Genes important for growth of Pseudomonas stutzeri PDA on chlorate were identified using a randomly DNA barcoded transposon mutant library. During chlorate reduction, mutations in genes encoding the chlorate reductase clrABC, predicted molybdopterin cofactor chaperon clrD, molybdopterin biosynthesis, and two genes of unknown function (clrE, clrF) had fitness defects in pooled mutant assays (Bar-seq). Markerless in-frame deletions confirmed that clrA, clrB, and clrC were essential for chlorate reduction, while clrD, clrE, and clrF had less severe growth defects. Interestingly, the key detoxification gene cld was essential for chlorate reduction in isogenic pure culture experiments, but showed only minor fitness ... More
Genes important for growth of Pseudomonas stutzeri PDA on chlorate were identified using a randomly DNA barcoded transposon mutant library. During chlorate reduction, mutations in genes encoding the chlorate reductase clrABC, predicted molybdopterin cofactor chaperon clrD, molybdopterin biosynthesis, and two genes of unknown function (clrE, clrF) had fitness defects in pooled mutant assays (Bar-seq). Markerless in-frame deletions confirmed that clrA, clrB, and clrC were essential for chlorate reduction, while clrD, clrE, and clrF had less severe growth defects. Interestingly, the key detoxification gene cld was essential for chlorate reduction in isogenic pure culture experiments, but showed only minor fitness defects in Bar-seq experiments. We hypothesized this was enabled through chlorite dismutation by the community, as most strains in the Bar-seq library contained an intact cld. In support of this, Δcld grew with wild-type PDA or ΔclrA, and purified Cld also restored growth to the Δcld mutant. Expanding on this, wild-type PDA and a Δcld mutant of the perchlorate reducer Azospira suillum PS grew on perchlorate in co-culture, but not individually. These results demonstrate that co-occurrence of cld and a chloroxyanion reductase within a single organism is not necessary and raises the possibility of syntrophic (per)chlorate respiration in the environment.
SIGNIFICANCE:
Numerous chlorate-reducing bacteria have been isolated, but no genetic study has been performed to understand the metabolism on a genome-wide scale. After expanding the genomics of chlorate reduction in the genus Pseudomonas, we built genetic tools for both targeted gene deletions and barcoded transposon mutagenesis in P. stutzeri PDA. We identified a set of genes essential for chlorate reduction in PDA, as well as two genes encoding conserved hypothetical proteins that we name clrE and clrF based on their chlorate-specific phenotypes. From discrepancies between Bar-seq and pure culture results, we also show that cld is dispensable when an exogenous chlorite detoxification system is available. This result counters the prevailing view that chloroxyanion respiration must be catalyzed within a single organism.
