The effect of hypochlorite on spores of Bacillus subtilis lacking small acid-soluble proteins

M.Z.H. SABLI, P. SETLOW AND W.M. WAITES. 1996. α/β-Type small acid-soluble proteins (SASP) bind to spore DNA and protect it against ultraviolet light, heat, hydrogen peroxide and freeze drying, making the spores much more resistant than vegetative cells to these agents. Spores of a mutant of Bacillus subtilis lacking the two major α/β-type SASP were almost 30 000-fold less resistant to hypochlorite than were wild-type spores. After treatment with hypochlorite, surviving spores of the mutant, but not those of the wild type, showed higher levels of mutation, suggesting that SASP contribute to hypochlorite resistance by protecting spore DNA.     

Analysis of deamidation of small, acid-soluble spore proteins from Bacillus subtilis in vitro and in vivo.

Deamidation of one specific asparagine residue in an alpha/beta-type small, acid-soluble spore protein (SASP) of Bacillus subtilis took place readily in vitro (time for 50% deamidation [t(1/2)], approximately 1 h at 70 degrees C), and the deamidated SASP no longer bound to DNA effectively. However, DNA binding protected against this deamidation in vitro. A mutant alpha/beta-type SASP in which the reactive asparagine was changed to aspartate also failed to bind to DNA in vitro, and this protein did not restore UV radiation and heat resistance to spores lacking the majority of their alpha/beta-type SASP. When expressed in Escherichia coli, where it is bound to DNA, the alpha/beta-type SASP deamidated with a t(1/2) of 2 to 3 h at 95 degrees C. However, the alpha/beta-type SASP was extremely resistant to deamidation within spores (t(1/2), >50 h at 95 degrees C). A gamma-type SASP of B. subtilis also deamidated readily in vitro (t(1/2) for one net deamidation, approximately 1 h at 70 degrees C), but this protein (which is not associated with DNA) deamidated fairly readily in spores (t(1/2), approximately 1 h at 95 degrees C). Total spore core protein also deamidated in vivo, although the rate was two- to threefold slower than that of deamidation of total protein in heated vegetative cells. These data indicate that protein deamidation is slowed significantly in spores, presumably due to the spore's environment. However, alpha/beta-type SASP are even more strongly protected against deamidation in vivo, presumably by their binding to spore DNA. Thus, not only do alpha/beta-type SASP protect spore DNA from damage; DNA also protects alpha/beta-type SASP.     

Folding of prokaryotic DNA. Isolation and characterization of nucleoids from Bacillus licheniformis

Intact and fast-sedimenting nucleoids of Bacillus licheniformis were isolated under low-salt conditions and without addition of detergents, polyamines or Mg2+. These nucleoids were partially unfolded by treatment with RNase and completely unfolded by treatments that disrupt protein-DNA interactions, like incubation with proteinase K, 0.1% sodium dodecyl sulphate and high ionic strength. Ethidium bromide intercalation studies on RNase-treated, proteinase-K-treated and non-treated nucleoids in combination with sedimentation analysis of DNase-I-treated nucleoids revealed that DNA is organized in independent, negatively supertwisted domains. In contrast to the DNA organization in bacterial nucleoids, isolated under high-salt conditions and in the presence of detergents (Stonington & Pettijohn, 1971; Worcel & Burgi, 1972), the domains of supertwisted DNA in the low-salt-isolated nucleoids studied here are restrained by protein-DNA interactions. A major role for nascent RNA in restraining supertwisted DNA was not observed. The superhelix density of B. licheniformis nucleoids calculated from the change of the sedimentation coefficient upon ethidium bromide intercalation, was of the same order of magnitude as that of other bacterial nucleoids and eukaryotic chromosomes, isolated under high-salt conditions: namely, -0.150 (corrected to standard conditions: 0.2 M-NaCl, 37 degrees C; Bauer, 1978). Electron microscopy of spread nucleoids showed relaxed DNA and regions of condensed DNA. Spreading in the presence of 100 micrograms ethidium bromide per ml revealed only condensed structures, indicating that nucleoids are intact. From spreadings of proteinase-K-treated nucleoids we infer that supertwisted DNA and the protein-DNA interactions, responsible for restraining the superhelical DNA conformation, are localized in the regions of condensed DNA.     

Effects of mutant small, acid-soluble spore proteins from Bacillus subtilis on DNA in vivo and in vitro.

alpha/beta-type small, acid-soluble spore proteins (SASP) of Bacillus subtilis bind to DNA and alter its conformation, topology, and photochemistry, and thereby spore resistance to UV light. Three mutations have been introduced into the B. subtilis sspC gene, which codes for the alpha/beta-type wild-type SASP, SspCwt. One mutation (SspCTyr) was a conservative change, as residue 29 (Leu) was changed to Tyr, an amino acid found at this position in other alpha/beta-type SASP. The other mutations changed residues conserved in all alpha/beta-type SASP. In one (SspCAla), residue 52 (Gly) was changed to Ala; in the second (SspCGln), residue 57 (Lys) was changed to Gln. The effects of the wild-type and mutant SspC on DNA properties were examined in vivo in B. subtilis spores and Escherichia coli as well as in vitro with use of purified protein. Both SspCwt and SspCTyr interacted similarly with DNA in vivo and in vitro, restoring much UV resistance to spores lacking major alpha/beta-type SASP, causing a large increase in plasmid negative supercoiling, and altering DNA UV photochemistry from cell type to spore type. In contrast, SspCAla had no detectable effect on DNA properties in vivo or in vitro, while SspCGln had effects intermediate between those of SspCAla and SspCwt. Strikingly, neither SspCAla nor SspCGln bound well to DNA in vitro. These results confirm the importance of the conserved primary sequence of alpha/beta-type SASP in the ability of these proteins to bind to spore DNA and cause spore UV resistance.     

Protection of DNA by alpha/beta-type small,acid-soluble proteins from Bacillus subtilis spores against cytosine deamination

Spores of Bacillus subtilis contain high levels of proteins, termed alpha/beta-type small, acid-soluble proteins (SASP), that protect the spore's DNA against different types of DNA damage. We tested one such protein, SspC, and two of its variants for their ability to protect plasmid DNA against hydrolytic deamination of cytosine to uracil. If unrepaired, such damage to DNA causes C to T mutations. We found that one SspC variant, SspC(Delta 11-D13K), protected DNA against cytosine deamination at two different temperatures (45 and 70 degrees C) and pH values (5.2 and 7.9), reducing the rate of deamination by as much as 10-fold. At 70 degrees C, pH 7.9, the wild-type SspC and its variant, SspC(Delta 11), provided little protection against deamination but were effective in protecting DNA at 45 degrees C, pH 7.9. Parallel studies of the abilities of these proteins to protect DNA against restriction digestion revealed that there was a good correlation between the abilities of the proteins to protect against restriction endonucleases and reductions in cytosine deaminations. These results show that the binding of SspC variants to DNA can prevent attack on DNA bases by water and suggest a new general mechanism by which DNA-binding proteins in cells may be able to protect chromosomes from endogenous and exogenous reactive chemicals by excluding them from the vicinity of DNA.     

Immunoelectron microscopic localization of small, acid-soluble spore proteins in sporulating cells of Bacillus subtilis.

Small, acid-soluble spore proteins SASP-alpha, SASP-beta, and SASP-gamma as well as a SASP-beta-lacZ gene fusion product were found only within the forespore compartment of sporulating Bacillus subtilis cells by using immunoelectron microscopy. The alpha/beta-type SASP were associated almost exclusively with the forespore nucleoid, while SASP-gamma was somewhat excluded from the nucleoid. These different locations of alpha/beta-type and gamma-type small, acid-soluble spore proteins within the forespore are consistent with the different roles for these two types of proteins in spore resistance to UV light.     

Transcriptional control of synthesis of acid-soluble proteins in sporulating Bacillus subtilis

The major acid-soluble spore proteins (ASSPs) isolated from mature spores of Bacillus subtilis are designated alpha, beta, and gamma (about 60, 60, and 100 amino acids in length, respectively). Alpha and beta are very similar, and gamma is very similar to a less predominant ASSP called delta (about 115 amino acids). A minor and very basic ASSP called epsilon is the same size as alpha and beta but is unrelated antigenically. These and several minor ASSPs comprise at least three related families of sporulation-specific gene products. Expression of the alpha and beta genes, detectable as functional mRNA in vitro, coincides with the time of synthesis of all of the major ASSPs in vivo. This apparently coordinate expression is dependent on at least the spo0A, spoIIA, and spoIIIA loci, but not on the spoIVA or spoVA loci, consistent with the late stage of this expression (initiating at 3.5 h after the start of sporulation and peaking at 5 h after start of sporulation). A few minor ASSPs may be asynchronously expressed.     

Isolation and characterization of histones and other acid-soluble chromosomal proteins from Physarum polycephalum

Chromosomal basic proteins were isolated from amoebal and plasmodial stages of the acellular slime mold Physarum polycephalum. Polyacrylamide electrophoresis on high resolution acid-urea gels separated the five histone fractions in the sequence H1, H2A, H2B, H3, and H4. Under these electrophoretic conditions Physarum histones migrated more like plant (rye) than animal (calf) histones. Furthermore, Physarum histones H1, H2A, and H2B have higher molecular weights on sodium dodecyl sulfate (SDS) gels than the corresponding calf fractions. No differences were detected between amoebal and plasmodial histones on either acid-urea or SDS-polyacrylamide gel electrophoresis. Amoebal basic proteins were fractionated by exclusion chromatography. The five histone fractions plus another major acid-soluble chromosomal protein (AS) were isolated. The Physarum core histones had amino acid compositions more closely resembling those of the calf core histones than of rye, yeast, or Dictyostelium. Although generally similar in composition to the plant and animal H1 histones, the Physarum H1 had a lower lysine content. The AS protein was extracted with 5% perchloric acid or 0.5 M NaCl, migrated between histones H3 and H4 on acid-urea polyacrylamide gels, and had an apparent molecular weight of 15 900 on SDS gels. It may be related to a protein migrating near H1. Both somewhat resembled the high mobility group proteins in amino acid composition.     

Site-directed mutagenesis with the ptsH gene of Bacillus subtilis. Isolation and characterization of heat-stable proteins altered at the ATP-dependent regulatory phosphorylation site

The codon for Ser-46 of the ptsH gene of Bacillus subtilis was modified by site-directed mutagenesis to the codons for Ala, Thr, Tyr, and Asp. The mutant genes were overexpressed, three of the corresponding proteins were purified to homogeneity with the exception for the Asp derivative, which could not be detected, although the gene had the desired nucleotide sequence. The phosphotransferase activity of the altered proteins was determined to be 20-35% of wild type activity, which correlates well with the slow phosphorylation of heat-stable protein (HPr) by enzyme I and phosphoenolpyruvate. The ATP-dependent HPr kinase, which previously was shown to be involved in the regulation of carbohydrate uptake of Gram-positive bacteria by covalent phosphorylation of Ser-46 of HPr, is entirely inactive toward the OH group of Thr-46 and Tyr-46 proteins. In addition, we constructed a strain of B. subtilis, where the altered gene coding for the Ala-46 derivative of HPr was introduced into the bacterial chromosome. The physiological properties of this mutant are described.     

Isolation and characterization of the lytic enzyme from Bacillus subtilis 797

The isolation procedure and some properties of the lytic enzyme produced by Bacillus subtilis 797 and capable of hydrolyzing the E. coli K-12 cells are described. Using hydrophobic chromatography on DNP-hexamethylene diamine Sepharose 4B and ion-exchange chromatography on DEAE-cellulose, a highly purified enzyme preparation with mol. weight of 28000, pI 8.2 has been obtained. The amino acid composition of the enzyme has been determined: Asp--37, Thr--17, Ser--34, Glu--15, Pro--14, Gly--17, Ala--36, Val--28, Met--4, Ile--11, Leu--17, Tyr--9, Phe--4, Lys--18, His--5, Arg--4. The enzyme is inhibited by a specific inhibitor of serine proteinases--benzylsulfonylfluoride, and is insensitive to EDTA and iodoacetic acid. The lytic enzyme has a proteolytic activity and splits the peptide substrate of bacterial serine proteinases--p-nitroanilide benzyloxycarbonyl-L-analyl-L-alanyl-L-leucine; the maxima for both activities lie within the pH range of 7.8-8.5. The lytic protease has the highest stability at pH 6-10. In some of its properties the enzyme is similar to serine proteinase from Bac. subtilis, i. e. subtilisins.     

Essential role of small, acid-soluble spore proteins in resistance of Bacillus subtilis spores to UV light.

Bacillus subtilis strains containing deletions in the genes coding for one or two of the major small, acid-soluble spore proteins (SASP; termed SASP-alpha and SASP-beta) were constructed. These mutants sporulated normally, but the spores lacked either SASP-alpha, SASP-beta, or both proteins. The level of minor SASP did not increase in these mutants, but the level of SASP-alpha increased about twofold in the SASP-beta- mutant, and the level of SASP-beta increased about twofold in the SASP-alpha- mutant. The growth rates of the deletion strains were identical to that of the wild-type strain in rich or poor growth media, as was the initiation of spore germination. However, outgrowth of spores of the SASP-alpha(-)-beta- strain was significantly slower than that of wild-type spores in all media tested. The heat resistance of SASP-beta- spores was identical to that of wild-type spores but slightly greater than that of SASP-alpha- and SASP-alpha(-)-beta- spores. However, the SASP-alpha- and SASP-alpha(-)-beta- spores were much more heat resistant than vegetative cells. The UV light resistances of SASP-beta- and wild-type spores were also identical. However, SASP-alpha(-)-beta- spores were slightly more sensitive to UV light than were log-phase cells of the wild-type or SASP-alpha(-)-beta- strain (the latter have identical UV light resistances); SASP-alpha- spores were slightly more UV light resistant than SASP-alpha(-)-beta- spores. These data strongly implicate SASP, in particular SASP-alpha, in the UV light resistance of B. subtilis spores.     

Properties of Bacillus megaterium and Bacillus subtilis mutants which lack the protease that degrades small, acid-soluble proteins during spore germination.

During germination of spores of Bacillus species the degradation of the spore's pool of small, acid-soluble proteins (SASP) is initiated by a protease termed GPR, the product of the gpr gene. Bacillus megaterium and B. subtilis mutants with an inactivated gpr gene grew, sporulated, and triggered spore germination as did gpr+ strains. However, SASP degradation was very slow during germination of gpr mutant spores, and in rich media the time taken for spores to return to vegetative growth (defined as outgrowth) was much longer in gpr than in gpr+ spores. Not surprisingly, gpr spores had much lower rates of RNA and protein synthesis during outgrowth than did gpr+ spores, although both types of spores had similar levels of ATP. The rapid decrease in the number of negative supertwists in plasmid DNA seen during germination of gpr+ spores was also much slower in gpr spores. Additionally, UV irradiation of gpr B. subtilis spores early in germination generated significant amounts of spore photoproduct and only small amounts of thymine dimers (TT); in contrast UV irradiation of germinated gpr+ spores generated almost no spore photoproduct and three to four times more TT. Consequently, germinated gpr spores were more UV resistant than germinated gpr+ spores. Strikingly, the slow outgrowth phenotype of B. subtilis gpr spores was suppressed by the absence of major alpha/beta-type SASP. These data suggest that (i) alpha/beta-type SASP remain bound to much, although not all, of the chromosome in germinated gpr spores; (ii) the alpha/beta-type SASP bound to the chromosome in gpr spores alter this DNA's topology and UV photochemistry; and (iii) the presence of alpha/beta-type SASP on the chromosome is detrimental to normal spore outgrowth.     

Expression of Bacillus megaterium and Bacillus subtilis small acid-soluble spore protein genes during stationary-phase growth of asporogenous B. subtilis mutants

The small acid-soluble spore proteins alpha and beta were not detected during stationary-phase growth of asporogenous Bacillus subtilis mutants blocked in stages 0, II, or III, but mutants blocked in stages IV or V accumulated nearly wild-type levels of these small acid-soluble spore proteins. Similar results were obtained when production of Bacillus megaterium C protein (also a small acid-soluble spore protein), as well as alpha and beta, were monitored in these mutants containing a recombinant plasmid carrying the B. megaterium C protein gene. The only exception was a spo0H mutant which synthesized a small amount of C protein, but no alpha or beta.     

Isolation and characterization of a novel extracellular metalloprotease from Bacillus subtilis.

We have isolated and characterized two minor extracellular proteases from culture supernatants of a strain of Bacillus subtilis containing deletion mutations of the genes for the extracellular proteases subtilisin (apr) and neutral protease (npr) and a minor extracellular protease (epr) as well as intracellular serine protease-I (isp-1). Characterization studies have revealed that one of these enzymes is the previously described protease bacillopeptidase F. The second enzyme, the subject of this report, is a novel metalloprotease, which we designate Mpr. Mpr is a unique metalloprotease that has been purified to apparent homogeneity by using both conventional and high-performance liquid chromatography procedures. Mpr has a molecular mass of approximately 28 kilodaltons on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a basic isoelectric point of 8.7. The enzyme showed maximal activity against azocoll at pH 7.5 and 50 degrees C. Mpr was inhibited by dithiothreitol and a combination of beta-mercaptoethanol and EDTA. Activity was moderately inhibited by beta-mercaptoethanol and EDTA alone as well as by cysteine and citrate and only marginally by phosphoramidon 1,10-phenanthroline and N-[N-(L-3-trans-carboxyoxiran-2-carbonyl)-L-leucyl]-agmatine. Mpr was not inhibited by phenylmethylsulfonyl fluoride. In addition, Mpr showed esterolytic but not collagenolytic activities. Our studies suggest that Mpr is a secreted metalloprotease containing cysteine residues that are required for maximal activity.     

The Bacillus subtilis HBsu protein modifies the effects of alpha/beta-type, small acid-soluble spore proteins on DNA

HBsu, the Bacillus subtilis homolog of the Escherichia coli HU proteins and the major chromosomal protein in vegetative cells of B. subtilis, is present at similar levels in vegetative cells and spores ( approximately 5 x 10(4) monomers/genome). The level of HBsu in spores was unaffected by the presence or absence of the alpha/beta-type, small acid-soluble proteins (SASP), which are the major chromosomal proteins in spores. In developing forespores, HBsu colocalized with alpha/beta-type SASP on the nucleoid, suggesting that HBsu could modulate alpha/beta-type SASP-mediated properties of spore DNA. Indeed, in vitro studies showed that HBsu altered alpha/beta-type SASP protection of pUC19 from DNase digestion, induced negative DNA supercoiling opposing alpha/beta-type SASP-mediated positive supercoiling, and greatly ameliorated the alpha/beta-type SASP-mediated increase in DNA persistence length. However, HBsu did not significantly interfere with the alpha/beta-type SASP-mediated changes in the UV photochemistry of DNA that explain the heightened resistance of spores to UV radiation. These data strongly support a role for HBsu in modulating the effects of alpha/beta-type SASP on the properties of DNA in the developing and dormant spore.     

Base-change mutations induced by various treatments of Bacillus subtilis spores with and without DNA protective small,acid-soluble spore proteins

Previous work has shown that spores of wild-type Bacillus subtilis are more resistant to killing by dry and wet heat, low vacuum lyophilization and hydrogen peroxide than are spores lacking the majority of their DNA protective alpha/beta-type small, acid-soluble spore proteins (SASP) (termed alpha(-)beta(-) spores). These four treatments kill alpha(-)beta(-) spores in large part by DNA damage with accompanying mutagenesis, but only dry heat kills wild-type spores by DNA damage and mutagenesis. DNA sequence analysis of nalidixic acid-resistant (nal(r)) mutants generated by these treatments has now shown that the nal(r) mutations are base changes in the gyrA gene that encodes one subunit of DNA gyrase. Analysis of the DNA sequence of the gyrA gene in a large number of nal(r) mutants also indicates that: (1) base changes induced by hydrogen peroxide and wet heat in alpha(-)beta(-) spores are similar to those in spontaneous nal(r) mutants with only a few notable differences; (2) base changes induced by dry heat in wild-type spores and low vacuum lyophilization of alpha(-)beta(-) spores are similar, and include a high level of a tandem base change seen previously only in spores treated with very high vacuum and (3) base changes induced by lyophilization and dry heat are very different from those in spontaneous mutants in wild-type and alpha(-)beta(-) spores, which exhibit only one significant difference. While the initial DNA damage generated in spores by dry heat, lyophilization or high vacuum is almost certainly different than that generated by hydrogen peroxide or wet heat, the precise nature of the DNA damage remains to be determined.     

Isolation and characterization of a unique protease from sporulating cells of Bacillus subtilis

Two proteases, designated I and II, have been isolated from sporulating cells of Bacillus subtilis. They were partially purified by ammonium sulfate fractionation, Sephadex chromatography and affinity columns. Protease I was found to be similar to an already characterized B. subtilis protease. Protease II is trypsin-like in its substrate specificity and is distinct from protease I in its pH optimum, pH stability, molecular weight, substrate specificity, heat stability and sensitivity to various inhibitors. While both enzymes were produced primarily during sporulation, they attained maximum levels of activity at different times. Distinct functions for these proteases in post exponential B. subtilis are likely.