Expression of yeast mitochondrial aconitase in Bacillus subtilis

Abstract

Expression of yeast mitochondrial aconitase (Aco1) in a Bacillus subtilis aconitase null mutant restored aconitase activity and glutamate prototrophy but only partially restored sporulation. Late sporulation gene expression in the Aco1-expressing strain was delayed.

FIG. 1.

Genotypic description of strains. The citB locus was either wild type or contained a spectinomycin resistance gene insertion, which results in a B. subtilis aconitase null mutant. The amyE locus was either wild type or contained various integrated DNAs, either the yeast mitochondrial aconitase gene (aco1) and an antibiotic resistance gene or an antibiotic resistance gene alone. unk represents an unlinked chromosomal mutation required for Aco1 function in B. subtilis.

FIG. 2.

Immunoblots with antibodies to yeast mitochondrial aconitase, Aco1, or to B. subtilis aconitase. Growth was at 30°C in DSM (A and C) (7) or minimal medium (B). Cells were isolated upon entry into stationary phase, and cell extracts were analyzed by immunoblotting with Aco1 antibody (A and B) or with antibody to B. subtilis aconitase (C). Lanes 1 and 4 show S. cerevisiae cell extract. The remaining lanes contain cell extracts from B. subtilis: lane 2, MAB160 (citB::spc); lane 3, AWS141 (citB::spc Pspac*-aco1 unk); lane 5, wild type (citB⁺); lane 6, AWS141(citB::spc Pspac*-aco1 unk); lane 7, AWS41 (citB::spc Δ aco1 unk); lane 8, JH642 (citB⁺); lane 9, AWS141 (citB::spc Pspac*-aco1 unk); lane 10, MAB160 (citB::spc). B. subtilis aconitase is a protein of 99 kDa but has the mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of a protein of approximately 120 kDa (6).

FIG. 3.

Kinetics of appearance of aconitase activity during stationary phase. Strains JH642, AWS141, MAB160, and AWS41 were grown in DSM, and cells were isolated at the time points indicated, in hours (T = 0, time of entry into stationary phase). Cell extracts were prepared and analyzed for aconitase activity (6), reported as units per milligram of protein.