Characterization of a mutant of Lactococcus lactis with reduced membrane-bound ATPase activity under acidic conditions.
A mutant of Lactococcus lactis subsp. lactis C2 with reduced membrane-bound ATPase activity was characterized to clarify its acid sensitivity. The cytoplasmic pH of the mutant was measured in reference to the parental strain under various pH conditions. At low pH, the mutant could not maintain its cytoplasmic pH near neutral, and lost its viability faster than the parental strain. The ATPase activities of cells cultured under neutral and acidic conditions using pH-controlled jar fermentors were measured. The relative ATPase activity of the mutant at pH 7.0 was 42% of the parental strain. At pH 4.5, the parental strain showed an ATPase activity 2.8-fold higher than that at pH 7.0 while the level of increase in the mutant was only 1.6. Northern and Western blot analyses found that at pH 7.0 the transcriptional level and the amount of F1 beta subunit were similar in both strains, suggesting that the mutant has a defective ATPase structural gene. On the other hand, at pH 4.5 the transcriptional level and the amount of F1 beta subunit were found to be significantly higher in both strains than those at pH 7.0. From these results, it was suggested that the mutant has a normal regulation system for ATPase gene expression. It was concluded that the mutant is acid sensitive due to its inability to extrude protons out of the cell with defective ATPase under acidic conditions.
Amachi
S
S
Department of Bioscience and Chemistry, Faculty of Agriculture, Hokkaido University, Sapporo, Japan.
Ishikawa
K
K
Toyoda
S
S
Kagawa
Y
Y
Yokota
A
A
Tomita
F
F
D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002462:Cell Membrane / Q000201:enzymology; D003593:Cytoplasm / Q000737:chemistry / Q000502:physiology; D015342:DNA Probes / Q000737:chemistry; D004269:DNA, Bacterial / Q000737:chemistry; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005285:Fermentation; D015964:Gene Expression Regulation, Bacterial; D006863:Hydrogen-Ion Concentration; D013294:Lactococcus lactis / Q000201:enzymology* / Q000235:genetics / Q000648:ultrastructure; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012329:RNA, Bacterial / Q000737:chemistry; D011817:Rabbits; D012588:Scintillation Counting; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D015342:DNA Probes; D004269:DNA, Bacterial; D012329:RNA, Bacterial; D006180:Proton-Translocating ATPases