An α/β-Type, Small, Acid-Soluble Spore Protein Which Has Very High Affinity for DNA Prevents Outgrowth of
            <i>Bacillus subtilis</i>
            Spores

Hayes, Christopher S.; Setlow, Peter

doi:10.1128/jb.183.8.2662-2666.2001

Cited by 29 publications

(30 citation statements)

References 25 publications

(26 reference statements)

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“…An essential feature of a/b-type SASPs is their specific cleavage by spore proteases during germination (Hayes & Setlow, 2001). GPR was first detected in B. megaterium (Setlow, 1976), and it was expected for a long time to be the only activity responsible for site-specific cleavage of a/b-type SASPs (Setlow et al, 1980).…”

Section: Discussionmentioning

confidence: 99%

Small acid-soluble spore proteins of Clostridium acetobutylicum are able to protect DNA in vitro and are specifically cleaved by germination protease GPR and spore protease YyaC

Wetzel

Fischer

2015

Microbiology

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Section: Discussionmentioning

confidence: 99%

Small acid-soluble spore proteins of Clostridium acetobutylicum are able to protect DNA in vitro and are specifically cleaved by germination protease GPR and spore protease YyaC

Wetzel

Fischer

2015

Microbiology

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“…All downstream primers contained a BamHI restriction site (underlined) after the translation stop codon (boldface), as well as flanking sequence to aid PCR and subsequent restriction enzyme digestion. The sspC ⌬N11-D13K-C3 gene was generated with the sspC ⌬N11-D13K upstream primer (5Ј CCATGGCTAAATTACTA ATTCCTCAAGCAG 3Ј) (8) containing an NcoI site (underlined) at the translation initiation codon (boldface; note that this mutant sspC gene was previously called sspC ⌬11-D13K , but we have renamed it sspC ⌬N11-D13K to distinguish between the N-and C-terminal mutations) and the sspC C3 downstream primer. Hot-start PCR was performed using Taq DNA polymerase and 30 cycles of the following program: 94°C for 1 min, 47°C for 1 min, and 72°C for 2 min, finishing with a 10-min extension step at 72°C.…”

Section: C3mentioning

confidence: 99%

“…The DNA-free ␣/␤-type SASP are then rapidly cleaved by the germination protease (GPR) and are eventually degraded to amino acids. At least one variant of SspC wt with significantly enhanced affinity for DNA appears not to dissociate readily from DNA upon spore germination, and thus this protein is not degraded rapidly by GPR (8). As a consequence, normal DNA function is not restored early in the germination of these spores and many of them die (8).…”

mentioning

confidence: 99%

“…Previous studies focusing on the N-terminal region of SspC wt identified some key residues affecting DNA binding (11,33). The culmination of these studies was the production of the SspC ⌬N11-D13K variant, in which 11 residues at the N terminus were deleted while the positive charge in this region of the protein was retained; these changes resulted in a greater-than-10-fold improvement in the protein's affinity for pUC19 DNA over that of SspC wt (4,8). The SspC ⌬N11-D13K variant also provided far greater protection to DNA against deamination and restriction enzyme digestion in vitro than did SspC wt (34).…”

mentioning

confidence: 99%

“…At least one variant of SspC wt with significantly enhanced affinity for DNA appears not to dissociate readily from DNA upon spore germination, and thus this protein is not degraded rapidly by GPR (8). As a consequence, normal DNA function is not restored early in the germination of these spores and many of them die (8). The potentially deleterious effect of ␣/␤-type SASP binding to DNA has also been seen in Escherichia coli, as shortly after induction of SspC wt synthesis in E. coli and binding of the protein to the cell's chromosome, cell growth and DNA replication cease, and in some genetic backgrounds, the cells die (25,37).…”

mentioning

confidence: 99%

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Effects of Carboxy-Terminal Modifications and pH on Binding of a Bacillus subtilis Small, Acid-Soluble Spore Protein to DNA

Kosman

Setlow

2003

J Bacteriol

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Variants of the wild-type Bacillus subtilis ␣/␤-type small, acid-soluble spore protein (SASP) SspC wt were designed to evaluate the contribution of C-terminal residues to these proteins' affinity for DNA. SspC variants lacking one to three C-terminal residues were similar to SspC wt in DNA binding, but removal of six C-terminal residues greatly decreased DNA binding. In contrast, a C-terminal extension of three residues increased SspC's affinity for DNA 5-to 10-fold. C-terminal and N-terminal changes that independently caused large increases in SspC-DNA binding affinity were combined and produced an additive effect on DNA binding; the affinity of the resulting variant, SspC ⌬N11-D13K-C3 , for DNA was increased >20-fold over that of SspC wt . For most of the SspC variants tested, lowering the pH from 7 to 6 improved DNA binding two-to sixfold, although the opposite effect was observed with variants having additional C-terminal basic residues. In vitro, the binding of SspC ⌬N11-D13K-C3 to DNA suppressed the formation of cyclobutane-type thymine dimers and promoted the formation of the spore photoproduct upon UV irradiation to the same degree as the binding of SspC wt . However, B. subtilis spores lacking major ␣/␤-type SASP and overexpressing SspC ⌬N11-D13K-C3 had a 10-foldlower viability and far less UV and heat resistance than spores overexpressing SspC wt . This apparent lack of DNA protection by SspC ⌬N11-D13K-C3 in vivo is likely due to the twofold-lower level of this protein in spores compared to the level of SspC wt , perhaps because of effects of SspC ⌬N11-D13K-C3 on gene expression in the forespore during sporulation. The latter results indicate that only moderately strong binding of ␣/␤-type SASP to DNA is important to balance the potentially conflicting requirements for these proteins in DNA transcription and DNA protection during spore formation, spore dormancy, and spore germination and outgrowth.

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Effect of Glutamate Synthase (GOGAT) Activity on Bacillus subtilis Spore Properties

Ruzal

Sánchez-Rivas

2003

Current Microbiology

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The role of glutamate as osmoprotector was investigated through the study of a mutation in its biosynthetic pathway. A glt::Tn917-lacZ-cat insertion mutant (N1) conferring glutamate auxotrophy and enhanced beta-galactosidase expression on high-salt media was selected. Co-transformation experiments and PCR analysis allowed locating the insertion into the gltB gene corresponding to the small unit of the glutamate synthase (GOGAT). The N1 mutant strain presented a glutamate requirement for growth and a tenfold decrease in GOGAT activity. Transcriptional activity of GOGAT, measured as beta-galactosidase from the transposon fusion, correlated with enzymatic activity; expression was enhanced at the stationary phase and in high-ionic-strength media. However, osmotolerance of cultures of N1 mutant were as wild-type (wt), at least in semi-rich medium. In contrast, sporulation was slightly reduced (75% of wt), and spores were less resistant to UV, heat, and osmolarity, properties linked to the content of small, acid-soluble proteins (SASP). The content of these proteins was, in fact, reduced, in particular the SASP-gamma type. The peptidoglycan-cortex, however, was not impaired since spores maintained lysozyme resistance. Addition of glutamate during sporulation partially rescued spore resistance, but germination and outgrowth remained impaired. Deficiencies in germination and outgrowth were also observed with spores from a gltA mutant strain. Taken together, these results pointed to the importance of GOGAT activity during sporulation, in particular for the synthesis SASPs.

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An α/β-Type, Small, Acid-Soluble Spore Protein Which Has Very High Affinity for DNA Prevents Outgrowth of Bacillus subtilis Spores

Cited by 29 publications

References 25 publications

Small acid-soluble spore proteins of Clostridium acetobutylicum are able to protect DNA in vitro and are specifically cleaved by germination protease GPR and spore protease YyaC

Small acid-soluble spore proteins of Clostridium acetobutylicum are able to protect DNA in vitro and are specifically cleaved by germination protease GPR and spore protease YyaC

Effects of Carboxy-Terminal Modifications and pH on Binding of a Bacillus subtilis Small, Acid-Soluble Spore Protein to DNA

Effect of Glutamate Synthase (GOGAT) Activity on Bacillus subtilis Spore Properties

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