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In vivo roles of the germination-specific lytic enzymes of Bacillus subtilis 168

Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK¹

Author for correspondence: Simon J. Foster. Tel: +44 114 2224411. Fax: +44 114 2728697. e-mail: s.foster{at}sheffield.ac.uk

Germination of endospores of Bacillus subtilis involves the activities of several germination-specific lytic enzymes, including glucosaminidase and lytic transglycosylase. Another non-hydrolytic activity, likely to be due to an epimerase, also occurs. The effect of pH on enzyme activities and the overall germination rate was measured. Optimal germination occurred between pH 7–9; however, optimum glucosaminidase and epimerase activities were noted at pH 5. Conversely, the lytic transglycosylase activity was greatest at pH 8. Treatment of spores (15 min) with heat (90 °C) or NaOH (0·25 M) led to impaired cortex hydrolysis/modification, but with <20% loss in viability. Analysis of muropeptides in the germination exudate revealed a reduction of >85% in glucosaminidase and epimerase products, when compared to untreated spores. Conversely, lytic transglycosylase activity was increased by alkali or heat treatment, which was possibly due to increased substrate availability. FB101 (sleB) spores, which lack lytic transglycosylase activity, showed 90-fold greater loss in viability than the wild-type after 1 h at 90 °C. Similarly, 97% of FB101 (sleB) spores were unable to form a colony on nutrient agar after 130 min exposure to 0·25 M NaOH at 4 °C, whereas the wild-type was unaffected. This demonstrates the crucial role of the lytic transglycosylase in cortex hydrolysis of damaged spores. The respective targets of heat and alkali in spores and their role during germination are discussed.

Keywords: spores, peptidoglycan, cortex, resistance, lytic transglycosylase

Abbreviations: AGFK, a mixture of L-asparagine, glucose, fructose and KCl; A₂pm, diaminopimelic acid; GSLE, germination-specific lytic enzyme

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