Complex Regulation of Arsenite Oxidation in Agrobacterium tumefaciens

Abstract

Seminal regulatory controls of microbial arsenite [As(III)] oxidation are described in this study. Transposon mutagenesis of Agrobacterium tumefaciens identified genes essential for As(III) oxidation, including those coding for a two-component signal transduction pair. The transposon interrupted a response regulator gene (referred to as aoxR), which encodes an ntrC-like protein and is immediately downstream of a gene (aoxS) encoding a protein with primary structural features found in sensor histidine kinases. The structural genes for As(III) oxidase (aoxAB), a c-type cytochrome (cytc 2 ), and molybdopterin biosynthesis (chlE) were downstream of aoxR. The mutant could not be complemented by aoxSR in trans but was complemented by a clone containing aoxS-aoxR-aoxA-aoxB-cytc 2 and consistent with reverse transcriptase (RT) PCR experiments, which demonstrated these genes are cotranscribed as an operon. Expression of aoxAB was monitored by RT-PCR and found to be up-regulated by the addition of As(III) to cell cultures. Expression of aoxAB was also controlled in a fashion consistent with quorum sensing in that (i) expression of aoxAB was absent in As(III)-unexposed early-log-phase cells but was observed in As(III)-unexposed, late-log-phase cells and (ii) treating As(III)-unexposed, early-log-phase cells with ethyl acetate extracts of As(III)-unexposed, late-log-phase culture supernatants also resulted in aoxAB induction. Under inducing conditions, aoxS expression was readily observed in the wild-type strain but significantly reduced in the mutant, indicating that AoxR is autoregulatory and at least partially controls the expression of the aox operon. In summary, regulation of A. tumefaciens As(III) oxidation is complex, apparently being controlled by As(III) exposure, a two-component signal transduction system, and quorum sensing.Arsenic (As) is a known carcinogen, occurring in the environment primarily as the oxyanion arsenate (H 2 AsO 4 Ϫ , HAsO 4 2Ϫ ) [As(V)] and arsenite (H 3 AsO 3 0 ) [As(III)]. As(III) is more toxic and generally the more mobile species (reviewed in reference 6), and hence, there is significant interest in understanding the factors that control As speciation in the environment. Both abiotic and biotic factors are involved; however, it is now generally viewed that microbial As redox transformations are important, if not principal, drivers controlling As speciation in soils, sediments, and natural waters (6, 20).Microbial As(III) oxidation and As(V) reduction activities are cellular strategies for either detoxification or for generating energy. A fairly detailed model explaining the genetics, regulation, and function of detoxification-based As(V) reduction is in place (32), but beyond that little is known. Genes encoding respiratory As(V) reductases have been cloned and enzyme characterizations are under way (recently reviewed in reference 32). Likewise, genes encoding As(III) oxidase have also recently been cloned and characterized (17,29), and extensive enzyme characterization has been achieved (...

Involvement of RpoN in Regulating Bacterial Arsenite Oxidation

“…The precisely targeted mutation and complementation experiments in the present study provide definitive evidence that the rpoN gene product is essential for aioBA expression and, hence, As III oxidation. This observation also allows a more definitive understanding of the As III oxidase-negative phenotype of the aioR:: Tn5-B22 mutant described previously (28). Specifically, the Tn5-B22 insertion in the extreme 3= region of aioR in the aioS-aioRaioB-aioA operon likely disrupted the DNA binding component of the regulator AioR and is consistent with AioR participating as a bacterial enhancer binding protein in conjunction with RpoN to facilitate the induction of aioBA.…”

“…Subsequent follow-up characterizations of this organism and this process failed to materialize; however, approximately 2 decades later, Santini et al (52) described the isolation and initial characterization of a Rhizobiumlike bacterium (strain NT-26) that could grow chemolithoautotrophically with As III as a sole electron donor for energy generation and with CO 2 as a sole carbon source. Soon thereafter, and in part stimulated by the massive arsenic poisoning disaster in Bangladesh (2), a series of studies initiated the characterization of microbial As III oxidation in natural environments, including geothermal springs (9,11,12,17,19,24,25,35,51) and soils (41); in mining-contaminated environments (6,13,40); and, most recently, in anoxic photosynthesis (21,33 (28,31) indicated the role and importance of the sensor kinase AioS and its putative regulatory partner AioR (a bacterial enhancer binding protein), direct proof of these two proteins working together as part of a putative As III signal perception and transduction cascade was just recently provided by Sardiwal et al (54), who demonstrated the autophosphorylation of an AioS component and the AioS-specific phosphorylation of AioR. Recently, our work has expanded this regulatory model to now include a third component, AioX, which is a periplasmic As III binding protein that is also essential for aioBA expression (39 …”

Structural and functional genomics of plasmid pSinA of Sinorhizobium sp. M14 encoding genes for the arsenite oxidation and arsenic resistance

“…The predicted aminoacid sequences of particular proteins encoded by the AIO cluster exhibit 91-100% identity in the amino-acid sequence to the gene products of arsenite oxidation modules of the facultatively chemolithoautotrophic Rhizobium sp. NT26 (Santini and van den Hoven, 2004), and the heterotrophic strains Agrobacterium tumefaciens strain 5A (Kashyap et al, 2006) and Ochrobactrum tritici SCII24 (Branco et al, 2009). Furthermore, the genetic organization of the plasmid pSinA AIO module (aioSRABcytCmoeA) is identical to that of the arsenite oxidase genes of Rhizobium sp.…”

Arsenate toxicity and stress responses in the freshwater ciliate Tetrahymena pyriformis

“…(Stock et al 2000). Previous studies have revealed that the two-component signal transduction system could regulate arsenite oxidation in bacteria (Kashyap et al 2006). Here under arsenate stress, the two-component sensor histidine kinase (spot 14) was also over-expressed in T. pyriformis.…”