Regulation and characterization of a newly deduced cell wall hydrolase gene (cwlJ) which affects germination of Bacillus subtilis spores

Abstract

The predicted amino acid sequence of Bacillus subtilis ycbQ (renamed cwlJ) exhibits high similarity to those of the deduced C-terminal catalytic domain of SleBs, the specific cortex-hydrolyzing enzyme of B. cereus and the deduced one of B. subtilis. We constructed a cwlJ::lacZ fusion in the B. subtilis chromosome. The beta-galactosidase activity and results of Northern hybridization and primer extension analyses of the cwlJ gene indicated that it is transcribed by EsigmaE RNA polymerase. cwlJ-deficient spores responded to both L-alanine and AGFK, the A580 values of spore suspensions decreased more slowly than in the case of the wild-type strain, and the mutant spores released less dipicolinic acid than did those of the wild-type strain during germination. However, the mutant spores released only slightly less hexosamine than did the wild-type spores. In contrast, B. subtilis sleB spores did not release hexosamine at a significant level. While cwlJ and sleB spores were able to germinate, CJSB (cwlJ sleB) spores could not germinate but exhibited initial germination reactions, e.g., partial decrease in A580 and slow release of dipicolinic acid. CJSB spores became slightly gray after 6 h in the germinant, but their refractility was much greater than that of sleB mutant spores. The roles of the sleB and cwlJ mutations in germination and spore maturation are also discussed.

FIG. 1

Alignment of the deduced amino acid sequences of B. subtilis CwlJ (Bs-CwlJ; YcbQ) (25), B. subtilis (Bs-SleB; deduced B. subtilis cortex-hydrolyzing amidase) (22), and B. cereus SleB (Bc-SleB; B. cereus cortex-hydrolyzing amidase) (21). Amino acid identities are indicated by shading, and amino acids identical in the three proteins are indicated by asterisks. Amino acids are numbered from the N termini of the proteins, and dashes indicate the introduction of gaps in the alignment. The arrowhead and arrows indicate a signal sequence cleavage site and repeated sequences, respectively. The nucleotide sequence G₂₅₆GC₂₅₈ (numbering with respect to the first A of the translational start codon of cwlJ) under GSDB, EMBL, DDBJ, and NCBI accession no. D30808 should be corrected to CGG; thus, the corresponding amino acid, G₈₆ (numbered with respect to the N-terminal amino acid) should be corrected to R.

FIG. 2

Time course of the production of the cwlJ-lacZ fusion protein (A) and Northern blot analysis of the cwlJ region (B). (A) cwlJ-directed β-galactosidase activity of strain cbQ was determined at the indicated times after the onset of sporulation. Squares, cell growth at an optical density of 600 nm (OD₆₀₀); diamonds, β-galactosidase activity. (B) Northern hybridization performed with the cwlJ-specific RNA probe as described in Materials and Methods. The lanes contained 5 μg of RNA from B. subtilis 168 at t₋₂ (−2), t₀ (0), t₂ (2), or t₄ (4), and B. subtilis 1S86 (SigF⁻), 1S60 (SigE⁻), SpoIIIGΔ1 (SigG⁻), and 1S38 (SigK⁻) at t_1.5 (1.5), t₃ (3), t_4.5 (4.5), or t₆ (6). 0.47 indicates the size (in kilobases) of the hybridizing RNA in comparison with the migration of 23S and 16S RNAs.

FIG. 3

Determination of the transcriptional start sites by primer extension analysis (A) and nucleotide sequence of the upstream region of cwlJ (B). (A) RNA (5 μg) from B. subtilis 168 at t_1.5 (lane 1), t_4.5 (lane 2), or t_7.5 (lane 3), 1S86 (SigF⁻) at t_4.5 (lane 4), 1S60 (SigE⁻) at t_4.5 (lane 5), SpoIIIGΔ1 (SigG⁻) at t_4.5 (lane 6), or 1S38 (SigK⁻) at t_4.5 (lane 7) or t_7.5 (lane 8) was hybridized with a labeled cbQPEX1 primer, which is complementary to nucleotides 27 to 44 in the sequence in panel B. The primer-extended products obtained with reverse transcriptase were subjected to electrophoresis in 12% (wt/vol) polyacrylamide sequencing gels, followed by autoradiography with an imaging plate (BAS-MP; Fuji). The dideoxy-DNA sequencing reaction mixtures with the cbQPEX1 primer were electrophoresed in parallel (lanes G, A, T, and C). The positions of the products are indicated by arrowheads. The boxed area indicates the −10 region of the ς^E promoter. In panel B, the promoter regions (−35 and −10) of ς^E and the transcriptional start position are indicated by underlines and arrowheads, respectively. The deduced rho-independent terminator is indicated by opposing arrows.

FIG. 4

Spore germination of B. subtilis 168, cbQ (cwlJ), 168SB (sleB), and CJSB (cwlJ sleB). The germination of spores of the B. subtilis strains was monitored at A₅₈₀ at the indicated times after the addition of l-alanine and is expressed as relative absorbance. The released dipicolinic acid and reducing groups in the supernatants of the spore suspensions were also measured. Squares, diamonds, circles, and triangles indicate B. subtilis 168, cbQ, 168SB, and CJSB, respectively.

FIG. 5

Phase-contrast microscopy of B. subtilis 168, cbQ, 168SB, and CJSB spores treated with l-alanine. Spores were germinated at 37°C for 6 h as described for Fig. 4, with the addition of germination buffer (10 mM l-alanine, 10 mM Tris-HCl [pH 8.4]), and then observed by phase-contrast microscopy. Bar, 5 μm.