Title

Identification of a new protein involved in Bacillus subtilis spore germination and study of its gene expression, localization and cross-linking into the spore coat

Date of Completion

January 2004

Keywords

Biology, Molecular|Biology, Microbiology|Chemistry, Biochemistry

Degree

Ph.D.

Abstract

Bacillus subtilis is a gram-positive, soil organism that when starved for one or more nutrients can initiate the developmental program of sporulation that eventually leads to the production of metabolically dormant spores. These spores can survive for long periods of time in the environment. However, in response to particular nutrients, termed germinants, the spores can come back to life through the process of germination. It was reported many years ago that in addition to nutrients, a 1:1 chelate of Ca2+ -pyridine-2, 6-dicarboxylic acid [dipicolinic acid (DPA)] can also trigger spore germination. The DPA is present in the core region of the dormant spores and is released early in germination. Previous studies have shown that the Ca2+-DPA that is released early in spore germination can activate the cortex lytic enzyme, CwlJ. The purpose of this work was to isolate mutants of B. subtilis spores that are unable to germinate with Ca2+-DPA. A random mutagenesis was followed by mapping and sequencing of the identified genes. This work identified a mutant allele of CwlJ and two other mutants, in which an unknown gene, ywdL, was prematurely terminated. Subsequent work showed that the YwdL (GerQ) protein is required for CwlJ function, at least because it is needed for assembly of CwlJ into the spore. Further analysis showed that GerQ is a spore coat protein that is synthesized early in sporulation and assembles into the spore coat later in development. Moreover, GerQ is cross-linked into complexes that are resistant to extraction and their formation was found to be dependent on the activity of a transglutaminase. GerQ is the first identified substrate for the transglutaminase, although the functional significance of GerQ cross-linking is not yet clear. Cross-linking of GerQ was found to occur very late in sporulation, after the release of the spore from the mother cell. Further study of GerQ may prove useful for learning more about spore coat protein cross-linking and the activity of the transglutaminase. ^