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Journal of Bacteriology, April 1999, p. 2373-2378, Vol. 181, No. 8
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Expression of a Germination-Specific Amidase, SleB, of Bacilli in the Forespore Compartment of Sporulating Cells and Its Localization on the Exterior Side of the Cortex in Dormant Spores

Ryuichi Moriyama,1,* Hideyuki Fukuoka,1 Shigeru Miyata,1 Sachio Kudoh,1 Atsuhiko Hattori,1 Satoshi Kozuka,2 Yoko Yasuda,2 Kunio Tochikubo,2 and Shio Makino1

Department of Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Aichi 464-8601,1 and Department of Microbiology, Nagoya City University Medical School, Nagoya, Aichi 467-8601,2 Japan

Received 21 September 1998/Accepted 26 January 1999

A germination-specific amidase of bacilli is a major spore-lytic enzyme that is synthesized with a putative signal sequence and hydrolyses spore cortex in situ. The sleB gene encoding this amidase in Bacillus subtilis and Bacillus cereus was expressed in the forespore compartment of sporulating cells under the control of sigma G, as shown by Northern blot and primer extension analyses. The forespore-specific expression of B. subtilis sleB was further indicated by the forespore-specific accumulation of a SleB-green fluorescent protein fusion protein from which a putative secretion signal of SleB was deleted. Immunoelectron microscopy with anti-SleB antiserum and a colloidal gold-immunoglobulin G complex showed that the enzymes from both Bacillus species are located just inside the spore coat layer in the dormant spore, and in the dormant spore, the amidases appear exist in a mature form lacking a signal sequence. These results indicate that SleB is translocated across the forespore's inner membrane by a secretion signal peptide and is deposited in cortex layer synthesized between the forespore inner and outer membranes. The peripheral location of the spore-lytic enzymes in the dormant spore suggests that spore germination is initiated at the exterior of the cortex.

* Corresponding author. Mailing address: Department of Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Aichi 464-8601, Japan. Phone: 81 (52) 789-4134. Fax: 81 (52) 789-4120. E-mail: moriyama{at}agr.nagoya-u.ac.jp.

Journal of Bacteriology, April 1999, p. 2373-2378, Vol. 181, No. 8
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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