Characterization of Escherichia coli ATP synthase .beta.-subunit mutations using a chromosomal delection strain

Abstract

(1) We constructed Escherichia coli strain JP17 with a deletion in the ATP synthase beta-subunit gene. JP17 is completely deficient in ATP synthase activity and expresses no beta-subunit. Expression of normal beta-subunit from a plasmid restores haploid levels of ATP synthase in membranes. JP17 was shown to be efficacious for studies of beta-subunit mutations. Site-directed mutants were studied directly in JP17. Randomly generated chromosomal mutants were identified by PCR and DNA sequencing, cloned, and expressed in JP17. (2) Eight novel mutations occurring within the putative catalytic nucleotide-binding domain were characterized with respect to their effects on catalysis and structure. The mutations beta C137S, beta G152D, beta G152R, beta E161Q, beta E161R, and beta G251D each impaired catalysis without affecting enzyme assembly or oligomeric structure and are of interest for future studies of catalytic mechanism. The mutations beta D301V and beta D302V, involving strongly conserved carboxyl residues, caused oligomeric instability of F1. However, growth characteristics of these mutants suggested that neither carboxyl side chain is critical for catalysis. (3) The mutations beta R398C and beta R398W rendered ATP synthase resistant to aurovertin, giving strong support to the view that beta R398 is a key residue in the aurovertin-binding site. Neither beta R398C or beta R398W impaired catalysis significantly.

Random Insertion of a TetR-Inducing Peptide Tag into Escherichia coli Proteins Allows Analysis of Protein Levels by Induction of Reporter Gene Expression

“…In contrast to the atpD ϩ strain, WH851 displayed no growth on agar plates containing minimal medium with succinate as the sole carbon source (data not shown). Since growth on this medium depends on a functional oxidative phosphorylation system, this phenotype is consistent with a nonfunctional ATP synthase (14,18). This assumption was further supported by the growth phenotype in liquid minimal medium supplemented with limiting amounts of glucose as the sole carbon source (18,34); in this medium, WH851 reached a maximal optical density of 0.3, in contrast to an optical density of 1.0 for the atpD ϩ reference strain (data not shown), again indicating the absence of oxidative phosphorylation.…”

Studies of nucleotide binding to the catalytic sites of Escherichia coli βY331W-F 1 -ATPase using fluorescence quenching

“…To generate ␤Y331W mutation (27), a mutant construct p3U␤Y331W was obtained using mutagenic oligonucleotide 5Ј-TGGGTATCTGGCCGGCCGTT, with bases in italic generating the mutation and p3U ϩ plasmid (51) as a template and expressed in JP17 strain (52).…”

Genetic and Biochemical Characterization of the F-ATPase Operon from S treptococcus sanguis 10904

“…We examined the ability of the cloned ATPase genes to complement defects in E. coli strain JP17, an atpD deletion mutant (38). Strain JP17 was transformed with selected plasmids containing streptococcal DNA (Table 1); the resulting strains were then assayed for their ability to release inorganic phosphate from ATP.…”