Amino Acid Residues in the GerAB Protein Important in the Function and Assembly of the Alanine Spore Germination Receptor of Bacillus subtilis 168

Abstract

The paradigm gerA operon is required for endospore germination in response to l-alanine as the sole germinant, and the three protein products, GerAA, GerAB, and GerAC are predicted to form a receptor complex in the spore inner membrane. GerAB shows homology to the amino acid-polyamine-organocation (APC) family of single-component transporters and is predicted to be an integral membrane protein with 10 membrane-spanning helices. Site-directed mutations were introduced into the gerAB gene at its natural location on the chromosome. Alterations to some charged or potential helix-breaking residues within membrane spans affected receptor function dramatically. In some cases, this is likely to reflect the complete loss of the GerA receptor complex, as judged by the absence of the germinant receptor protein GerAC, which suggests that the altered GerAB protein itself may be unstable or that the altered structure destabilizes the complex. Mutants that have a null phenotype for l-alanine germination but retain GerAC protein at near-normal levels are more likely to define amino acid residues of functional, rather than structural, importance. Single-amino-acid substitutions in each of the GerAB and GerAA proteins can prevent incorporation of GerAC protein into the spore; this provides strong evidence that the proteins within a specific receptor interact and that these interactions are required for receptor assembly. The lipoprotein nature of the GerAC receptor subunit is also important; an amino acid change in the prelipoprotein signal sequence in the gerAC1 mutant results in the absence of GerAC protein from the spore.

INTRODUCTION

In the model endospore-former Bacillus subtilis, endospore germination can be induced by exposure of the spores to l-alanine. This is mediated via a germinant receptor, a likely complex of three proteins, encoded by the gerA operon (31). The GerAA and GerAC components of this complex have been found in the inner membranes of the spores by Western blot analysis of spore fractions (14). The coexpressed GerAB protein, a predicted integral membrane protein with 10 membrane spans, would locate with the other two proteins, forming a membrane-associated receptor that initiates germination in response to l-alanine concentrations of ≥10 μM.

Homologous gene clusters, often organized as tricistronic operons, can be identified in the genomes of sporeformers across the genus Bacillus and in most clostridial species. The germinant specificity of various receptors has been elucidated in several species, and in all such cases, these receptors seem to be involved in the triggering of spore germination, individually or in combination. In B. subtilis, four other receptor operons have been identified as well as gerA. The gerB and gerK operons encode receptors that together mediate the germination response to the cogerminant mixture AGFK (l-asparagine, d-glucose, d-fructose, and K⁺ ions) (8). It has been suggested that the GerK receptor has a prominent role in the interaction with sugars, while GerB binds l-amino acids, but not sufficiently to induce germination without adjuncts (1, 18). Two other homologous operons of B. subtilis, yndDEF and yfkQRT, have also been identified in the genome, but nothing is known of their function, and they do not seem to be involved in germination in response to standard nutrients, as they do not contribute to germination on rich laboratory media (22).

The failure so far to successfully overexpress germinant receptor proteins GerAA or GerAB or to isolate these low-abundance proteins from spores has meant that defining the configuration of these proteins individually or as complexes in the spore membrane has not been possible. The manner in which these receptors interact with their germinant and how this interaction activates the process of germination are also undefined. The GerAB protein family, comprising equivalent homologs in other germinant receptor operons, forms one branch of the phylogenetic grouping of the amino acid-polyamine-organocation (APC) single-component membrane transporter family in bacteria, archaea, and yeasts (16) and are a likely site of germinant binding, though probably for signal transduction rather than for bulk transport of germinant (19). Recent analysis of the behavior of a receptor in Bacillus megaterium QM B1551 whose germinant specificity varies with alternative GerUB and GerVB proteins reinforces this interpretation of the role of GerAB family proteins (4, 5).

One advantage to studying the GerA receptor of B. subtilis is that l-alanine can stimulate as the sole germinant and only one receptor complex is needed to trigger this germination response. This means that the influence of other germinant receptors on phenotype is limited, allowing a more conclusive analysis of germination receptor function. This article describes the characterization of the germination behavior of spores carrying a variety of site-directed mutations in the gerAB gene. Some alterations in GerAB, probably those that disrupt the stability of GerAB itself, interfere with the assembly or stability of the GerA germinant receptor complex as a whole, presenting arguably the best evidence thus far that the proteins form a receptor complex. Spores of a gerAC1 mutant, with a defective prelipoprotein processing sequence, also lack GerAC protein, demonstrating that the retention of GerAC protein in the spore is also dependent on its lipoprotein status.

MATERIALS AND METHODS

Strains and culture conditions.

The laboratory Bacillus subtilis 168 strain 1604 (trpC2) was used as the background for all the mutants constructed. The routine culture media were L broth and agar for Escherichia coli strains XL10-Gold (Stratagene) and DH5α. Oxoid nutrient broth and agar were used for B. subtilis. Antibiotic resistance was selected as appropriate; antibiotic resistance in E. coli was selected with ampicillin (100 μg ml⁻¹) and kanamycin (10 μg ml⁻¹), and in B. subtilis, it was selected with kanamycin (5 μg ml⁻¹). Sodium d,l-lactate (used at 0.5% [wt/vol]) was from Fisher Scientific.

Spore preparation.

To prepare washed spore suspensions, gerAB mutant cultures were grown in SG medium (17), with shaking (220 rpm) at 37°C for 48 h. Spores of the parent strain, strain 1604, were always prepared in parallel to act as a control. The harvest, washing, and storage of spore preparations were as previously described (20). Spores were stored at −20°C and tested for germination between 2 and 6 weeks after preparation. For each mutant, two independent spore preparations were examined for their germination properties, and at least two replicate assays were carried out.

Mutant construction.

gerAB mutant alleles AB203, AB205, and AB206 were constructed as described by Mongkolthanaruk et al. (21a), using a Stratagene QuikChange II XL kit to introduce mutations in plasmid pAAM201 and then transferring them to the chromosome of the citG::kan strain AM1651, selecting CitG⁺ transformants on minimal medium containing lactate and then screening them for cotransformation of the mutant allele, which was only 8 to 12% for these gerAB mutations. All other mutations were generated by the more-efficient method described in that paper, using plasmid pAAM210, which carries a larger insert from the citG-gerA region, for the initial mutagenesis. The mutations were then transferred, by transformation with linearized DNA, to the chromosome of strain AM1720 Δ(citG-gerA)::kan, where the citG-gerA region, extending from an NcoI site 28 bp into the citG gene to a BglII site 526 bp into the gerAC gene, has been replaced by a kanamycin resistance cassette, introduced as a NcoI-BglII fragment from pDG792 (6). This deletion leaves ca. 1 kb of citG and 460 bp of gerAC to provide homology for the double crossover from pAAM210. Details of the oligonucleotides and the precise base substitutions used in the mutagenesis procedures (6) are available on request. Cit⁺ transformants were selected, purified, and checked for kanamycin sensitivity to confirm replacement of the cassette with a complete gerA operon. DNA sequencing was used to check that the mutations introduced into pAAM210 were as expected, and the gerAB gene was resequenced after transfer of this region to the chromosome.

Spore germination.

Spores were heat activated at 70°C for 30 min and cooled in ice before the addition of germinants. Spores were germinated at an optical density at 600 nm (OD₆₀₀) of 0.6 in 1 mM l-alanine, 20 mM KCl, and 10 mM Tris-HCl (pH 7.6) unless stated otherwise. To measure the dependence on concentration, concentrations of l-alanine from 1 μM to 100 mM were used. Germination was carried out at 37°C, and the change in OD₄₉₀ of the suspension was measured every 2 min for 100 min using a VICTOR² 1420 multilabel counter (Wallac). Spores tested (mutants and wild type [WT]) were prepared at the same time under identical conditions of harvesting and washing, and germination experiments were carried out in parallel.

Assessment of germination rates.

The OD of a spore suspension will decrease to approximately half of its initial level if all the spores in the population germinate. The measurement of germination in this way reflects a heterogeneous time to response of the individual spores in the population. The study of germination in single spores has allowed differentiation of the period of microlag, the time interval after the addition of germinant before any germination response (such as loss of refractility or dipicolinic acid [DPA] release) can be detected, and microgermination, the time for the actual germination-associated change to occur. There is no correlation between the length of these events for a single spore (7, 27). Once triggered, germination proceeds very quickly, making microlag the rate-limiting step (13, 29). In general, a slower maximum germination rate, as measured by OD loss, reflects that the microlag is longer and more asynchronous in the population, and it often correlates with a lower overall percentage of germination. The germination rates of B. subtilis spore suspensions do not always fit Michaelis-Menten kinetics, as double-reciprocal plots (i.e., 1/germinant concentration versus 1/ΔA₄₉₀ min⁻¹) often give nonlinear graphs (23). Instead, we report the maximum rate of germination (G_max) and the germinant concentration needed to achieve 50% of G_max (C₅₀). These values were determined graphically by plotting the maximum germination rate against germinant concentration.

Western blotting of proteins from crude spore extracts.

Spores (10 mg [dry weight]) were suspended in 0.5 ml prechilled breakage buffer (50 mM Tris-HCl [pH 7.5] containing 0.5 mM EDTA and 1 mM phenylmethylsulfonyl fluoride [PMSF]) and broken by shaking with glass beads (Fast-Prep blue tubes) in a Fast-Prep shaker (FP120; Anachem). A further 0.5 ml of buffer was added and mixed, and the samples were allowed to stand for 1 min to allow the glass beads to settle. The broken-spore suspension was removed and stored at −80°C. Samples (10 μg protein) in 6.5 μl breakage buffer were mixed with 2.5 μl NuPAGE LDS sample buffer (Invitrogen) and 1 μl NuPAGE sample reducing agent, heated at 95°C for 10 min, and cooled on ice for 2 min before loading on SDS-polyacrylamide gels. NuPAGE 4 to 12% Bis-Tris gels (Invitrogen) were used with morpholinepropanesulfonic acid (MOPS) running buffer, containing antioxidant in the upper chamber, according to the manufacturer's instructions. Separated proteins were blotted onto Hybond P. Primary rabbit polyclonal anti-GerAC antibody (14) was pretreated with immobilized E. coli lysate (Pierce) before use. Horseradish peroxidase (HRP)-conjugated anti-rabbit IgG was used as the secondary antibody and detected using an ECL Plus Western blotting detection kit (GE Healthcare).

RESULTS

Introduction of amino acid substitutions in the GerAB protein.

A ClustalW2 alignment of GerAB and several functional homologs was constructed (Fig. 1). GerBB (32% amino acid identity) and GerKB (24% identity) were chosen for comparison, as they are also encoded by genes in the B. subtilis genome. GerLB (25% identity) and GerIB (25% identity) both contribute to the l-alanine germination response in Bacillus cereus. GerQB (22% identity) is a component of the GerQ receptor of B. cereus, which along with GerI is required for a germination response to inosine.

Fig. 1.

ClustalW2 multiple-sequence alignment of GerAB and homologs from Bacillus species. GenBank accession numbers are listed. Residues with an asterisk below the sequences are identical in all sequences, while residues indicated by two dots and single dots represent more-conservative or less-conservative substitutions, respectively. Heavy lines above the sequences represent positions of the TOPCONS-predicted transmembrane helices of GerAB. Residues in boldface type and underlined were altered in the current study or are point mutants (L24, T192) from a previous study, discussed in the text. Gaps introduced to maximize alignment are indicated by dashes.

Of the residues targeted for substitution, GerAB residues G25, W45, P178, E202, F268, and R271 were identical in all six proteins, while Y136, L176, and F272 were highly conserved, showing identity in all but one of the sequences. F272 was also chosen as a residue of interest, because a F-to-I substitution at the equivalent position in GerBB allowed the GerB receptor to respond to l-amino acids, albeit slowly, without the influence of the GerK receptor (1, 2).

Structure prediction programs (SOSUI, TMHMM, TopPred, and TOPCONS) all predict that the GerAB protein and its homologs will have 10 transmembrane α-helices and predict that both termini are located in the cytoplasm. Some difference in helix boundaries was seen between predictions; however, these differences were minor. Charged residues within a hydrophobic membrane environment are frequently important to structure and function. The conserved E202, in potential membrane span 6, and the nonconserved E51, in potential membrane span 2, were therefore both included as targets for alteration. Residues altered in predicted loop regions included L176 and P178 in the loop between predicted membrane helices 5 and 6, the conserved residues F268, R271, and F272 in the loop between predicted membrane helices 7 and 8, and the nonconserved D326, D327, and N329 in the short loop between predicted membrane helices 9 and 10. All the residues chosen for substitution and their predicted positions in the GerAB protein are indicated in Fig. 1.

Range of mutant phenotypes.

gerAB mutants could be subdivided, in a somewhat arbitrary fashion, into four groups on the basis of their germination response to l-alanine. The mutants are listed in these groups, with a phenotype summary, in Table 1, and examples of more-detailed germination properties are shown in Fig. 2. Group I mutants germinate as well as wild type in 10 mM l-alanine. AB213(F272I) and AB215(D326A) mutants (Fig. 2A) have phenotypes that are essentially identical to that of the parent strain, strain 1604. The AB217(N329A) mutant is included in group I, as it will germinate as fast as the parent in excess germinant, but it does show a small (4-fold) decrease in the population's sensitivity to l-alanine (Fig. 2A).

Table 1.

Germination behavior of mutants with site-directed mutations by phenotypic group

Strain (gerAB mutant allele)	Groupa	Residue change	Gmax (% OD loss min−1)b	C50 of l-Ala (× 10−5 M)c	% OD loss (100 mM l-Ala, 100 min)d	d-Ala/l-Ala ratio for 50% inhibitione	GerAC protein levelf
1604 (wild type)			2.00	8	40	1:3	+++
AM1702 (AB213)	I	F272I	1.75	8	40	1:4	+
AM1704 (AB215)	I	D326A	1.80	8	41	1:2	+++
AM1706 (AB217)	I	N329A	2.00	30	41	1:8	+
AM1697 (AB208)	II	L176A	1.00	13	23	1:2	++
AM1705 (AB216)	II	D327A	1.33	15	31	1:2	+++
AM1703 (AB214)	III	F272A	1.00	20	31	1:11	+++
AM1694 (AB205)	III	W45L	0.75	35	23	1:2	+++
AM1700 (AB211)	III	F268A	1.33	70	37	1:10	+++
AM1692 (AB203)	IV	G25V			5		−
AM1695 (AB206)	IV	E51L			<1		+++
AM1696 (AB207)	IV	Y136F			2		+/−
AM1698 (AB209)	IV	P178A			10		+
AM1699 (AB210)	IV	E202A			<1		−
AM1701 (AB212)	IV	R271A			12		+

^aMutants have been allocated to phenotypic groups I to IV.

^bG_max, maximum rate of germination (ΔOD₄₉₀ min⁻¹).

^cC₅₀ is the concentration of the l-alanine germinant required to achieve 50% of the maximum germination rate.

^dThe proportion of spores germinating within 100 min at 100 mM l-Ala was calculated after subtraction of OD loss in the absence of germinant (0 to 5%) and is reported to demonstrate the eventual level of germination achieved.

^eThe effectiveness of inhibition by d-Ala is presented as the relative ratio of d-Ala to l-Ala that reduces by 50% the maximum germination rate achieved in 1 mM l-Ala in the absence of inhibitor.

^fThe level of GerAC protein is a qualitative estimate from Western blot data. Symbols; −, no GerAC protein; +, 10 to 30% of wild-type level; ++, 30 to 50% of wild-type level; +++, >50% of wild-type level.

Fig. 2.

Dependence of the germination rate on the concentration of l-alanine in the wild type and mutants. The maximum slope of each germination curve (ΔOD min⁻¹) for a spore suspension is plotted against the concentration of germinant. (A) Bacillus subtilis strain 1604 (wild type) (●) and D326A (I) (○), N329A (I) (□), D327A (II) (■), and L176A (II) (▵) mutants are shown. The roman numerals in parentheses indicate phenotypic groups. (B) B. subtilis strain 1604 (●) and F272A (III) (○), W45L (III) (■), and F268A (III) (□) mutants are shown.

Populations of mutant spores in group II, AB208(L176A) and AB216(D327A) mutants, show a different pattern of response (Fig. 2A). For these mutants, some spores in the population germinate in low concentrations of germinants; for this fraction of the population, the C₅₀ is not very different—at most twice that of the wild type. However, about half of the spores do not respond within 100 min, even at the highest germinant concentrations tested (Table 1). The G_max rates for the fraction of the population that do germinate, if corrected for this, could then be similar to those of wild type and group I mutants. Unusually, for the AB216(D327A) mutant, the germination rate and magnitude did improve close to group I levels after the spores had been stored for about 6 months.

Group III mutants, AB205(W45L), AB211(F268A), and AB214(F272A) mutants, also show fractional germination, reflected in lower maximum rates (G_max). They require rather higher concentrations of l-alanine (over 100 μM for the W45L and F268A mutants) to elicit any germination response (Fig. 2). Finally, the six group IV mutants show very little germination response, even at the highest concentrations of l-alanine, although some mutants do show very limited germination by 100 min (Table 1). Addition of a combination of glucose and fructose to l-alanine did not stimulate the germination rate or magnitude of any of the mutants, except for the F268A mutant and, to a small extent, the R271A mutant (data not shown).

Spores were also tested for loss of heat resistance during germination in l-alanine. This early event followed the same germination kinetics as the decrease in OD, confirming that it was the initial reaction that was affected in a proportion of the mutants, resulting in fractional germination in group II and group III mutants and that the reduced rate in the decrease in OD was not due to a defect in downstream events (such as activation of cortex lytic enzymes) in the entire population. It was also noted that the plating efficiency of dormant spores was significantly reduced (by at least 4-fold) for four of the group IV mutants, namely, the E51L, G25V, Y136F, and E202A mutants.

Competition between d-alanine and l-alanine in the mutants.

Spores of mutants in groups I to III were germinated in 1 mM l-alanine in the presence of various concentrations of d-alanine, which is a competitive inhibitor. Table 1 lists the relative ratio of d-Ala to l-Ala that reduces by 50% the maximum germination rate achieved in 1 mM l-Ala in the absence of inhibitor. Four of the mutants showed a 50% reduction in germination rate at a ratio that was very similar to that of the parental 1604 strain, suggesting that, for the fraction of spores that respond to l-alanine, the relative effectiveness of d- and l-isomers was unchanged. Exceptions to this were the N329A, F268A, and F272A mutants, in which d-alanine competed better with l-alanine, inhibiting at a lower relative concentration.

A defect in processing of the prelipoprotein GerAC results in failure to retain GerAC in the spore.

Another protein of the predicted germinant receptor is GerAC, a predicted lipoprotein. A mutation affecting the cleavage site of the prelipoprotein signal sequence in GerAC or in GerBC (15) interferes with the function of the cognate receptor, and the point mutation gerAC1 (20) which has a severe effect on l-Ala germination, would also affect prelipoprotein cleavage (21a). Western blotting data (Fig. 3) with an anti-GerAC antibody (14) demonstrate that the GerAC protein is absent from mature spores of the gerAC1 mutant, in which the consensus prelipoprotein processing site (LSGC) is altered by a G17E change. As this antibody was reported to show some cross-reaction with GerBC (14), a double mutant was constructed and checked (Fig. 3), but in fact, no cross-reaction was observed. There is a precedent for the loss of a germination protein if its lipoprotein status is compromised; a mutation affecting the prelipoprotein cleavage sequence of GerD resulted, similarly, in the failure to retain GerD in the spore (21). Given the data on the loss of GerAC in mutants defective in the other two subunits described below, it would be interesting to know whether there is any consequence for GerAA and GerAB assembly into the inner spore membrane if GerAC is missing, as is the case in the gerAC1 mutant, but we do not currently have the reagents to test this.

Fig. 3.

ECL detection of GerAC in crude spore extracts by Western blotting. Western blot of extracts from broken spores separated on a Nu-PAGE gel (Invitrogen) were probed with anti-GerAC antibody and detected using ECL Plus detection kit (GE Healthcare). The GerAC band runs at 42 kDa under the conditions used. Lanes 1, MagicMark protein standards (20 to 120 kDa; Invitrogen); 2, B. subtilis strain 1604; 3, gerAC1 ΔgerB mutant; 4, gerAC1 mutant; 5, strain FB20 (gerA::spc).

Effects of gerAB mutations on the level of GerAC protein in spores.

One fundamental issue to address on interpreting loss-of-function mutations, such as those in group IV above, is whether the protein encoded is present but nonfunctional or whether the change has interfered with the stability of the protein and the protein is therefore depleted in the mutant. Ideally, the solution would be to measure the levels of GerAB protein in the spore, but no antibody is available. As we have available a relatively sensitive anti-GerAC polyclonal antibody of proven specificity, we considered using this instead as a test of receptor, rather than GerAB, stability. The presence or absence of the GerAC receptor subunit would report indirectly on the consequences of the GerAB amino acid change for the stability of the overall complex.

Proteins in spore extracts were separated by SDS-PAGE and probed with anti-GerAC antibody, detected using enhanced chemiluminescence (ECL). Figure 4 shows the results of a typical Western blot. Proteins from extracts of wild-type and mutant spores were loaded at 10 μg protein in each well. The loading level was confirmed in gels loaded in parallel and then stained with Coomassie blue (data not shown).

Fig. 4.

Detection of GerAC in crude spore extracts of B. subtilis gerAB mutants. Proteins were separated on NuPAGE 4 to 12% Bis-Tris gels and transferred to polyvinylidene difluoride (PVDF) membranes, and then GerAC protein was detected with antibody using an ECL Plus detection kit (GE Healthcare). Lanes: 1, Magic Mark protein standards (20 to 120 kDa; Invitrogen); 2, B. subtilis strain 1604 (wild type); 3, AB203(G25V) mutant; 4, AB205(W45L) mutant; 5, AB206(E51L) mutant; 6, AB207(Y136F) mutant; 7, AB208(L176A) mutant; 8, AB209(P178A) mutant; 9, AB210(E202A) mutant; 10, AB211(F268A) mutant; 11, AB212(R271A) mutant; 12, AB213(F272I) mutant; 13, AB214(F272A) mutant; 14, AB215(D326A) mutant; 15, AB216(D327A) mutant; 16, AB217(N329A) mutant. (Lanes 1 to 9 and 10 to 16 are from separate gels). The phenotypic group of each mutant is indicated by the roman numeral below the gel.

No GerAC band was ever seen in extracts from AB203(G25V) mutants (Fig. 4, lane 3) or AB210(E202A) mutants (lane 9). Levels of GerAC comparable to those of the wild-type parent (lane 2) were detected for the AB205(W45L) mutant (lane 4), AB206(E51L) mutant (lane 5), AB208(L176A) mutant (lane 7), AB211(F268A) mutant (lane 10), AB214(F272A) mutant (lane 13), AB215(D326A) mutant (lane 14), and AB216(D327A) mutant (lane 15) (Fig. 4). Of those that gave a lower GerAC signal, the AB207(Y136F) mutant (lane 6) and the AB213(F272I) mutant (lane 12) gave slightly stronger bands in other, replicate experiments, so they may contain GerAC at low but significant levels. The AB209(P178A) mutant (Fig. 4, lane 8) did not give a detectable GerAC band at all in two other experiments, so the level of GerAC is definitely very low. The AB212(R271A) mutant (lane 11) and AB217(N329A) mutant (lane 16) consistently gave bands that were approximately one-third to one-quarter as strong as the wild type. Extracts of AB213(F272I) spores (Fig. 4, lane 12) appear depleted in GerAC, but the levels were higher in another experiment.

As expected, all of the mutants in groups I, II, and III show the presence of GerAC, albeit in some cases at lower levels than that of the WT. GerAC is detected in spores of the group IV gerAB mutant AB206(E51L), and at a lower level in group IV mutants [AB207(Y136F) and AB212(R271A) mutants]; therefore, it is unlikely that their l-alanine germination deficiency is solely due to receptor instability. However, the lack of any GerAC in the spore extracts of the group IV gerAB mutants AB203(G25V) and AB210(E202A) and the very little detected in the AB209(P178A) mutant suggest that the l-alanine germination deficiency seen for these mutants could be explained entirely by receptor instability. While the presence of GerAC protein does not prove that the GerA receptor complex remains intact, it suggests that this is likely. An absence of GerAC demonstrates that in some mutants, the receptor is certainly not intact and that some amino acid changes in GerAB can have consequences for the retention of GerAC in the spore.

DISCUSSION

Although the absence of structural information on the GerAB protein limits the interpretation of the outcomes of this mutagenesis study, a number of conclusions can be drawn from the work. The range of phenotypes obtained reflect a variety of effects on the function of the alanine germinant receptor, highlighting the importance of some residues for overall function and revealing that overall receptor assembly may be disrupted by defects in one subunit.

A number of the gerAB mutants described here show a reduction in receptor function, but only one of the amino acid substitutions described here (N329A) increases the concentration of germinant required (though only by 4-fold) while retaining a complete response of the whole spore population at high germinant concentrations. Two random point mutations that altered the C₅₀ for l-alanine, the gerAB38 and gerAB44 mutations, described some years ago (23), result in L24F and T192I substitutions, respectively (19). These substitutions, located in potential transmembrane helices 1 and 6, increased the requirement for l-alanine by 8-fold and 100-fold, respectively, but would allow complete germination of the spore population within 75 min at saturating l-alanine concentrations. In contrast, several of the site-directed mutants described here require higher concentrations of germinant for response, but not all of the spores are responsive, even to the higher concentration; this fractional germination likely reflects a heterogeneity in the population, but it would require further study of the separated germinated and nongerminated populations, as in reference 12, to address this question. Under certain circumstances, such as limiting germinant concentrations, only a fraction of wild-type spores will germinate. Such fractional germination can be correlated with relatively superdormant spores in the population (11) that behave as physiologically distinct spores (12), rather than involving only random, stochastic processes during exposure to germinant. Perhaps the reduced overall amount of GerAC, reflecting overall receptor, detected by Western blotting of spore extracts results from a variable amount of receptor complex in different spores.

The detailed interpretation of the consequences of all of these amino acid changes for germinant receptor function is impossible without structural information on the protein. However, the organization of GerAB as an integral membrane protein with 10 membrane-spanning helices is strongly predicted.

The most extreme group IV mutants lack GerAC protein. The gerAB203(G25V) mutation alters a conserved glycine within the first predicted membrane-spanning helix. The change to a larger valine residue was engineered to retain hydrophobicity in this region of the protein, while losing the potential for helix bending. The AB210(E202A) mutation alters a conserved charged residue in predicted membrane-spanning helix 6 of GerAB; therefore, the environments of the G25 and E202 residues in the overall structure of GerAB seem important for maintaining structure of the GerA complex as a whole. Two more group IV mutants, the AB207(Y136F) and AB209(P178A) mutants, showed a reduction in GerAC levels, particularly in the latter case. In GerAB and its homologs, the conserved P178 residue is predicted to be in the outer loop between helices 5 and 6, and it could be important for the conformation of this loop or the following helix 6. The Y136F change, in a largely conserved residue within membrane-spanning helix 4, may also affect the stability of the GerA complex, although it is harder to rationalize how loss of a single side chain hydroxyl group might have such a dramatic effect on structure. Y136 may also be functionally important, as the presumed low residual level of GerA receptor in these spores seems to be nonfunctional. Of the remaining group IV mutants, the AB206(E51L) mutant showed wild-type levels of GerAC; therefore, it is assumed that the entire receptor complex is present, albeit nonfunctional. This acidic residue in helix 2, which is not conserved in GerAB homologs, is therefore of functional rather than structural importance. The level of GerAC in the AB212(R271A) mutant is lower than the WT level, but the remaining level appears to be sufficient in some other mutants to give significant germination rates; therefore, this highly conserved residue in the outer loop between helices 7 and 8 is also likely to be of functional significance.

Group I, II, and III mutants all have significant levels of GerAC protein. For some of these mutants, it is likely that the amino acid changes that reduce germination rates in the population and result in a significant fraction of the spores failing to germinate at all reflect functional defects rather than simply reduced receptor levels.

GerAB and its homologs, all proteins with 10 predicted membrane spans, represent a branch of the APC (amino acid/polyamine/organocation) superfamily of membrane transporters (16). It is unlikely that germinant receptors engage in bulk transport of germinants (24), but there is a precedent within the APC superfamily for a signal transduction role, as some members of this family in lower and higher eukaryotes can act as direct sensors rather than, or as well as, transporters of extracellular substrates (9, 26). The recent determination of three-dimensional (3D) structures of several members of the APC family, including AdiC (10) and ApcT (25), have highlighted common structural elements in these transporters, shared with a similarly organized LeuT (28). These all include a core of transmembrane (TM) helices 1 to 10, organized as two five-helix repeats, with TM helices 11 and 12 contributing to dimerization of the protein. Most notably, TM helices 1 and 6 have interruptions within their helical structure, with two or four conserved residues, respectively, in extended conformations. Substrate binding in AdiC involves side chains and unwound regions of TM helices 1 and 6, as well as side chains in other TM helices, such as TM helices 3, 8, and 10; substrate binding also leads to a significant conformational change in the protein, especially of TM helix 6a, which would also be dependent on the flexibility about the unwound region (10). A recent mutational analysis of the GerVB receptor in B. megaterium (3), focusing on residues in TM helix 6 of the GerVB glucose receptor protein, and informed by the structural data on these transporters, identified effects on the concentration of glucose, and sometimes of other germinants, required to stimulate germination.

The most dramatic mutations here that result in loss of function and apparent total loss of receptor, as judged by the absence of GerAC protein, would affect residues G25V and E202A, which would lie in the putative extended regions of TM helices 1 and 6, respectively, if the analogy with the structures of the APC transport proteins is a meaningful one. The previously reported gerAB38(L24F) and gerAB44(T192I) mutations described above, also located in the same helices 1 and 6, highlight further the importance of these regions for alanine binding. Failure to detect the GerAC protein would certainly explain a complete loss of function of the receptor, as it is already known that this subunit is required for alanine-dependent germination (30).

We do not yet understand how the receptor is assembled in the inner spore membrane, and this is highlighted by the unexpected observation that single-amino-acid changes in the GerAB subunit of the germinant receptor can influence levels of GerAC, in some circumstances resulting in the complete absence of GerAC. As the operon is in single copy at the normal location in the mutants and the base change would not induce polar effects, the level of expression would likely remain that of wild type. The failure to retain GerAC is presumed, therefore, to result from a failure to assemble a stable receptor complex in the inner membrane, although the fate of GerAC protein during sporulation in such mutants should be explored. The GerAB protein may be required to stabilize the GerAC lipoprotein in the membrane via protein-protein interactions. Although it is possible that amino acid changes may interfere with direct interactions between the two proteins, the frequency of the effect suggests that commonly, a reduction in GerAC levels may result from a more general destabilization of the structure of the GerAB protein. Similar experiments with gerAA mutants in the accompanying paper (21a) demonstrate that the presence of GerAA protein is also required for GerAC retention in the spore, and thus receptor assembly or stability. This evidence of interaction between the subunits is persuasive but not complete; it will be important in the future to demonstrate the levels of all three proteins, GerAA, GerAB, and GerAC, in such mutants. It does, however, highlight the importance of considering effects on the receptor protein complex when considering the phenotypes of mutants altered in individual genes.