GerN, an Antiporter Homologue Important in Germination of Bacillus cereus Endospores

Abstract

A homologue of the grmA spore germination gene of Bacillus megaterium and of a NaH-antiporter gene (napA) of Enterococcus hirae has been identified in Bacillus cereus 569 (ATCC 10876). The putative protein product has 58 and 43% amino acid identity with GrmA and NapA, respectively. Insertional inactivation of this B. cereus gene, named gerN, did not affect vegetative growth or sporulation. The null mutant spores were 30-fold slower to germinate in inosine (5 mM) but germinated almost normally in response to L-alanine (10 mM). The null mutant spores germinated after several hours with inosine as the sole germinant, but germination was asynchronous and the normal order of germination events was perturbed. At a suboptimal germinant concentration (50 M), inosine germination was completely blocked in the mutant, while the rate of germination in 50 M L-alanine was reduced to one-third of that of the wild type. The requirement for GerN function in the response to a particular germinant suggests that a germination receptor may have a specifically associated antiporter, which is required at the initiation of germination and which, in the case of the inosine receptor, is GerN. Since germination in suboptimal concentrations of L-alanine shows a delay, additional germination transporters may be required for optimal response at low germinant concentrations.Under certain nutrient stresses Bacillus species undergo a complex differentiation process resulting in the formation of highly resistant endospores, which subsequently, when favorable growth conditions return, germinate back to vegetative cells and grow and divide. The molecular genetics of sporulation (7, 25) and germination (14, 15) of Bacillus species have been reviewed. Analysis of Bacillus subtilis mutants which are defective in response to one or both of their germinants-L-alanine or asparagine with glucose, fructose, and K ϩ (AGFK)-suggests that the germinants interact with separate germinant-specific receptor complexes within the spore (14). Mutations within the gerA operon of B. subtilis specifically block germination initiated by L-alanine (34). The predicted amino acid sequences of the three GerA proteins encoded by the gerA operon suggest that they may be membrane associated, and they are the most likely candidates for the germinant receptor for L-alanine. Mutations in the gerB operon (18), responsible for AGFK germination in B. subtilis, allowed recognition of the novel germinant D-alanine; this strongly reinforces the argument that such gerA homologues encode the germinant receptor complexes.B. cereus endospores have been shown to germinate in response to inosine and L-alanine; a combination of these germinants elicits the most rapid response (32). Operons encoding putative receptor complexes for the germinants have been named gerI (6) and gerL (3), respectively, and these operons are members of the gerA family identified in B. subtilis. How the germinant receptors are activated and how this leads to the global changes of spore germination have, however, not...

Analysis of spore cortex lytic enzymes and related proteins in Bacillus subtilis endospore germination

“…The earliest events in spore germination involve receptor proteins encoded by homologues of the H. Chirakkal and others GerAA, AB and AC proteins (Paidhungat & Setlow, 1999 ;Moir et al, 2002), which are located in the inner membrane (Hudson et al, 2001 ;). Receptor-germinant interaction results in the triggering of a cascade of biochemical events, including loss of heat resistance and ion movements from the core (Thackray et al, 2001 ;Southworth et al, 2001). Hydrolysis of spore cortex by cortex-specific lytic enzymes is a later event in germination (Popham et al, 1996 ;Boland et al, 2000).…”

GerO, a Putative Na + /H + -K + Antiporter, Is Essential for Normal Germination of Spores of the Pathogenic Bacterium Clostridium perfringens

“…Indeed, a member of the CPA-2 monovalent cation-proton antiporter family of membrane transport proteins (27), GrmA, is essential for germination of B. megaterium ATCC 12872 spores (34), since grmA inactivation makes spores unable to release their DPA and complete germination with a variety of germinants. Similarly, in Bacillus cereus ATCC 10876, a GrmA-type homologue, GerN, is essential for spore germination with inosine but not L-alanine (35), and studies with everted vesicles have shown that GerN possesses electrogenic Na ϩ /H ϩ -K ϩ antiporter activity (32). The GerN homolog, GerT, also plays a minor role in B. cereus spore germination with inosine, as well as a major role in spore outgrowth under some conditions (29).…”

Functional Consequences of Amino Acid Substitutions to GerVB, a Component of the Bacillus megaterium Spore Germinant Receptor

“…Despite this extreme dormancy, spores retain the ability to initiate vegetative metabolism rapidly upon exposure to appropriate nutrients via the process of germination (20,30). Significant advances have been made in recent years through the application of mainly genetic techniques to characterize the molecular apparatus involved in spore germination, identifying structural genes that encode germinant receptors (4,8,14,23,37), ion channels (31,35), and hydrolytic enzymes involved in the degradation of the cortical peptidoglycan that surrounds the spore protoplast (6,11,16,21). Cytological observations on spore coat degradation during germination (29) have also recently been extended by the application of atomic force microscopy to provide insights to the structural basis for the disassembly of the outer layers that provide the primary barrier to environmental insult (26).…”