The biosynthesis of poly d-glutamic acid, the capsular material of Bacillus anthracis.

Abstract

SUMMARY Capsules of Bacillus anthracis only begin to be formed towards the end of exponential growth and appear first at the extremities of the cells. Once begun, capsule formation is not inhibited by tetracycline, so that capsular polypeptide is not synthesized like protein. Cultures were grown in broth + albumin in the presence of 0.015 M-HCO3—and 5 vol. CO2 + 95 vol. air until capsulation began, and were then incubated in air in tetracycline broth (to inhibit subsequent enzyme formation): capsules continued to increase in size, which suggested that HCO3—made capsular synthesis possible but was not required for the formation of the polypeptide itself. Cultures transferred from air to adequate concentrations of CO2 did not immediately become capsulated, whatever their stage of growth. Mutants with altered nutritional requirements for capsulation were selected by phage α from wild-type strains grown either (1) on charcoal agar in air, so selecting for CO2-independence (D mutants), or (2) on bicarbonate agar incubated in CO2. so selecting for independence of an absorbent (F mutants). Both classes formed capsules in air (≃ 0.0001 M-HCO3-) and the capsular polymer apparently had the same chemical structure as that of their parent. No evidence was found that the mutants differed from their parent in being able to fix HCO3—more efficiently or in being able to utilize compounds normally derived from HCO3—that were present in the medium. They may therefore arise following mutation in regulatory genes controlling capsular synthesis. Some D mutants grew very slowly in CO2 but yielded CO2-resistant mutants of which 66 were examined: 44 resembled the parental strain 2160s, and 22 were rough (C—). CO2-sensitive D mutants also gave rise to derivatives which formed rough colonies in air: some resembled strain 2160s; others were C—; but some had a new phenotype in being rough in air, fully capsulated in 0.006-0-015 M-HCO3—and inhibited by 0.03 M-HCO3 -. Absorbents are required by wild-type strains to inactivate the long-chain fatty acids that occur in nutrient media. These acids are thought to interfere with the uptake or utilization of HCO3—, rather than with a later stage in capsular synthesis, because (1) they do not inhibit capsulation of D or F mutants, (2) they enable CO2-sensitive D mutants to grow in CO2, and (3) they probably interfere with toxin formation, which also requires CO2. As toxin is protein it must be synthesized differently from capsular polypeptide, and the only stage common to both pathways is therefore likely to be HCO3—uptake.