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

Journal Article · · Applied and Environmental Microbiology
DOI:https://doi.org/10.1128/aem.00401-22· OSTI ID:1889276
 [1]; ORCiD logo [1];  [1];  [1];  [1];  [1];  [1]; ORCiD logo [2];  [3];  [3];  [1]
  1. Univ. of Oklahoma, Norman, OK (United States)
  2. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
  3. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Univ. of California, Berkeley, CA (United States)
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Rhodanobacter has been found as the dominant genus in aquifers contaminated with high concentrations of nitrate and uranium in Oak Ridge, TN, USA. The in situ stimulation of denitrification has been proposed as a potential method to remediate nitrate and uranium contamination. Among the Rhodanobacter 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 R. denitrificans strains. First, we optimized the growth conditions, tested antibiotic resistance, and determined appropriate transformation parameters in 10 Rhodanobacter strains. We then deleted the upp gene, which encodes uracil phosphoribosyltransferase, in R. denitrificans strains FW104-R3 and FW104-R5. The resulting strains were designated R3_Δupp and R5_Δupp and used as host strains for mutagenesis with 5-fluorouracil (5-FU) resistance as the counterselection marker to generate markerless deletion mutants. Further, to test the developed protocol, the narG gene encoding nitrate reductase was knocked out in the R3_Δupp and R5_Δupp host strains. As expected, the narG 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 R. denitrificans strains, which will allow for future functional genomic studies in these strains furthering our understanding of the metabolic and resistance mechanisms present in Rhodanobacter species.

Research Organization:
Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)
Sponsoring Organization:
USDOE Office of Science (SC), Biological and Environmental Research (BER)
Grant/Contract Number:
AC02-05CH11231
OSTI ID:
1889276
Journal Information:
Applied and Environmental Microbiology, Vol. 88, Issue 14; ISSN 0099-2240
Publisher:
American Society for MicrobiologyCopyright Statement
Country of Publication:
United States
Language:
English

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Related Subjects

59 BASIC BIOLOGICAL SCIENCES
Rhodanobacter denitrificans
nitrate-reducing bacterium
low-pH resistance
mutagenesis
in-frame deletion
upp