Correlation between cytochrome aa3 concentrations and streptomycin accumulation in Bacillus subtilis

Abstract

Accumulation of aminoglycosides by Bacillus subtilis appears to require specific components of the electron transport chain. These components include cytochromes and the lipophilic quinone vitamin K2. The present study concerns the importance of cytochrome aa3, a terminal oxidase, in the uptake of streptomycin. Growth conditions have been established such that the concentration of cytochrome aa3 can be modified over a wide range; on defined minimal salts agar, the wild-type strain (RB1) and an strC mutant (RB95) synthesized cytochrome aa3 only when adequate amounts of Casamino Acids (Difco Laboratories, Detroit, Mich.) were present. A positive correlation between cytochrome aa3 levels and streptomycin accumulation was observed. The same correlation was seen when cytochrome aa3 was measured in relation to growth susceptibility. These correlations suggest that cytochrome aa3 Is necessary for accumulation of streptomycin by B. subtilis.

Structure and expression of the cytochrome aa3 regulatory gene ctaA of Bacillus subtilis

“…The strC2 mutation reduces the level of ctaA'-lacZ fusion activity. strC mutants were isolated as spontaneous streptomycin-resistant colonies (25) and have been found to contain only 40% of the wild-type complement of cytochrome aa3 (16,27). The streptomycin-resistant phenotype has been correlated with the unique deficiency in cytochrome aa3 (3,16).…”

“…strC mutants were isolated as spontaneous streptomycin-resistant colonies (25) and have been found to contain only 40% of the wild-type complement of cytochrome aa3 (16,27). The streptomycin-resistant phenotype has been correlated with the unique deficiency in cytochrome aa3 (3,16). To examine any possible relationship between strC and ctaA, the ctaA'-lacZ transcriptional fusion was integrated into strain RB95 (strC2), creating strain RB1021, and ,Bgalactosidase activities were measured in 2 x NS broth during growth and sporulation.…”

Isolation and sequence of ctaA, a gene required for cytochrome aa3 biosynthesis and sporulation in Bacillus subtilis

“…Growth conditions have been established that repress or enhance the cellular levels of the enzyme (28). Systematic increases in cytochrome aa3 levels brought about by the addition of vitamin-free Casamino Acids (Difco) to growth media result in a corresponding increase in growth inhibition by streptomycin and the rate of uptake of the antibiotic (28). Cytochrome aa3 deficiency has been observed in a specific class of streptomycin-resistant (Strr) mutants (strC) of B. slubtilis (28,43,48).…”

“…A selection strategy to isolate cytochrome aa3-deficient mutants of B. siubtilis was based on the observation that B. subtilis strains phenotypically deficient in cytochrome aa3 do not accumulate streptomycin (2,28). Growth conditions have been established that repress or enhance the cellular levels of the enzyme (28).…”

“…Cytochromes aa3 and o function as terminal oxidases (13,15), although the physiological roles played by the two oxidase-containing branches of the respiratory chain are not clear. In addition, the existence of a cytochrome d in B. subtilis has been suggested by detection of an absorbance maximum at 617 nm (28,47). Cytochrome aa3 catalyzes electron transfer from cytochrome c to dioxygen (13,15) and has been purified to homogeneity and extensively characterized (10)(11)(12).…”