Genome Sequence of Brevundimonas sp., an Arsenic Resistant Soil Bacterium

Brevundimonas sp. is a bacteria able to grow in metal(loid) contaminated soil from Puchuncaví Valley, central Chile. This study has isolated a bacterial strain capable of growth under high doses of arsenic (As) (6000 mg L−1), and a draft genome sequence was generated. Additionally, real-time PCR was performed to examine the effect of As on some genes related to As resistance. Results demonstrated a total of 3275 predicted annotated genes with several genes related to the ars operon, metal(loid) resistance-related genes, metal efflux pumps, and detoxifying enzymes. Real-time PCR showed that the arsB involved in the efflux of As was down-regulated, whereas arsR, arsH, and ACR3 did not show differences with the addition of As. Our study provides novel evidence of diverse As regulating systems in tolerant bacteria that will lead to a better understanding of how microorganisms overcome toxic elements and colonize As contaminated soils and to the possible use of their specific properties in bioremediation.

An arsRB resistance operon confers tolerance to arsenite in the environmental isolate Terribacillus sp. AE2B 122

Terribacillus sp. AE2B 122 is an environmental strain isolated from olive-oil agroindustry wastes. This strain displays resistance to arsenic, one of the most ubiquitous carcinogens found in nature. Terribacillus sp. AE2B 122 possesses an unusual ars operon, consisting of the transcriptional regulator (arsR) and arsenite efflux pump (arsB) but no adjacent arsenate reductase (arsC) locus. Expression of arsR and arsB was induced when Terribacillus was exposed to sub-lethal concentrations of arsenate. Heterologous expression of the arsB homologue in Escherichia coli ∆arsRBC demonstrated that it conferred resistance to arsenite and reduced the accumulation of arsenic inside the cells. Two members of the arsC-like family (Te3384 and Te2854) found in the Terribacillus genome were not induced by arsenic, but their heterologous expression in E. coli ∆arsC and ∆arsRBC increased the accumulation of arsenic in both strains. We found that both Te3384 and Te2854 slightly increased resistance to arsenate in E. coli ∆arsC and ∆arsRBC, possibly by chelation of arsenic or increasing the resistance to oxidative stress. Finally, arsenic speciation assays suggest that Terribacillus is incapable of arsenate reduction, in agreement with the lack of an arsC homologue in the genome.

Identification of A Novel Arsenic Resistance Transposon Nested in A Mercury Resistance Transposon of Bacillus sp. MB24

A novel TnMERI1-like transposon designated as TnMARS1 was identified from mercury resistant Bacilli isolated from Minamata Bay sediment. Two adjacent ars operon-like gene clusters, ars1 and ars2, flanked by a pair of 78-bp inverted repeat sequences, which resulted in a 13.8-kbp transposon-like fragment, were found to be sandwiched between two transposable genes of the TnMERI1-like transposon of a mercury resistant bacterium, Bacillus sp. MB24. The presence of a single transcription start site in each cluster determined by 5′-RACE suggested that both are operons. Quantitative real time RT-PCR showed that the transcription of the arsR genes contained in each operon was induced by arsenite, while arsR2 responded to arsenite more sensitively and strikingly than arsR1 did. Further, arsenic resistance complementary experiments showed that the ars2 operon conferred arsenate and arsenite resistance to an arsB-knocked out Bacillus host, while the ars1 operon only raised arsenite resistance slightly. This transposon nested in TnMARS1 was designated as TnARS1. Multi-gene cluster blast against bacteria and Bacilli whole genome sequence databases suggested that TnMARS1 is the first case of a TnMERI1-like transposon combined with an arsenic resistance transposon. The findings of this study suggested that TnMERI1-like transposons could recruit other mobile elements into its genetic structure, and subsequently cause horizontal dissemination of both mercury and arsenic resistances among Bacilli in Minamata Bay.

Gene expression of the arsenic resistance operon in Chromobacterium violaceum ATCC 12472

Chromobacterium violaceum ATCC 12472 presents an arsRCB-type operon, which is involved in arsenic resistance. The regulating protein of this resistance system (ArsR) does not have the small conserved site (ELCVDCL) to link to the metalloid, as observed in Escherichia coli , and is thus considered to be an atypical ArsR protein, like that observed in Acidithiobacillus ferrooxidans . In the present study, the gene expression profile of the ars operon under induction at different concentrations of arsenite — As(III) — was obtained via real-time PCR (TaqMan), by correlating the threshold cycle (Ct) values of induced and uninduced (control) samples. Through linear regression analysis (R2 = 0.9926), the gene expression profile of the ars operon showed clearly that the 0.125 μmol/L concentration of As(III) was sufficient to provoke a 4-fold increase in the resistance system, and a further increase in concentration resulted in an increase of up to 53-fold in transcription rates. The relation between resistance and induction of the ars operon indicates that the increased resistance to As(III) is associated with the increase in the number of transcripts.

The ars Operon in the skinElement of Bacillus subtilis Confers Resistance to Arsenate and Arsenite

ABSTRACT The Bacillus subtilis skin element confers resistance to arsenate and arsenite. The ars operon in theskin element contains four genes in the orderarsR, ORF2, arsB, andarsC. Three of these genes are homologous to thearsR, arsB, and arsC genes from the staphylococcal plasmid pI258, while no homologs of ORF2have been found. Inactivation of arsR, arsB, orarsC results in either constitutive expression ofars, an arsenite- and arsenate-sensitive phenotype, or an arsenate-sensitive phenotype, respectively. These results suggest that ArsR, ArsB, and ArsC function as a negative regulator, a membrane-associated protein need for extrusion of arsenite, and arsenate reductase, respectively. Expression of the arsoperon was induced by arsenate, arsenite, and antimonite. Northern hybridization and primer extension analysis showed that synthesis of a full-length ars transcript of about 2.4 kb was induced by arsenate and that the ars promoter contains sequences that resemble the −10 and −35 regions of promoters that are recognized by EςA.

Expression of theEscherichia colichromosomalarsoperon

A chromosomally located operon (ars) of Escherichia coli has been previously shown to be functional in arsenic detoxification. DNA sequencing revealed three open reading frames homologous to the arsR, arsB, and arsC open reading frames of plasmid-based arsenic resistance operons isolated from both E. coli and staphylococcal species. To examine the outline of transcriptional regulation of the chromosomal ars operon, several transcriptional fusions, using the luciferase-encoding luxAB genes of Vibrio harveyi, were constructed. Measurement of the expression of these gene fusions demonstrated that the operon was rapidly induced by sodium arsenite and negatively regulated by the trans-acting arsR gene product. Northern blotting and primer extension analyses revealed that the chromosomal ars operon is most likely transcribed as a single mRNA of approximately 2100 nucleotides in length and processed into two smaller mRNA products in a manner similar to that found in the E. coli R773 plasmid-borne ars operon. However, transcription was found to initiate at a position that is relatively further upstream of the initiation codon of the arsR coding sequence than that determined for the E. coli R773 plasmid's ars operon.Key words: arsenic resistance, Escherichia coli, transcription, gene fusions.