Biological function of gramicidin: studies on gramicidin-negative mutants.

Abstract

By the use of a rapid radioautographic screening rocedure, two mutants of Bacillus brevis ATCC 8185 that have lost the ability to produce gramicidin have been isolated. These mutants produced normal levels of tyrocidine and sporulated at the same frequency as the parent strain. Their spores, however, were more heat-sensitive and had a reduced 4ipicolinic acid content. Gramicidin-producing revertants occurred at a relatively high frequency among tie survivors of prolonged heat treatment and had also regained the ability to produce heat-resistant spores. A normal spore phenotype could also be restored by the addition of gramicidin to cultures of the mutant strain at the end of exponential growth. On the other hand, the addition of dipicolinic acid could not cure the spore defect. These results provide strong evidence that the inability to produce gramicidin is directly responsible for the observed spore defects. Indeed, they unambiguously demonstrate a function of a peptide antibiotic in bacterial sporulation. The possibility that this function consists of the regulation of transcription during the transition from growth to sporulation is discussed.The biological function of peptide antibiotics has been the subject of much speculation and debate, the hypotheses proposed ranging from antibacterial defense (1, 2) and detoxication of unwanted metabolites (3,4) to the regulation of bacterial sporulation (5, 6). We favor a regulatory role in sporulation, which is consistent with the observations that peptide antibiotics are produced.during the transition from vegetative growth to sporulation and that they inhibit vegetative growth of the producing organism. More specifically, we have postulated that peptide antibiotics regulate RNA synthesis during the early stages of sporulation by selectively inhibiting the transcription of genes that function only during vegetative growth (6, 7).This hypothesis has been examined in some detail in Bacillus brevis ATCC 8185, which produces two kinds of antibiotics: gramicidin, a family of pentadecapeptides with blocked aminoand carboxyl-termini, and tyrocidine, a family of cyclic decapeptides (8). Studies with purified RNA polymerase have revealed that both gramicidin (6, 7, 9) and tyrocidine (6, 9, 10) inhibit transcription at concentrations at which they are produced early during sporulation. Moreover, gramicidin was found to inhibit specifically the interaction of RNA polymerase with DNA (7), consistent with the regulation of promoter recognition. Nevertheless, experiments of this kind cannot provide conclusive proof for a regulatory function of antibiotics-this must come from the study of mutants that have a specific lesion in antibiotic synthesis.In this paper, we describe the isolation and characterization of mutants of B. brevis that have lost the ability to produce gramicidin. Our results show that these mutants produce defective spores, and that this defect can be cured by the addition of gramicidin during the early stages of sporulation.EXPERIMENTAL PROCEDURE Bacter...

Interfacial properties of gramicidin and gramicidin-lipid mixtures measured with static and dynamic monolayer techniques

“…Linear gramicidins are polypeptide antibiotics produced by Bacillus brevis (ATCC 8185) (1). These peptides have been proposed to be involved in bacterial sporulation by interaction with the transcriptional process during sporogenesis (2)(3)(4)(5). Despite this intriguing biological function gramicidins are especially known for their ability to specifically facilitate passive diffusion of protons and alkali cations through membranes (6,7).…”

Peptide Antibiotics and Sporulation: Induction of Sporulation in Asporogenous and Peptide-negative Mutants of Bacillus brevis

“…Attempts have been made to isolate such mutants, but the mutants obtained in both the laboratory of H. Paulus (personal communication) and in our laboratory were, in most cases, unstable and reverted easily. However, Mukherjee & Paulus (1977) were the 0022-1287/84/000l-1366 $02.00 0 1984 SGM first to report a correlation between anti biotic synthesis and sporulation by using gramicidinnegative mutants. They showed that these mutants failed to produce intact spores unless the cells were treated with gramicidin within a critical time period of growth.…”

srfA is an operon required for surfactin production, competence development, and efficient sporulation in Bacillus subtilis

“…Certain antibiotic-negative mutants had been shown either to be defective in sporulation or to produce heat-sensitive spores (17,19). In each case, the sporulation defect could be corrected by the addition of the antibiotics exogenously.…”