Cooperativity between different nutrient receptors in germination of spores of Bacillus subtilis and reduction of this cooperativity by alterations in the GerB receptor

Abstract

The GerA nutrient receptor alone triggers germination of Bacillus subtilis spores with L-alanine or L-valine, and these germinations were stimulated by glucose and K+ plus the GerK nutrient receptor. The GerB nutrient receptor alone did not trigger spore germination with any nutrients but required glucose, fructose, and K+ (GFK) (termed cogerminants) plus GerK for triggering of germination with a number of L-amino acids. GerB and GerA also triggered spore germination cooperatively with l-asparagine, fructose, and K+ and either L-alanine or L-valine. Two GerB variants (termed GerB*s) that were previously isolated by their ability to trigger spore germination in response to D-alanine do not respond to D-alanine but respond to the same L-amino acids that stimulate germination via GerB plus GerK and GFK. GerB*s alone triggered spore germination with these L-amino acids, although GerK plus GFK stimulated the rates of these germinations. In contrast to l-alanine germination via GerA, spore germination via L-alanine and GerB or GerB* was not inhibited by D-alanine. These data support the following conclusions. (i) Interaction with GerK, glucose, and K+ somehow stimulates spore germination via GerA. (ii) GerB can bind and respond to L-amino acids, although normally either the binding site is inaccessible or its occupation is not sufficient to trigger spore germination. (iii) Interaction of GerB with GerK and GFK allows GerB to bind or respond to amino acids. (iv) In addition to spore germination due to the interaction between GerA and GerK, and GerB and GerK, GerB can interact with GerA to trigger spore germination in response to appropriate nutrients. (v) The amino acid sequence changes in GerB*s reduce these receptor variants' requirement for GerK and cogerminants in their response to L-amino acids. (vi) GerK binds glucose, GerB interacts with fructose in addition to L-amino acids, and GerA interacts only with L-valine, L-alanine, and its analogs. (vii) The amino acid binding sites in GerA and GerB are different, even though both respond to L-alanine. These new conclusions are integrated into models for the signal transduction pathways that initiate spore germination.

FIG. 1.

Germinants and cogerminants for different germinant receptors and d-alanine inhibition of these receptors in B. subtilis spores as known at the beginning of this work. Abbreviations used for germinant receptors (boxed letters) are as follows: A, GerA; B, GerB; B*, GerB*; K, GerK. Abbreviations for germinants, cogerminants, and inhibitors are as follows: A, l-alanine; dA, d-alanine; f, d-fructose; g, d-glucose; k, K⁺; N, l-asparagine; V, l-valine. Germinants are defined as molecules that alone will trigger spore germination. Cogerminants are molecules that stimulate germination with germinants but do not trigger germination alone. The symbol “/” indicates “or,” and there is disagreement in the literature over the identity of the cogerminants in parentheses. Question marks indicate that information is not available. For d-alanine inhibition, y (yes) and n (no) are under the germinant for which inhibition data are available. Note that GFK and l-alanine or l-asparagine are all listed as cogerminants for GerB plus GerK, as none of these compounds alone trigger germination via these two receptors. The designation of glucose as a cogerminant for GerB* and GerK with a question mark adjacent to K is because the GerK requirement for the effect of glucose had not yet been tested (data for this figure are from references , , , and 27).

FIG. 2.

Spore germination via GerA, GerBA*, or GerBB* as a function of the concentration of l-alanine. Spores of strains FB87 (has only GerA), PS3710 (has only GerBA*), and PS3665 (has only GerBB*) were germinated with l-alanine, and rates of spore germination were determined as described in Materials and Methods. The symbols for the spores used are as follows: •, FB87 (has only GerA); ▴, PS3710 (has only GerBA*); and ○, PS3665 (has only GerBB*).

FIG. 3.

Dependence of the rate of germination of gerBB* spores on the concentrations of various amino acids. Spores of strain PS3665 (has only GerBB*) were heat shocked and germinated with various concentrations of different amino acids, and the rates of spore germination were determined as described in Materials and Methods. Maximum rates of spore germination were taken as those at 10 mM amino acid. The symbols for the amino acids used are as follows: ○, l-alanine; •, l-asparagine; ▪, l-serine; ▴, d-alanine; □, glycine; and ▾, l-threonine.

FIG. 4.

Amino acid sequences in regions of GerA, GerB, and GerK in which amino acid changes give GerB* variants. The amino acid sequence changes in B. subtilis GerBA* and GerBB* were described previously (20). Residues that are identical in the GerA, GerB, and GerK receptors are shown in boldface type.

FIG. 5.

Alternative models for signal processing in B. subtilis spore germination based on both previous and current work. (A) The receptors can operate either alone or when complexed with other receptors to give a particular rate of spore germination. While only complexes of two receptors are shown, all three receptors could be in a complex in this model. The short arrows beneath GerB and GerK indicate that these receptors alone give little if any germination. (B) The signals from individual receptors, which may be very different in intensity, are fed into a hypothetical signal integrator that integrates and/or sums all signals to generate an output that is a particular rate of spore germination. The specific role of K⁺ is not shown in either A or B, since K⁺ may interact with all three receptors. Abbreviations used for receptors are in boxes and are as follows: A, GerA; B, GerB; B*, GerB*; K, GerK. Abbreviations used for germinants or cogerminants are as follows: A, l-alanine; AA, l-alanine, l-asparagine, l-serine, or l-threonine; f, fructose; g, glucose; V, l-valine. The symbol “/” indicates “or.” Cogerminants are shown in parentheses above the affected receptor; germinants are also shown above the affected receptor.