Identification of new proteins that modulate the germination of spores of bacillus species

Abstract

A number of operons encoding the nutrient germinant receptors (GRs) in dormant spores of Bacillus megaterium and Bacillus subtilis species have small open reading frames (ORFs) of unknown function within or immediately adjacent to the operons. Inactivation of the genes in these ORFs, encoding proteins now termed D proteins, either significantly increased or decreased spore germination via the associated GR but had no effects on germination via non-GR-dependent germinants. These effects on GR-dependent germination were complemented by ectopic expression of the appropriate D gene (gene encoding D protein). However, substitution of noncognate D genes in two GR operons resulted in inhibition of germination via the GR manipulated, although ectopic overexpression of a D gene had no effect on overall GR-dependent germination. The various D genes studied were expressed in the forespore during sporulation in parallel with the associated GR operon, and transcription of a B. subtilis D gene was controlled by RNA polymerase sigma factor σ(G). These results indicate that proteins encoded by small ORFs within or adjacent to operons encoding GRs play major roles in modulating GR function in spores of Bacillus species. In B. subtilis, deletion of a D gene (B. subtilis gerKD [gerKDbs]) adjacent to the gerK operon encoding the GerK GR or ectopic expression or overexpression of gerKDbs had no major effect on the levels of GR subunits or of two other germination proteins.

Fig 1

Locations of putative D genes in or adjacent to operons that encode GRs in B. megaterium (A) and B. subtilis (B). Scales below the genes are in kilobase pairs.

Fig 2

Rates of germination of wild-type and ΔgerKDbs B. subtilis spores with AGFK (A) and l-valine (B). Spores of B. subtilis strains PS533 (wild type) and PS4256 (ΔgerKDbs) were germinated with various concentrations of l-valine (A) or l-asparagine with d-glucose and d-fructose constant at 10 mM, and K⁺ constant at ∼15 mM (B), and rates of spore germination were assessed by Tb-DPA fluorescence and given in arbitrary units (AU) as described in Materials and Methods. Symbols: ○, wild-type (PS533) spores; ●, ΔgerKDbs (PS4256) spores; △, ΔgerKDbs amyE::gerKDbs (PS4313) spores; ▲, amyE::PsspB-gerKDbs (PS4314) spores.

Fig 3

Germination of spores of various B. subtilis strains with CaDPA (A) and dodecylamine (B). Spores of B. subtilis strains PS533 (wild type), PS4256 (ΔgerKDbs), PS4313 (ΔgerKDbs amyE::gerKDbs), and PS4314 (amyE::PsspB-gerKDbs) germinated in the presence of CaDPA (A) or dodecylamine (B), and spore germination with these two agents was measured as described in Materials and Methods. The dormant spores of these four strains had essentially the same amounts of DPA. Symbols: ○, PS533 spores; ●, PS4256 spores; △, PS4313 spores; ▲, PS4314 spores.

Fig 4

Western blot analysis of germination proteins in total lysates of spores of various B. subtilis strains. Total spore lysates from spores of B. subtilis strains PS533 (wild type) (lanes 1 and 5), PS4256 (ΔgerKDbs) (lanes 2 and 6), PS4213 (amyE::gerKDbs ΔgerKDbs) (lanes 3 and 7), and PS4314 (amyE::PsspB-gerKDbs) (lanes 4 and 8) were isolated, and samples with identical amounts of protein from ∼7 × 10⁶ spores (lanes 1 to 4) or 1.8 × 10⁶ spores (lanes 5 to 8) (with amounts of protein adjusted appropriately based on preliminary SDS-PAGE and Coomassie blue staining) were run on SDS-polyacrylamide gels and subjected to Western blot analysis with antisera against various germination proteins as described in Materials and Methods. The strips probed for the various antigens were from several different blots.

Fig 5

Western blot analysis of germination proteins in the isolated inner membrane fraction from spores of various B. subtilis strains. The inner membrane fraction was isolated from the total spore lysates analyzed in Fig. 4 from spores of B. subtilis strains PS533 (wild type) (lanes 1 and 5), PS4256 (ΔgerKDbs) (lanes 2 and 6), PS4213 (amyE::gerKDbs ΔgerKDbs) (lanes 3 and 7), and PS4314 (amyE::PsspB-gerKDbs) (lanes 4 and 8). Samples with identical amounts of inner membrane protein from ∼2.3 × 10⁸ spores (lanes 1 to 4) or 5.6 × 10⁷ spores (lanes 5 to 8) (with amounts of protein adjusted appropriately based on preliminary SDS-PAGE and Coomassie blue staining) were run on SDS-polyacrylamide gels and subjected to Western blot analysis with antisera against various germination proteins as described in Materials and Methods. The strips probed for the various antigens were from several different blots.

Fig 6

Potential transcription and translation signals upstream of the gerKDbs and gerUD coding sequences. The perfect B. subtilis mRNA ribosome binding site (rbs) (28) and consensus −10 and −35 sequences recognized by B. subtilis RNA polymerase with σ^G and the rbs are shown above the gerKDbs sequence. The X in the −35 consensus sequence denotes a position that is any nucleotide, the Y in the −10 sequence denotes residues that are most often A or C, and the Z in the −10 sequence denotes a position that is most often A or T. The translation-initiating ATG codons are underlined. In the sequences shown, the values in parentheses between the −35 and −10 sequences, the −10 sequence and the rbs, and the rbs and the ATG codon are the consensus spacings between these elements (3, 27). The dashes indicate positions that are any nucleotide.

Fig 7

Levels of β-galactosidase and DPA during sporulation of strains with a gerKDbs-lacZ fusion and with or without sporulation-specific RNA polymerase σ factors. Various B. subtilis strains carrying gerKDbs-lacZ were sporulated by resuspension, and equal aliquots of cultures were assayed for β-galactosidase (○, ●, △, ▲, □) or DPA (■) as described in Materials and Methods. The strains analyzed were PS4290 (has all sigma factors) (○, ■), PS4293 (no σ^E) (●), PS4294 (no σ^F) (△), PS4295 (no σ^G) (▲), and PS4296 (no σ^K) (□). Strain PS533 that lacks a lacZ fusion was also sporulated by resuspension in parallel, and the levels of β-galactosidase from this strain were subtracted from those of other samples taken at the same time. The maximum value of these subtracted values was <10% of the maximum value obtained with the wild-type strain carrying gerKDbs-lacZ.

Fig 8

RT-PCR analysis of expression of B. megaterium GR A- and D-protein genes during sporulation. RT-PCR was conducted using gene-specific primers designed to amplify 230- to 300-bp fragments of gerUA (A), gerUD (B), gerKAbm (C), and gerKDbm (D) from RNA isolated from sporulating cultures as described in Materials and Methods. The numbers above the lanes refer to the time (in hours) after entry into sporulation. Positive-control reactions (+) (i.e., genomic DNA) and negative-control reactions (−) (i.e., no template DNA/RNA) reactions are indicated above the lanes. Lanes M, molecular weight markers (M).