Involvement of RpoN in Regulating Bacterial Arsenite Oxidation

Abstract

In this study with the model organism Agrobacterium tumefaciens, we used a combination of lacZ gene fusions, reverse transcriptase PCR (RT-PCR), and deletion and insertional inactivation mutations to show unambiguously that the alternative sigma factor RpoN participates in the regulation of As III oxidation. A deletion mutation that removed the RpoN binding site from the aioBA promoter and an aacC3 (gentamicin resistance) cassette insertional inactivation of the rpoN coding region eliminated aioBA expression and As III oxidation, although rpoN expression was not related to cell exposure to As III . Putative RpoN binding sites were identified throughout the genome and, as examples, included promoters for aioB, phoB1, pstS1, dctA, glnA, glnB, and flgB that were examined by using qualitative RT-PCR and lacZ reporter fusions to assess the relative contribution of RpoN to their transcription. The expressions of aioB and dctA in the wild-type strain were considerably enhanced in cells exposed to As III , and both genes were silent in the rpoN::aacC3 mutant regardless of As III . The expression level of glnA was not influenced by As III but was reduced (but not silent) in the rpoN::aacC3 mutant and further reduced in the mutant under N starvation conditions. The rpoN::aacC3 mutation had no obvious effect on the expression of glnB, pstS1, phoB1, or flgB. These experiments provide definitive evidence to document the requirement of RpoN for As III oxidation but also illustrate that the presence of a consensus RpoN binding site does not necessarily link the associated gene with regulation by As III or by this sigma factor. E vidence of microbial arsenite (As III ) oxidation was first reported nearly a century ago (18). Subsequent progress has been sporadic, with work that identified some organisms capable of As III oxidation (46,48,60) and then a study of a Pseudomonas arsenitoxidans strain reported to grow chemolithoautotrophically with As III as a sole electron donor (23). 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). Likewise, progress has been made in the understanding of the biochemistry of the As III oxidase enzyme (1,14,37 oxidase structural genes were later cloned from the above-mentioned Rhizobium NT-26 organism (53). The symbols for genes coding for functions associated with As III oxidation have...

Arsenite Oxidase Also Functions as an Antimonite Oxidase

“…3B). Since the Sb(III) oxidation activity was shown to be depleted in the aioA::Tn5-B22 mutant but restored by the aioSRBA-cytC fragment, the evidence clearly argues that there must be a separate, active promoter somewhere within this fragment that influences aioBA expression other than the As(III)-sensitive promoter upstream of aioB that involves RpoN and AioR (46,47). We suggest that this promoter is upstream of aioS based on the following observations.…”

“…Escherichia coli strain S17-1 (45) was used as a conjugation donor to introduce the transposon or for mutant complementation work. We previously described the construction of the aioB::lacZ reporter that allows for tracking expression of the aioBA genes transcribed from the aioB proximal promoter that involves AioR and RpoN (46,47). A. tumefaciens strain 5A contains four arsR genes (48) that are well known to be induced by both metalloids (49); thus, as an experimental control, we also selected one of these genes, arsR4, to examine As(III) and Sb(III) regulatory effects.…”

An ArsR/SmtB Family Member Is Involved in the Regulation by Arsenic of the Arsenite Oxidase Operon in Thiomonas arsenitoxydans

“…As in the other arsenite oxidizers (10,24,(42)(43)(44), the T. arsenitoxydans aioBA operon is transcribed from a 54 -dependent promoter (26) (Fig. 5).…”

Biorremediación de arsénico mediada por microorganismos genéticamente modificados.

“…La subunidad pequeña (Aoxa) con un centro de [2Fe-2S], es una proteína Rieske que desempeña un papel en la transferencia de electrones de As III a un citocromo o azurina y cuenta con un péptido señal TAT para el transporte de la enzima hacia el citoplasma (Anderson et al, 1992;Duval et al, 2010;Muller et al, 2003). En 1992 se purif icó la primera arsenito oxidasa de Alcaligenes faecalis, pero los genes que codif ican para AOX se identif icaron por primera vez en Herminiimonas arsenoxydans (Anderson et al, 1992;Lièvremont et al, 2009;Kang et al, 2012). Desde entonces, muchas alfa, beta y gama proteobacteria, crenarchaeota y Deinococcus Thermus se han estudiado debido a la relación f ilogenética de sus enzimas AOX (Lebrun et al, 2003;Muller et al, 2003Muller et al, , 2006.…”

“…También responde a señales "quorum sensing" que pueden ser emitidas por genes lux o un sistema de dos componentes, donde una proteína transmembranal o dominio receptor recibe un estímulo extracelular (como la exposición al arsénico) y se fosforila de modo que un dominio activo permita la activación del gen (Slyemi et al, 2013;Kang et al, 2012;Barba-Ostria, 2014).…”