Title: 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 Cu²⁺ and Zn²⁺ resistance systems in Pseudomonas stutzeri strain RCH2 are compared with those experimentally implicated in Cu²⁺and Zn²⁺resistance, as determined by using a DNA-barcoded transposon mutant library. Mutant fitness data obtained under denitrifying conditions are combined with regulon predictions to yield a much more comprehensive picture of Cu²⁺and Zn²⁺resistance in strain RCH2. The results not only considerably expand what is known about well-established metal ion exporters (CzcCBA, CzcD, and CusCBA) and their accessory proteins (CzcI and CusF), they also reveal that isolates with mutations in some predicted Cu²⁺ resistance systems do not show decreased fitness relative to the wild type when exposed to Cu²⁺. In addition, new genes are identified that have no known connection to Zn²⁺(corB,corC, Psest_3226, Psest_3322, and Psest_0618) or Cu²⁺resistance (Mrp antiporter subunit gene, Psest_2850, and Psest_0584) but are crucial for resistance to these metal cations. Growth of individual deletion mutants lackingcorB,corC, Psest_3226, or Psest_3322 confirmed the observed Zn-dependent phenotypes. Notably, to our knowledge, this is the first time a bacterial homolog of TMEM165, a human gene responsiblemore » for a congenital glycosylation disorder, has been deleted and the resulting strain characterized. Finally, the fitness values indicate Cu²⁺- and Zn²⁺ -based inhibition of nitrite reductase and interference with molybdenum cofactor biosynthesis for nitrate reductase. These results extend the current understanding of Cu²⁺ and Zn²⁺ efflux and resistance and their effects on denitrifying metabolism. Here in this study, genome-wide mutant fitness data in P. stutzeriRCH2 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 Ca²⁺/H⁺antiport and a human congenital glycosylation disorder, CorB and CorC, which were previously linked to Mg²⁺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.« less

Authors:

Vaccaro, Brian J. ^[1]; Lancaster, W. Andrew ^[1]; Thorgersen, Michael P. ^[1]; Zane, Grant M. ^[2]; Younkin, Adam D. ^[2]; Kazakov, Alexey E. ^[3]; Wetmore, Kelly M. ^[3]; Deutschbauer, Adam ^[3]; Arkin, Adam P. ^[3]; Novichkov, Pavel S. ^[3]; Wall, Judy D. ^[2]; Adams, Michael W. W. ^[1]

---

+ Show Author Affiliations

1. Univ. of Georgia, Athens, GA (United States). Dept. of Biochemistry and Molecular Biology
2. Univ. of Missouri, Columbia, MO (United States). Dept. of Biochemistry
3. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Environmental Genomics and Systems Biology Division

Publication Date:

Fri Jul 29 00:00:00 EDT 2016

Research Org.:

Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Sponsoring Org.:

USDOE Office of Science (SC), Biological and Environmental Research (BER)

OSTI Identifier:

1378758

Grant/Contract Number:  

AC02-05CH11231

Resource Type:

Accepted Manuscript

Journal Name:

Applied and Environmental Microbiology

Additional Journal Information:

Journal Volume: 82; Journal Issue: 19; Journal ID: ISSN 0099-2240

Publisher:

American Society for Microbiology

Country of Publication:

United States

Language:

English

Subject:

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; 59 BASIC BIOLOGICAL SCIENCES