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Volume 163, Issue 11

Markerless deletion of putative alanine dehydrogenase genes in using a -based counterselection technique Free

Abstract

strains are used for the large-scale production of industrial exoenzymes from proteinaceous substrates, but details of the amino acid metabolism involved are largely unknown. In this study, two chromosomal genes putatively involved in amino acid metabolism of were deleted to clarify their role. For this, a convenient counterselection system for markerless in-frame deletions was developed for . A deletion plasmid containing up- and downstream DNA segments of the chromosomal deletion target was conjugated to and integrated into the genome by homologous recombination. Thereafter, the counterselection was done by using a cassette. The presence of cytosine deaminase and cytosine permease exerted a conditionally lethal phenotype on cells in the presence of the cytosine analogue 5-fluorocytosine. Thereby clones were selected that lost the integrated vector sequence and the anticipated deletion target after a second recombination step. This method allows the construction of markerless mutants in strains in iterative cycles. MW3 derivatives lacking either one of the ORFs BL03009 or BL00190, encoding a putative alanine dehydrogenase and a similar putative enzyme, respectively, retained the ability to grow in minimal medium supplemented with alanine as the carbon source. In the double deletion mutant MW3 ΔBL03009 ΔBL00190, however, growth on alanine was completely abolished. These data indicate that the two encoded enzymes are paralogues fulfilling mutually replaceable functions in alanine utilization, and suggest that in MW3 alanine utilization is initiated by direct oxidative transamination to pyruvate and ammonium.

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Keyword(s): 5-fluorocytosine , Bacillus licheniformis , codBA , counterselection , cytosine deaminase and markerless deletion
© 2017 The Authors
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2017-11-01
2024-04-24
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Markerless deletion of putative alanine dehydrogenase genes in Bacillus licheniformis using a codBA-based counterselection technique
Microbiology 163, 1532 (2017); https://doi.org/10.1099/mic.0.000544
/content/journal/micro/10.1099/mic.0.000544
/content/journal/micro/10.1099/mic.0.000544
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