Development of a Markerless Deletion Mutagenesis System in Nitrate-Reducing Bacterium Rhodanobacter denitrificans

• Published in: Applied and environmental microbiology Vol. 88; no. 14; p. e0040122
• Main Authors: Tao, Xuanyu, Zhou, Aifen, Kempher, Megan L, Liu, Jiantao, Peng, Mu, Li, Yuan, Michael, Jonathan P, Chakraborty, Romy, Deutschbauer, Adam M, Arkin, Adam P, Zhou, Jizhong
• Format: Journal Article
• Language: English
• Published: United States American Society for Microbiology 26.07.2022
• Subjects: 5-Fluorouracil; Antibiotic resistance; Antibiotics; Aquifers; Bacteria - genetics; BASIC BIOLOGICAL SCIENCES; Bioremediation; Contamination; Deletion mutant; Denitrification; Ecosystem; Environmental Microbiology; Gammaproteobacteria; Gene deletion; Genetic transformation; Genetics and Molecular Biology; Genome editing; Groundwater; Growth conditions; Heavy metals; in-frame deletion; low-pH resistance; Mutagenesis; Mutants; NarG gene; Nitrate reductase; nitrate-reducing bacterium; Nitrates; pH effects; Phosphoribosyltransferase; Reductases; Rhodanobacter; Rhodanobacter denitrificans; Species; Strains (organisms); upp; Upp gene; Uracil; Uracil phosphoribosyltransferase; Uranium; low-pH resistance; nitrate-reducing bacterium; upp; in-frame deletion; mutagenesis; Rhodanobacter denitrificans
• ISSN: 0099-2240; 1098-5336
• DOI: 10.1128/aem.00401-22

has been found as the dominant genus in aquifers contaminated with high concentrations of nitrate and uranium in Oak Ridge, TN, USA. The stimulation of denitrification has been proposed as a potential method to remediate nitrate and uranium contamination. Among the species, Rhodanobacter denitrificans strains have been reported to be capable of denitrification and contain abundant metal resistance genes. However, due to the lack of a mutagenesis system in these strains, our understanding of the mechanisms underlying low-pH resistance and the ability to dominate in the contaminated environment remains limited. Here, we developed an in-frame markerless deletion system in two strains. First, we optimized the growth conditions, tested antibiotic resistance, and determined appropriate transformation parameters in 10 strains. We then deleted the gene, which encodes uracil phosphoribosyltransferase, in strains FW104-R3 and FW104-R5. The resulting strains were designated R3_Δ and R5_Δ and used as host strains for mutagenesis with 5-fluorouracil (5-FU) resistance as the counterselection marker to generate markerless deletion mutants. To test the developed protocol, the gene encoding nitrate reductase was knocked out in the R3_Δ and R5_Δ host strains. As expected, the mutants could not grow in anoxic medium with nitrate as the electron acceptor. Overall, these results show that the in-frame markerless deletion system is effective in two strains, which will allow for future functional genomic studies in these strains furthering our understanding of the metabolic and resistance mechanisms present in species. Rhodanobacter denitrificans is capable of denitrification and is also resistant to toxic heavy metals and low pH. Accordingly, the presence of species at a particular environmental site is considered an indicator of nitrate and uranium contamination. These characteristics suggest its future potential application in bioremediation of nitrate or concurrent nitrate and uranium contamination in groundwater ecosystems. Due to the lack of genetic tools in this organism, the mechanisms of low-pH and heavy metal resistance in strains remain elusive, which impedes its use in bioremediation strategies. Here, we developed a genome editing method in two strains. This work marks a crucial step in developing as a model for studying the diverse mechanisms of low-pH and heavy metal resistance associated with denitrification.