Extremely variable conservation of γ-type small, acid-soluble proteins from spores of some species in the bacterial order Bacillales

Abstract

γ-Type small, acid-soluble spore proteins (SASP) are the most abundant proteins in spores of at least some members of the bacterial order Bacillales, yet they remain an enigma from both functional and phylogenetic perspectives. Current work has shown that the γ-type SASP or their coding genes (sspE genes) are present in most spore-forming members of Bacillales, including at least some members of the Paenibacillus genus, although they are apparently absent from Clostridiales species. We have applied a new method of searching for sspE genes, which now appear to also be absent from a clade of Bacillales species that includes Alicyclobacillus acidocaldarius and Bacillus tusciae. In addition, no γ-type SASP were found in A. acidocaldarius spores, although several of the DNA-binding α/β-type SASP were present. These findings have elucidated the phylogenetic origin of the sspE gene, and this may help in determining the precise function of γ-type SASP.

Fig. 1.

Comparison of amino acid sequences of γ-type SASP from spore-forming Bacillales species. The sequences are given in the one-letter code for the following species: Afl, Anoxybacillus flavithermus; Bam, Bacillus amyloliquefaciens; Ban, Bacillus anthracis; Bat, Bacillus atrophaeus; Bav, Bacillus aminovorans; Bbr, Brevibacillus brevis; Bce, Bacillus cereus; Bcl, Bacillus clausii; Bcy, Bacillus cytotoxis; Bfi, Bacillus firmus; Bha, Bacillus halodurans; Bli, Bacillus licheniformis; Bme, Bacillus megaterium; Bmy, Bacillus mycoides; Bpf, Bacillus pseudofirmus; Bps, Bacillus pseudomycoides; Bpu, Bacillus pumilus; Bsu, Bacillus subtilis; Bth, Bacillus thuringiensis; Bwc, Bacillus weihenstephanensis chromosome; Bwp, B. weihenstephanensis plasmid; GC5, Geobacillus sp. C56-T3; Gka, Geobacillus kaustophilus; Gst, Geobacillus stearothermophilus; Gth, Geobacillus thermodenitrificans; GWC, Geobacillus sp. WCH70; GY1, Geobacillus sp. Y412MC61; Pym, Paenibacillus sp. Y412MC10; Hha, Halobacillus halophilus (originally Sporosarcina halophila); Lsp, Lysinibacillus sphaericus; Oih, Oceanobacillus iheyensis; Pcu, Paenibacillus curdlanolyticus; Pjd, Paenibacillus sp. JDR-2; Pot, Paenibacillus sp. oral taxon 786 strain D14; Ppo, Paenibacillus polymyxa strain E681; Sur, Sporosarcina ureae; and Tth, Thermoactinomyces thalpophilus. For species whose abbreviations are underlined, the amino acid sequences were from the sspE gene cloned from this species (18, 25, 33); all other sequences were from the species' completed genome sequences. The seven-residue sequences shaded in red are the sites for recognition and cleavage by the SASP-specific protease GPR during spore outgrowth, with cleavage between the bold residues (29, 32). The sequence in the N-terminal region of Ppo in purple is the protein sequence determined in this work, and the yellow blocks of sequences in Ppo and Bsu represent two long blocks of repeated sequences in each of these proteins.

Fig. 2.

Comparison of amino acid sequences of α/β-type SASP from A. acidocaldarius, B. subtilis, B. tusciae, G. kaustophilus, and Paenibacillus species. The sequences are shown in the one-letter code, with the asterisk in the Btu3 sequence denoting a stop codon and the dashes introduced to maximize sequence alignments. The sequences are from the following species: Bsu, B. subtilis; Gka, G. kaustophilus; Aac, A. acidocaldarius; Btu, B. tusciae; Pym, Paenibacillus sp. Y412MC10; Pcu, P. curdlanolyticus; Pjd, Paenibacillus sp. JDR-2; Pot, Paenibacillus sp. oral taxon 786 strain D14; and Ppo, P. polymyxa. The sequences above the break are those from species that are more distantly related to those whose sequences are listed below this break. The long regions of sequence shaded in red and yellow are sequences that each form long α-helices that are important structural elements in these proteins' binding to DNA (16). The site of cleavage of these proteins during spore outgrowth by the SASP-specific protease is between the bold residues in the red-shaded regions. The sequence blocks shaded in green seem to be duplications of the GPR cleavage site region, and in all of the proteins with this duplication, the spacing between the red and yellow blocks of sequence is greatly increased. The Ppo1, Ppo2, Aac1, and Aac2 sequences in purple were obtained by protein sequence analysis in this work, although the complete sequences of the P. polymyxa proteins are from strain E681 and the sequences of the A. acidocaldarius proteins are from strain DSM 446.

Fig. 3.

Polyacrylamide gel electrophoresis at low pH of acetic acid extracts from spores of P. polymyxa ATCC 842 (lanes 1 and 2) and A. acidocaldarius NRS 1662 (lane 3). P. polymyxa spores were isolated and purified, and ∼5 mg (dry weight) was disrupted before or after germination; the dry powder was extracted, dialyzed and lyophilized; aliquots were run via polyacrylamide gel electrophoresis at a low pH; and the gel was stained as described in Materials and Methods. The samples run in lanes 1 and 2 are from dormant P. polymyxa spores (lane 1) and germinated P. polymyxa spores (lane 2). Bands labeled 1 and 3 in lane 1 are the Ppo1 and Ppo2 α/β-type SASP, respectively (Fig. 2), while band 2 is the product of an sspE-like gene (Fig. 1), as determined by amino-terminal sequence analysis of these protein bands as described in the text. Bands labeled a to h are ones that were largely or completely removed by decoating treatment, while bands 1 to 3 were not (data not shown). (Lane 3) Dormant A. acidocaldarius spores (12 mg [dry weight]) were ruptured and extracted, an aliquot from ∼4 mg spores was run via polyacrylamide gel electrophoresis at a low pH, proteins on the gel were transferred to a polyvinylidene difluoride membrane, and the membrane was stained as described in Materials and Methods. Bands labeled 1 and 2 in lane 3 are the A. acidocaldarius α/β-type SASP Aac1 and Aac2, respectively, as described in the text. Lanes 1 and 2 are from the same gel, while lane 3 is from a separate gel. The labeled horizontal arrows adjacent to lanes 1 and 3 denote the migration positions of B. subtilis SASP-α and -γ, which were determined by running an aliquot of an acetic acid extract of B. subtilis spores in lanes that are not shown but were adjacent to lanes 1 and 3.

Fig. 4.

Putative upstream and downstream regulatory regions for genes encoding α/β-type and γ-type SASP from various species. The genes from the various species are those in Fig. 1 and 2, and the names of the species are as follows: Aac, A. acidocaldarius; Btu, B. tusciae; Pym, Paenibacillus sp. Y412MC10; Pcu, P. curdlanolyticus; Pjd, Paenibacillus sp. JDR-2; Pot, Paenibacillus sp. oral taxon 786 strain D14; and Ppo, P. polymyxa. The upstream and downstream sequences of the genes are from the NCBI Microbial Genomes database. The optimal −10 and −35 promoter sequences and their spacing for strong σ^G promoters are listed above the upstream sequences and are from highly expressed σ^G-dependent genes of four Bacillus species, including those encoding α/β-type and γ-type SASP (22). Bold nucleotides in the −10 and −35 sequences are >90% conserved in these sequences, and nucleotides that are not bold are 50 to 70% conserved. Note that while the Bacillus genes are almost exclusively recognized by σ^G, the recognition sequence for σ^F overlaps that of σ^G to a significant extent (36), so the sequences shown upstream of genes in other species that do or may encode SASP might be recognized by σ^F rather than σ^G. A perfect RBS sequence for Bacillus mRNAs is also listed above the upstream sequences. For each gene, in the upstream sequences the putative −10 and −35 σ^G promoter sequences are highlighted in purple, the RBS in yellow, and the translation start codon in red; for the downstream sequences the translation stop codon is highlighted in green and an inverted repeat followed by a T-rich region is in cyan.

Fig. 5.

Phylogenetic tree for Firmicutes species. The tree was constructed using 16S rRNA sequences as described in the text. Organism names in green contain an sspE gene, organism names in red do not contain an sspE gene, and for organism names in black the completed genome sequence is not available and analysis of the presence or absence of the sspE gene has not been carried out. The red asterisk adjacent to B. selenitireducens indicates that this species almost certainly does not sporulate, as described in the text. The large green asterisk in the region between the ancestor of the Paenibacillus genus and the ancestor of the clade containing Alicyclobacillus species indicates the period in Firmicutes evolution when the sspE gene appeared. The numbers adjacent to interior branch points in the tree are bootstrap values.