Netropsin stimulates the formation of an extracellular proteinase and suppresses protein turnover in sporulating Bacillus megaterium

Abstract

Netropsin stimulated the rate of synthesis of an extracellular metalloproteinase in Bacillus megaterium incubated in a sporulation medium. The antibiotic delayed but did not suppress the decrease in the ability to synthesize the proteinase occurring at later sporulation stages. Netropsin also stimulated the synthesis of the proteinase when added to a growing culture; it inhibited the increase of protein turnover which was switched on between the 2nd and 3rd hour in the sporulating population. No refractile spores were developed during 6 h at 35°C in the antibiotic-treated culture. In the control 60% of sporulating cells were observed under similar conditions. INTRODUCTIONThe peptidic antibiotic netropsin inhibits sporulation in Bacillus subtilis at concentrations exerting no or negligible effect on growth [1]. The antibiotic seems to suppress the sporogenesis be-* To whom correspondence and reprint requests should be addressed.fore or at the beginning of stage II [2]. Netropsin interacts with the A + T-rich sequences of DNA but it is not intercalated into the DNA molecule as is the case with actinomycin D [3,4]. Netropsin binds within the minor groove of DNA by displacing the water molecules [5]. The antibiotic was found not to affect the synthesis of several enzymes considered to be related to the sporogenesis in B. subtilis (e.g. glucose dehydrogenase and alkaline phosphatase) and it stimulated slightly the formation of the aconitase. However, the excretion of proteinases was decreased and the formation of the coat protein and of the dipicolinic acid characteristic for the later sporulation phases, was suppressed [1,6,7]. The activity of a sporulationspecific form of the RNA polymerase was also decreased by netropsin [8]. B. megaterium synthesizes an extracellular metalloproteinase which is formed during growth as well as during sporulation [9], unlike the metalloand serine proteinases of B. subtilis. The formation of proteinase during growth of B. megaterium is controlled by repression with amino acids, the carbon and energy sources having only a slight effect. However, its synthesis during sporulation seems to be partially under developmental control [101.

Expression of dnaK and groESL operons during sporulation of Bacillus megaterium

“…3. In both cases sporulation was blocked during the reversible phase [10,12]. The culture was grown at 35³C in SYG supplemented with 1 Wg ml 3I netropsin.…”

“…1 h after netropsin addition, when the population had reached a density of 0.4^0.5 mg ml 3I dry mass, a portion of the population was exposed to a 15-min heat shock at 45³C. However, the growth-and sporulation-permissive temperature used in experiments with netropsin and the asporogenic mutant was increased to 35³C, used in previous studies with the antibiotic or this mutant [10,12]. Portions of the culture were treated with a 10-min heat shock at 45³C at T H , T P and T R .…”

“…Cultivation was carried out in growth (SYG) or sporulation (SM) medium [11] in a shaking water bath at 27 or 35 þ 0.5³C, as described in the text. Sporulation was inhibited by 1 Wg ml 3I netropsin (Serva) [12]. Sporulation was inhibited by 1 Wg ml 3I netropsin (Serva) [12].…”

Netropsin increases formation of mRNA coding for a neutral metalloproteinase in Bacillus megaterium

“…Electronmicroscopical analysis of sporulating cells indicates that the antibiotic may affect the onset of the sporulation process (BEAMAN et al 1980). We found that netropsin increased the formation of an extracellular proteinase whose synthesis takes place during the first hours of sporulation, and delayed its switching off at later sporulation phases (KUCEROVA et al 1986). The antibiotic also stimulated the synthesis of the proteinase mRNA during growth.…”

“…Netropsin is an antibiotic with cytostatic properties which inhibits sporulation in Bacillus subtilis and Bacillus megaterium without impairing substantially the growth (KEILMAN et al 1975, KUCEROVA et al 1986). Electronmicroscopical analysis of sporulating cells indicates that the antibiotic may affect the onset of the sporulation process (BEAMAN et al 1980).…”