Effects of overexpression of nutrient receptors on germination of spores of Bacillus subtilis

Abstract

The rates of germination of Bacillus subtilis spores with L-alanine were increased markedly, in particular at low L-alanine concentrations, by overexpression of the tricistronic gerA operon that encodes the spore's germinant receptor for L-alanine but not by overexpression of gerA operon homologs encoding receptors for other germinants. However, spores with elevated levels of the GerA proteins did not germinate more rapidly in a mixture of asparagine, glucose, fructose, and K(+) (AGFK), a germinant combination that requires the participation of at least the germinant receptors encoded by the tricistronic gerB and gerK operons. Overexpression of the gerB or gerK operon or both the gerB and gerK operons also did not stimulate spore germination in AGFK. Overexpression of a mutant gerB operon, termed gerB*, that encodes a receptor allowing spore germination in response to either D-alanine or L-asparagine also caused faster spore germination with these germinants, again with the largest enhancement of spore germination rates at lower germinant concentrations. However, the magnitudes of the increases in the germination rates with D-alanine or L-asparagine in spores overexpressing gerB* were well below the increases in the spore's levels of the GerBA protein. Germination of gerB* spores with D-alanine or L-asparagine did not require participation of the products of the gerK operon, but germination with these agents was decreased markedly in spores also overexpressing gerA. These findings suggest that (i) increases in the levels of germinant receptors that respond to single germinants can increase spore germination rates significantly; (ii) there is some maximum rate of spore germination above which stimulation of GerA operon receptors alone will not further increase the rate of spore germination, as action of some protein other than the germinant receptors can become rate limiting; (iii) while previous work has shown that the wild-type GerB and GerK receptors interact in some fashion to cause spore germination in AGFK, there also appears to be an additional component required for AGFK-triggered spore germination; (iv) activation of the GerB receptor with D-alanine or L-asparagine can trigger spore germination independently of the GerK receptor; and (v) it is likely that the different germinant receptors interact directly and/or compete with each other for some additional component needed for initiation of spore germination. We also found that very high levels of overexpression of the gerA or gerK operon (but not the gerB or gerB* operon) in the forespore blocked sporulation shortly after the engulfment stage, although sporulation appeared normal with the lower levels of gerA or gerK overexpression that were used to generate spores for analysis of rates of germination.

FIG. 1.

OD₆₀₀ values and levels of β-galactosidase from sspE-lacZ in sporulating cultures of various B. subtilis strains. B. subtilis strains were grown and sporulated in liquid 2×SG medium without antibiotics, the OD₆₀₀s were measured, and β-galactosidase was extracted and assayed as described in Materials and Methods. Open symbols indicate OD₆₀₀ values and filled symbols indicate β-galactosidase specific activities for strain PS3471 (PsspB::gerA sspE-lacZ) (triangles) and strain PS3469 (PsspB::gerB sspE-lacZ) (circles). The curves for strains carrying only sspE-lacZ (PS3468) or PsspB::gerK sspE-lacZ (PS3472) were essentially identical (within 20%) to those for the strain with PsspB::gerB sspE-lacZ or PsspB::gerA sspE-lacZ, respectively (data not shown). Values for the strain carrying PsspB::gerB* sspE-lacZ (PS3415) were essentially identical to those for strains PS3468 and PS3469 (data not shown).

FIG. 2.

Level of GerBA in spores of various B. subtilis strains. The inner membranes from spores of various strains were isolated, aliquots of membrane protein were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and GerBA was detected by Western blot analysis as described in Materials and Methods. The amounts and the identities of the membrane samples in the various lanes are 1 mg of wild-type spores (lane 1), 100 μg of PsspB::gerB* spores (strain PS3415) (lane 2), 10 μg of PsspB::gerB* spores (strain PS3415) (lane 3), 1 mg of PsspD::gerB* spores (strain PS3502) (lane 4), 40 μg of PsspD::gerB* spores (strain PS3502) (lane 5), and 1 mg of ΔgerB spores (strain FB60) (lane 6). The numbers and arrows to the left of the figure show the migration positions of molecular mass markers in kilodaltons, and the vertical bar on the right shows the position of the GerBA bands. Note that GerBA gives multiple bands, as seen previously (27).

FIG. 3.

Rate of loss in OD₆₀₀ values and DPA release during germination of spores with or without elevated levels of the GerA receptor. Spores of strain PS832 (wild type) (○, •) or PS3476 (PsspD::gerA) (▵, ▴) were germinated in 50 mM KPO₄ (pH 7.4) and 1 mM l-alanine at either 37°C (a) or 7°C (b), and the OD₆₀₀ values (○, ▵) and the DPA released (•, ▴) were measured as described in Materials and Methods. Note that not all of the data points taken in the experiment whose results are indicated in panel a are shown. t, time; t0, time zero.