biostudies-literatureTao XU.S. Department of Energye0040122https://www.ebi.ac.uk/biostudies/studies/S-EPMC9317963biostudies-literatureUnknown88(14)<i>Rhodanobacter</i> has been found as the dominant genus in aquifers contaminated with high concentrations of nitrate and uranium in Oak Ridge, TN, USA. The <i>in situ</i> stimulation of denitrification has been proposed as a potential method to remediate nitrate and uranium contamination. Among the <i>Rhodanobacter</i> 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 <i>R. denitrificans</i> strains. First, we optimized the growApplied and environmental microbiologyDevelopment of a Markerless Deletion Mutagenesis System in Nitrate-Reducing Bacterium Rhodanobacter denitrificans.PMC9317963DE-AC02-05CH11231Liu JLi YArkin APZhou AMichael JPDeutschbauer AMTao XChakraborty RPeng MKempher MLZhou JfalseDevelopment of a Markerless Deletion Mutagenesis System in Nitrate-Reducing Bacterium Rhodanobacter denitrificans.<i>Rhodanobacter</i> has been found as the dominant genus in aquifers contaminated with high concentrations of nitrate and uranium in Oak Ridge, TN, USA. The <i>in situ</i> stimulation of denitrification has been proposed as a potential method to remediate nitrate and uranium contamination. Among the <i>Rhodanobacter</i> 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 <i>R. denitrificans</i> strains. First, we optimized the grow2022-01-01T00:00:00Z2022 Jul2026-06-12T05:48:17.969Z2026-06-12T03:08:22.192ZS-EPMC93179633573780710.1128/aem.00401-22