Novel Metal Cation Resistance Systems from Mutant Fitness Analysis of Denitrifying Pseudomonas stutzeri

Abstract

Metal ion transport systems have been studied extensively, but the specificity of a given transporter is often unclear from amino acid sequence data alone. In this study, predicted Cu 2؉ IMPORTANCEIn this study, genome-wide mutant fitness data in P. stutzeri RCH2 combined with regulon predictions identify several proteins of unknown function that are involved in resisting zinc and copper toxicity. For zinc, these include a member of the UPF0016 protein family that was previously implicated in Ca 2؉ /H ؉ antiport and a human congenital glycosylation disorder, CorB and CorC, which were previously linked to Mg 2؉ transport, and Psest_3322 and Psest_0618, two proteins with no characterized homologs. Experiments using mutants lacking Psest_3226, Psest_3322, corB, corC, or czcI verified their proposed functions, which will enable future studies of these little-characterized zinc resistance determinants. Likewise, Psest_2850, annotated as an ion antiporter subunit, and the conserved hypothetical protein Psest_0584 are implicated in copper resistance. Physiological connections between previous studies and phenotypes presented here are discussed. Functional and mechanistic understanding of transport proteins improves the understanding of systems in which members of the same protein family, including those in humans, can have different functions.T he responses of microorganisms to metal toxicity have been well studied (1). In brief, metal ions can be toxic by binding to essential proteins or other molecules, causing them to become nonfunctional or function incorrectly. This occurs, for example, where the toxic metal ion binds to a binding site that requires a different metal ion to function. Toxic metal ions can also catalyze reactions that are detrimental to the cell, such as hydroxyl radical generation catalyzed by free iron (Fe 3ϩ/2ϩ ) (2) or iron-sulfur cluster degradation by copper (3).Understanding modes of toxicity and metal resistance systems is important, as the information gained from bacteria can be applied to human physiology and medicine. For example, Wilson's disease and Menkes disease are two human diseases caused by mutations in copper transporters (4). Likewise, acrodermatitis enteropathica, while less well-studied at a mechanistic level, is caused by a mutation of the SLC39A4 zinc transporter gene (5). Wilson's disease results in copper accumulation, which is known to cause tissue damage due to the generation of reactive oxygen species (4). Similarly, copper and zinc are known to be involved in neurodegenerative diseases, such as Alzheimer's and Parkinson's, again with a connection to oxidative stress, although their precise roles are still unclear (6). In addition, copper has long been used as

Sensitivity to the two peptide bacteriocin plantaricin EF is dependent on CorC, a membrane‐bound, magnesium/cobalt efflux protein

“…Putative Mg 2+ efflux proteins have been identified as CorB, CorC, and CorD in Salmonella (Gibson, Bagga, Miller, & Maguire, ) and Shigella (Zhang, Ren, Zhu, Li, & Wang, ) and YhdP in Bacillus (Akanuma et al, ). These proteins might also export other metals such as Zn 2+ , as indicated for CorB and CorC in Pseudomonas stutzeri (Vaccaro et al, ).…”

Multiple signaling systems target a core set of transition metal homeostasis genes using similar binding motifs

“…P. stutzeri RCH2 (hereafter referred to as RCH2) is emerging as a model organism to study denitrification and metal reduction under various redox regimes (Lalucat et al ., ; Thorgersen et al ., ). RCH2 is also of interest for its novel metal cation resistance genes (Vaccaro et al ., ). Although the annotated genome of RCH2 and other isolates from these environments often encode multiple signaling systems for response to a range of metals, these signal transduction cascades and their relation to each other is poorly delineated.…”

“…RCH2 contains a remarkable number of redundant metal homeostasis genes, and notably contains two paralogous signaling systems for copper response (Vaccaro et al ., ). A recent study using RCH2 RB‐Tnseq libraries has shown that in this bacterium numerous operons and regulators are involved in response to elevated concentrations of copper and zinc cations (Vaccaro et al ., ). At the same time, several known copper resistance genes (e.g., copA1 , copB1 , copA2 , copB2 ) did not demonstrate a copper‐specific phenotype in that study.…”

Nitrate-Utilizing Microorganisms Resistant to Multiple Metals from the Heavily Contaminated Oak Ridge Reservation

“…For example, in a study of denitrification rates in saltmarsh sediments, it was reported that initial denitrification rates were inhibited by several metals at 1 g/liter, including Pb, Ni, Cr, Zn, Cu, Fe, and Cd (24). Similarly, whole-genome fitness assays conducted on a model denitrifying organism, Pseudomonas stutzeri RCH2, grown under denitrifying conditions, revealed that disruptions to nitrate reductase and denitrification-related genes resulted in decreased fitness of the organism when grown in the presence of elevated concentrations of several different metals, including Cu, Zn, Cr, and U (39,40). The ORR organisms that we isolated must therefore have molecular and physical mechanisms of metal resistance that allow them to survive in the extreme ORR environment, and the nature of those mechanisms is currently under study.…”