Determining Roles of Accessory Genes in Denitrification by Mutant Fitness Analyses

Abstract

Enzymes of the denitrification pathway play an important role in the global nitrogen cycle, including release of nitrous oxide, an ozone-depleting greenhouse gas. In addition, nitric oxide reductase, maturation factors, and proteins associated with nitric oxide detoxification are used by pathogens to combat nitric oxide release by host immune systems. While the core reductases that catalyze the conversion of nitrate to dinitrogen are well understood at a mechanistic level, there are many peripheral proteins required for denitrification whose basic function is unclear. A bar-coded transposon DNA library from Pseudomonas stutzeri strain RCH2 was grown under denitrifying conditions, using nitrate or nitrite as an electron acceptor, and also under molybdenum limitation conditions, with nitrate as the electron acceptor. Analysis of sequencing results from these growths yielded gene fitness data for 3,307 of the 4,265 protein-encoding genes present in strain RCH2. The insights presented here contribute to our understanding of how peripheral proteins contribute to a fully functioning denitrification pathway. We propose a new low-affinity molybdate transporter, OatABC, and show that differential regulation is observed for two MoaA homologs involved in molybdenum cofactor biosynthesis. We also propose that NnrS may function as a membrane-bound NO sensor. The dominant HemN paralog involved in heme biosynthesis is identified, and a CheR homolog is proposed to function in nitrate chemotaxis. In addition, new insights are provided into nitrite reductase redundancy, nitric oxide reductase maturation, nitrous oxide reductase maturation, and regulation.

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

“…Co 2ϩ and Cd 2ϩ data were included where relevant to Zn 2ϩ transport. As previously described (24), the data are presented as gene fitness values (w), which reflect the growth of mutants with disruptions of individual genes relative to the overall mutant library population such that an increase in the relative abundance of mutants in which a gene is mutated shows positive fitness for that gene and a decrease shows negative fitness (30). Gene fitness values are interpreted as the effect of the absence of the gene product; however, in some cases, transposon insertion can affect the expression of downstream genes as well (so-called "polar effects") (42).…”

“…The P. stutzeri RCH2 RB-TnSeq mutant library, containing 166,448 single transposon insertions with known, sequence-identified genome locations (30), was grown as previously described with nitrate (20 mM) as the electron acceptor (24). Zn 2ϩ , Cu 2ϩ , Co 2ϩ , or Cd 2ϩ toxicity was induced by supplementing the growth medium with 500 M ZnCl 2 , 100 M CuCl 2 , 100 M CoCl 2 , or 6 M CdCl 2 .…”

“…P. stutzeri RCH2 was cultured anaerobically in 100-well plates as previously described (24). Lactate (20 mM) was used as a carbon source, with nitrate (20 mM) as the terminal electron acceptor.…”

“…Thus, transporters are not perfectly specific, due to similarities in the size, charge, and to some extent, coordination preferences of metal ions, particularly among the divalent late d-block metals (Fe-Zn). In some cases, lower-specificity transporters may even provide a competitive advantage (24,25). However, the specificity (or generality) of a given transporter is currently very difficult to determine bioinformatically.…”

“…Strain RCH2 was originally characterized for its chromate-reducing activity (29), and a random barcode transposon site sequencing (RB-TnSeq) library is now available (30). In this study, we used this library to determine which RCH2 efflux systems were truly involved in Cu 2ϩ and Zn 2ϩ resistance during denitrifying growth (24). Since some Zn 2ϩ transport systems can also transport Co 2ϩ and Cd 2ϩ (14), RB-TnSeq analysis was also conducted in the presence of toxic concentrations of each of these two metal ions for comparison.…”