Bacteriophage resistance of a deltathyA mutant of Lactococcus lactis blocked in DNA replication

• Published in: Applied and environmental microbiology Vol. 68; no. 6; pp. 3010 - 3023
• Main Authors: Pedersen, Martin B, Jensen, Peter R, Janzen, Thomas, Nilsson, Dan
• Format: Journal Article
• Language: English
• Published: United States 01.06.2002
• Subjects: Bacteriophages - physiology; Colony-Forming Units Assay; DNA Replication - physiology; DNA, Viral - physiology; Lactococcus lactis - genetics; Lactococcus lactis - physiology; Lactococcus lactis - virology; Mutation; Temperature; Thymidine - deficiency; Thymidine - metabolism; Thymidylate Synthase - deficiency; Thymidylate Synthase - genetics; Thymidylate Synthase - metabolism; Thymine Nucleotides - analysis; Virus Replication - physiology
• ISSN: 0099-2240

The thyA gene, which encodes thymidylate synthase (TS), of Lactococcus lactis CHCC373 was sequenced, including the upstream and downstream regions. We then deleted part of thyA by gene replacement. The resulting strain, MBP71 deltathyA, was devoid of TS activity, and in media without thymidine, such as milk, there was no detectable dTTP pool in the cells. Hence, DNA replication was abolished, and acidification by MBP71 was completely unaffected by the presence of nine different phages tested at a multiplicity of infection (MOI) of 0.1. Nonreplicating MBP71 must be inoculated at a higher level than CHCC373 to achieve a certain pH within a specified time. For a pH of 5.2 to be reached in 6 h, the inoculation level of MBP71 must be 17-fold higher than for CHCC373. However, by adding a limiting amount of thymidine this could be lowered to just 5-fold the normal amount, while acidification was unaffected with MBP71 up to an MOI of 0.01. It was found that nonreplicating MBP71 produced largely the same products as CHCC373, though the acetaldehyde production of the former was higher.