Isolation and characterization of new facultatively alkalophilic strains of Bacillus species

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Abstract
Four facultatively alkalophilic isolates were purified from enrichment cultures initiated with lime-treated garden soil. Four isolates, OF1, OF3, OF4, and OF6, were obligately aerobic, spore-forming, gram-positive, motile rods which were capable of growth at both pH 7.5 and pH 10.5. Strains OF1 and OF6 grew best at the lower pH value; and whereas growth of these strains at pH 10.5 was completely dependent on added Na+, growth at pH 7.5 was only partially dependent on added Na+. Strains OF3 and OF4 grew better at pH 10.5 than at pH 7.5, with strain OF3 growing modestly over its entire pH range, while OF4 grew well. Growth of OF3 and OF4 was completely dependent on added Na+ at both pH 7.5 and pH 10.5. DNA-DNA hybridization studies indicated that OF1 and OF6 are closely related strains but are not related to the other isolates, Bacillus subtilis, or two previously studied obligately alkalophilic bacilli. OF3 was unrelated to any of the other organisms examined in the study, whereas OF4 showed complete homology with obligately alkalophilic Bacillus firmus RAB. All four isolates maintained a cytoplasmic pH that was considerably lower than the external pH when the latter was 10.5. Although substantial transmembrane electrical potentials were observed, the total electrochemical proton gradient (delta mu H+) was low at pH 10.5 in all the strains. By contrast, delta mu H+ was substantial at pH 7.5 and at that pH was composed entirely of an electrical potential. These results are in contrast to previous findings that obligately alkalophilic bacilli generate only small electrical potentials at near neutral pH. All the isolates exhibited substantial rates of respiration as measured by oxygen consumption. Neither respiration nor NADH oxidation by everted membrane vesicles was significantly stimulated by Na+. Analyses of reduced versus oxidized difference spectra of membranes from OF4 showed that the total membrane cytochrome content was considerably higher in cells grown at pH 10.5 than at pH 7.5, with the levels of c- and a-type cytochromes exhibiting the largest pH-dependent differences. Initial examination of membrane protein profiles on gel electrophoresis also indicated a number of changes in pattern in each isolate, depending on the growth pH.