Biochemical aspects of germination and outgrowth of Bacillus brevis Nagano and control by gramicidin S [proceedings].

It has been demonstrated that gramicidin S inhibits the outgrowth of spores of the producer-organism Bacillus brevis Nagano (Nandi & Seddon, 1978). Once again we have used the antibiotic-negative mutants E-I and BI-9 (supplied by Professor Y. Saito) in an attempt to relate control by gramicidin S over outgrowth to respiration, RNA synthesis and protein synthesis of the spores. Spores were prepared and germinated in nutrient broth at 37°C with aeration as described previously (Nandi & Seddon, 1978), except that for the present studies a concentration of 2 x lo8 spores/ml was used. The gramicidin S-negative mutants were compared with wild-type spores with respect to biochemical parameters of outgrowth. Outgrowth of the antibiotic-negative mutants after 80min of incubation was accompanied by a simultaneous, rapid increase in respiration that continued to rise until at least 200min (monitored in a Clark oxygen electrode). This was followed by the rapid development of RNA synthesis at loomin, which reached a peak at 180-190min. Protein synthesis developed rapidly at 180min and continued to rise up to 250min. RNAand protein-synthesizing activities were monitored by pulse-labelling culture samples with 14C-labelled precursors (see legend to Table 1). The wild-type spores did not outgrow for at least 250min and respiration, RNA synthesis and protein synthesis remained insignificant during this time. This was also true of mutant spores that germinated in the presence of 1Opg of gramicidin S/ml. It was also discovered that if gramicidin S was removed from the wild-type spores, they would behave as mutant spores. This was effected by immersing an ethanol suspension of spores in a boiling-water bath for 2min and then removing the spores by centrifugation. For a concentration of 10'O spores in 2ml of ethanol, three such extractions were very effective at removing gramicidin S while leaving the viability of the spores relatively unimpaired. The washed, extracted spores would then outgrow after 80min in nutrient broth, and respiration, RNA synthesis and protein synthesis would develop in a similar time-course to the mutant spores. Addition of appropriate concentrations of gramicidin S or wild-type extracts once again restored inhibition of outgrowth and associated activities. Respiration and [14C]uracil uptake are normally well established after 180min and ['4C]lysine uptake is at 2lOmin. However, if gramicidin S is added, at physiological concentrations (10pg/2 x los spores), at or any time before 80min these activities fail to develop. As soon as outgrowth begins the culture becomes increasingly insensitive to gramicidin S. After 140min there is active cell division and the early events of outgrowth, respiration and RNA synthesis are now relatively unaffected by addition of gramicidin S, even at high concentrations (Table 1). In agreement with these observations, we find that gramicidin S does not inhibit ['4C]uracil uptake by vegetative cultures of B. brevis wild-type and E-I. Our studies were based on experiments of Sarkar & Paulus (1972) who found that tyrocidine inhibits RNA synthesis in B. brevis. Thus we have only evidence of a gramicidin S-effect on spores and no evidence for an effect on the vegetative cell. Although respiration is the earliest outgrowth-associated phenomenon that we have observed, it appears to become significant only shortly before the onset of outgrowth in mutant spores germinated in nutrient broth. However, B. brevis Nagano spores suspended in l0mM-potassium phosphate buffer, pH 7.4, are stimulated to germinate by 10mM-L-alanine. Spore germination follows a similar course to that in nutrient broth, but in this simple system it is easier to follow the development of respiration in the very early stages. Respiration can be detected within lOmin of initiation with L-alanine and rises steadily to a concentration of 6 8 nmol of 02/min per lo8 spores after