of Halobacterium sp. strain NRC-1 encodes genes homologous to those responsible for conferring resistance to arsenic. These genes occur on both the large extrachromosomal replicon pNRC100 (arsADRC and arsR2M) and on the chromosome (arsB). We studied the role of these ars genes in arsenic resistance genetically by construction of gene knockouts. Deletion of the arsADRC gene cluster in a Halobacterium NRC-1 ⌬ura3 strain resulted in increased sensitivity to arsenite and antimonite but not arsenate. In contrast, knockout of the chromosomal arsB gene did not show significantly increased sensitivity to arsenite or arsenate. We also found that knockout of the arsM gene produced sensitivity to arsenite, suggesting a second novel mechanism of arsenic resistance involving a putative arsenite(III)-methyltransferase. These results indicate that Halobacterium sp. strain NRC-1 contains an arsenite and antimonite extrusion system with significant differences from bacterial counterparts. Deletion analysis was facilitated by an improved method for gene knockouts/replacements in Halobacterium that relies on both selection and counterselection of ura3 using a uracil dropout medium and 5-fluoroorotic acid. The arsenite and antimonite resistance elements were shown to be regulated, with resistance to arsenic in the wild type inducible by exposure to a sublethal concentration of the metal. Northern hybridization and reverse transcription-PCR analyses showed that arsA, arsD, arsR, arsM, arsC, and arsB, but not arsR2, are inducible by arsenite and antimonite. We discuss novel aspects of arsenic resistance in this halophilic archaeon and technical improvements in our capability for gene knockouts in the genome.The halophilic archaeon (haloarchaeon) Halobacterium sp. strain NRC-1 is an excellent model for postgenomic analysis of heavy metal resistance. Its genome is completely sequenced, and a large number of genetic tools are available for characterization of this extreme halophile (3, 9, 10). It is easily grown in the laboratory in hypersaline medium containing about a 10-fold concentration of seawater (2), and its natural environment is usually rich in heavy metals, many of which are toxic to cells. The genome sequence of Halobacterium sp. strain NRC-1 revealed multiple putative metal ion transporter genes, including arsenic, cadmium, copper, cobalt, zinc, and iron (9), indicating an excellent ability to handle metal ions in its environment. However, none of these hypothetical genes has been shown to be functional, and very few studies have been directed at the understanding of heavy metal resistance in haloarchaea.The Halobacterium sp. strain NRC-1 genome contains a 2-Mb chromosome and two megaplasmids/minichromosomes, pNRC100 and pNRC200 (191 and 365 kb, respectively) (6-9).

Vertical stratification of microbial communities in the Red Sea revealed by 16S rDNA pyrosequencing

“…PCR amplification of the V3 region of the 16S rRNA gene The total DNA in replicate samples was pooled together for PCR using the forward primer U341F (5 0 -CCTACGGGRSGCAGCAG-3 0 ) (Hansen et al, 1998) and reverse primer R685 (5 0 -ATCTACGC ATTTCACCGCTAC-3 0 ) for bacteria (Wang et al, 2004), and the forward primer A344F (5 0 -AYGGGGY GCASCAGGSG-3 0 ) and reverse primer A519R (5 0 -GG TDTTACCGCGGCKGCTG-3 0 ) for archaea (Teske and Sorensen, 2007) in separate reactions. A set of sixnucleotide (nt) barcodes was designed and added to the 5 0 end of U341F, R685, A344F and A519R for the multiplexing of samples in the pyrosequencing runs.…”

Archaeal genetics — the third way

“…Such markers can be implemented in organisms that grow poorly on minimal medium, as complex media deficient in uracil (such as casamino acids) can be used. This has enabled the establishment of gene knock-out systems in Halobacterium 77,78 , Hfx. volcanii 79,80 , and T. kodakaraensis 53 (see Table 2).…”

Effects of microbial processes on the fate of arsenic in paddy soil

“…Widespread ArsM homologues in prokaryotic and eukaryotic microorganisms can catalyze this process, and many methylated intermediates and trimethylarsine as the end are produced in this process [53,[57][58][59]. It has been reported that bacteria, anaerobic archaea and halophiles in soils or sludges can methylate As [60][61][62][63]. Arsenic biomethylation has been observed in numerous cyanobacteria and algae as well [14,57].…”