The katX gene, which codes for the catalase in spores of Bacillus subtilis, is a forespore-specific gene controlled by sigmaF, and KatX is essential for hydrogen peroxide resistance of the germinating spore

Abstract

Previous work has shown that the katX gene encodes the major catalase in dormant spores of Bacillus subtilis but that this enzyme has no role in dormant spore resistance to hydrogen peroxide. Expression of a katX-lacZ fusion began at approximately h 2 of sporulation, and >75% of the katX-driven beta-galactosidase was packaged into the mature spore. A mutation in the gene coding for the sporulation-specific RNA polymerase sigma factor sigmaF abolished katX-lacZ expression, while mutations in genes encoding sigmaE, sigmaG, and sigmaK did not. Induction of sigmaF synthesis in vegetative cells also resulted in katX-lacZ expression, while induction of sigmaG expression did not; the katX-lacZ fusion was also not induced by hydrogen peroxide. Upstream of the in vivo katX transcription start site there are sequences with good homology to those upstream of known sigmaF-dependent start sites. These data indicate that katX is an additional member of the forespore-specific sigmaF regulon. A mutant in the katA gene, encoding the major catalase in growing cells, was sensitive to hydrogen peroxide during sporulation, while a katX mutant was not. However, outgrowth of katX spores, but not katA spores, was sensitive to hydrogen peroxide. Consequently, a major function for KatX is to protect germinating spores from hydrogen peroxide.

FIG. 1

Expression of katA- and katX-lacZ fusions in growing and stationary-phase cells. Strains PS2573 (katA-lacZ) and IB427 (katX-lacZ) were grown at 37°C in LB medium, and samples were taken for assay of β-galactosidase as described in Materials and Methods. Symbols: ○ and •, strain PS2573; ▵ and ▴, strain IB427; ○ and ▵, OD₆₀₀; • and ▴, β-galactosidase specific activity.

FIG. 2

Expression of the katX-lacZ fusion in spo mutants. Strains of the wild-type or spo strains carrying the katX-lacZ fusion were sporulated by the resuspension method, and samples were taken for assay of β-galactosidase as described in Materials and Methods. Time zero is the time of initiation of sporulation. Symbols: •, IB434 (wild type); ○, IB431 (spoIIIG); □, IB429 (spoIIAC); ◊, IB430 (spoIIGB); ▵, IB432 (spoIVCB) (ς^K).

FIG. 3

Induction of the katX-lacZ fusion in vegetative cells producing ς^F or ς^G. Strains IB435 (producing ς^F under P_spac control) and IB436 (producing ς^G under P_spac control) were grown in 2xYT medium. At an OD₆₀₀ of 0.25, each culture was split in half, and one half was made 2 mM in IPTG. Samples were taken subsequently for assay of β-galactosidase as described in Materials and Methods. Symbols: ○ and •, strain IB435; ▵ and ▴, strain IB436; • and ▴, without IPTG; ○ and ▵, with IPTG.

FIG. 4

Primer extension analysis of the katX transcription start site. RNA was isolated from sporulating cells, and primer extension analysis was carried out with primer katX-45 and products analyzed as described in Materials and Methods. Lanes A, G, C, and T, sequencing reactions with primer katX-45 and plasmid pIB449; lane 1, primer extension product with RNA from strain IB441 (amyE::katX-lacZ spoIIIG); lane 2, primer extension product with RNA from strain IB439 (amyE::katX-lacZ). Extension products are marked with an arrow, and the transcription start site in the katX upstream sequence to the left is marked with a dot.

FIG. 5

DNA sequence of the amino-terminal coding and upstream regions of katX. Sequences corresponding to −10 and −35 promoter elements are labeled and underlined; the important two G residues upstream of the −10 region are in boldface; the likely ribosome binding site (RBS) is underlined; the nucleotide at the transcription initiation site is in boldface and labeled +1. The amino-terminal coding regions of both katX and a possible divergently transcribed gene (yxlJ) are also shown, with the encoded amino acid given beneath the second nucleotide of each codon. The DNA sequence in this region is from reference .

FIG. 6

Effects of hydrogen peroxide on sporulating cells of various strains. Strains were sporulated by the resuspension method, hydrogen peroxide was added to 0.2 mM 1 h (A) or 3 h (B) after initiating sporulation (arrow), and the OD₆₀₀ of the culture was measured. Symbols: ○, PS832 (wild type); •, PS2488 (katA); ▵, PS2558 (katX); ▴, PS2664 (katA katX).

FIG. 7

Effects of hydrogen peroxide on spore outgrowth. Spores of strain PS832 (wild type) were germinated as described in Materials and Methods, various amounts of hydrogen peroxide were added 10 min after initiating germination (arrow), and the OD₆₀₀ of the culture was measured. Symbols: ○, no hydrogen peroxide; •, plus 0.2 mM hydrogen peroxide; ▵, plus 2 mM hydrogen peroxide; ▴, plus 10 mM hydrogen peroxide.

FIG. 8

Effects of hydrogen peroxide on outgrowth of spores of catalase mutants. Spores of various strains were germinated as described in Materials and Methods, hydrogen peroxide was added to 2 mM 10 min after initiation of germination (arrow), and the OD₆₀₀ of the culture was determined. Symbols: ○, PS832 (wild type); •, PS2488 (katA); ▵, PS2588 (katX); ▴, PS2664 (katA katX).

FIG. 9

Comparison of promoter regions of ς^F-dependent genes. Promoter sequences of ς^F genes were taken from primer extension analyses reported in references , , , , , and and this work. The −10 and −35 regions are labeled and underlined; the G residues upstream of the −10 region are in boldface, as are the transcription initiating nucleotides. The consensus sequence shown is from the seven gene sequences shown. Positions with single residues in the consensus have these residues in at least five genes; where there are two residues shown, each is present in at least two genes.