Genetic and biochemical characterization of the F-ATPase operon from Streptococcus sanguis 10904.
Oral streptococci utilize an F-ATPase to regulate cytoplasmic pH. Previous studies have shown that this enzyme is a principal determinant of aciduricity in the oral streptococcal species Streptococcus sanguis and Streptococcus mutans. Differences in the pH optima of the respective ATPases appears to be the main reason that S. mutans is more tolerant of low pH values than S. sanguis and hence pathogenic. We have recently reported the genetic arrangement for the S. mutans operon. For purposes of comparative structural biology we have also investigated the F-ATPase from S. sanguis. Here, we report the genetic characterization and expression in Escherichia coli of the S. sanguis ATPase operon. Sequence analysis showed a gene order of atpEBFHAGDC and that a large intergenic space existed upstream of the structural genes. Activity data demonstrate that ATPase activity is induced under acidic conditions in both S. sanguis and S. mutans; however, it is not induced to the same extent in the nonpathogenic S. sanguis. Expression studies with an atpD deletion strain of E. coli showed that S. sanguis-E. coli hybrid enzymes were able to degrade ATP but were not sufficiently functional to permit growth on succinate minimal media. Hybrid enzymes were found to be relatively insensitive to inhibition by dicyclohexylcarbodiimide, indicating loss of productive coupling between the membrane and catalytic subunits.
Kuhnert
Wendi L
WL
Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, USA.
Quivey
Robert G
RG
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D021901:DNA, Intergenic; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D023061:Gene Order; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009154:Mutation; D009876:Operon; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D012689:Sequence Homology, Nucleic Acid; D013295:Streptococcus mutans / Q000235:genetics; D013298:Streptococcus sanguis / Q000201:enzymology / Q000235:genetics*; D024363:Transcription Initiation Site
D021901:DNA, Intergenic; D004791:Enzyme Inhibitors; D004024:Dicyclohexylcarbodiimide; D006180:Proton-Translocating ATPases