Biomedical subjects

P Setlow

Publications and source records attributed to P Setlow.

At least 55 records · Page 3Linked to original sources

PubMed1999

Regulation of four genes encoding small, acid-soluble spore proteins in Bacillus subtilis.

Three genes (sspH, sspL, and tlp) encoding new, minor small, acid-soluble proteins (SASP) unique to spores of Bacillus subtilis are expressed only in the forespore compartment during sporulation of this organism. The sspH and sspL genes are monocistronic, whereas tlp is the second gene in an operon with a second small orf, which we have termed sspN. The sspH and sspL genes are recognized primarily by the forespore-specific sigma factor for RNA polymerase, sigmaG; the sspN-tlp operon is recognized equally well by sigmaG and the other forespore-specific sigma factor, sigmaF. Sequences centered 10 and 35nt upstream of the 5'-ends of sspH, sspL, and sspN mRNAs all show homology to -10 and -35 sequences recognized by sigmaF and sigmaG, which are generally quite similar. Mutations disrupting the sspH, sspL, sspN-tlp, or tlp loci cause a loss of the appropriate SASP from spores, but have no discernible effect on sporulation, spore properties, or spore germination.

Bacillus subtilis↗

PubMed1999

Structural studies of a novel germination protease from spores of Bacillus megaterium.

The amino acid sequence-specific protease (termed GPR) in the bacterium Bacillus megaterium initiates the rapid degradation of small, acid-soluble spore proteins during the germination of spores of this organism. GPR is synthesized during spore formation as an inactive zymogen termed P46, which later autoprocesses to a smaller active form termed P41, which acts during spore germination. However, GPR exhibits no obvious mechanistic or amino acid sequence similarity to any of the known classes of proteases. To initiate the determination of the mechanisms of P46 to P41 conversion, P46 inactivity, and P41 catalysis, B. megaterium GPR has been overexpressed in Escherichia coli and purified to homogeneity by anion-exchange and size exclusion chromatography, and crystals of both P46 and P41 have been obtained by the vapor diffusion method. P46 crystals diffracted x rays to 3.5 A but the crystals of P41 diffracted x rays to only 6.5 A. A native x-ray diffraction data set of P46 has been collected; the unit cell parameters are a = b = 76.8, c = 313.1 A, alpha = beta = gamma = 90 degrees; the space group is tetragonal P41212 or P43212. The asymmetric unit contains two monomeric molecules with a crystal volume per unit protein mass of 2. 85 A3/Da and a solvent content of about 57%. An isomorphous heavy atom derivative data set has also been obtained for P46 crystals with potassium dicyanoaurate (I).

Bacillus megaterium↗

PubMed1999

The katX gene of Bacillus subtilis is under dual control of sigmaB and sigmaF.

The gene katX, which encodes a catalase in Bacillus subtilis, is transcribed by EsigmaF in the pre-spore. This catalase is responsible for the resistance to hydrogen peroxide shown by germinating and outgrowing spores. We demonstrated that katX is also a sigmaB-dependent general stress gene, since it is strongly induced by heat, salt and ethanol stress, as well as by energy depletion. The -10 and -35 sequences of the sigmaB- and sigmaF-dependent promoters of katX overlap, and the transcriptional start points used by EsigmaB and EsigmaF differ by only one nucleotide. Our results indicate that the level of KatX level in outgrowing spores depends mainly on EsigmaF, because sigB mutants show normal KatX activity in dormant and outgrowing spores. katX mutants also develop the non-specific resistance to oxidative stress that is typical of glucose-starved cells. Therefore, the physiological role of sigmaB-dependent katX expression remains obscure. The results indicate an overlap between the sigmaB regulon and the sigmaF regulon, and the physiological implications of this overlap are discussed.

Arabidopsis Proteins↗

PubMed1999

Formaldehyde kills spores of Bacillus subtilis by DNA damage and small, acid-soluble spore proteins of the alpha/beta-type protect spores against this DNA damage.

Killing of wild-type spores of Bacillus subtilis with formaldehyde also caused significant mutagenesis; spores (termed alpha-beta-) lacking the two major alpha/beta-type small, acid-soluble spore proteins (SASP) were more sensitive to both formaldehyde killing and mutagenesis. A recA mutation sensitized both wild-type and alpha-beta- spores to formaldehyde treatment, which caused significant expression of a recA-lacZ fusion when the treated spores germinated. Formaldehyde also caused protein-DNA cross-linking in both wild-type and alpha-beta- spores. These results indicate that: (i) formaldehyde kills B. subtilis spores at least in part by DNA damage and (b) alpha/beta-type SASP protect against spore killing by formaldehyde, presumably by protecting spore DNA.

Bacillus subtilis↗

PubMed1999

Roles of low-molecular-weight penicillin-binding proteins in Bacillus subtilis spore peptidoglycan synthesis and spore properties.

The peptidoglycan cortex of endospores of Bacillus species is required for maintenance of spore dehydration and dormancy, and the structure of the cortex may also allow it to function in attainment of spore core dehydration. A significant difference between spore and growing cell peptidoglycan structure is the low degree of peptide cross-linking in cortical peptidoglycan; regulation of the degree of this cross-linking is exerted by D,D-carboxypeptidases. We report here the construction of mutant B. subtilis strains lacking all combinations of two and three of the four apparent D, D-carboxypeptidases encoded within the genome and the analysis of spore phenotypic properties and peptidoglycan structure for these strains. The data indicate that while the dacA and dacC products have no significant role in spore peptidoglycan formation, the dacB and dacF products both function in regulating the degree of cross-linking of spore peptidoglycan. The spore peptidoglycan of a dacB dacF double mutant was very highly cross-linked, and this structural modification resulted in a failure to achieve normal spore core dehydration and a decrease in spore heat resistance. A model for the specific roles of DacB and DacF in spore peptidoglycan synthesis is proposed.

Bacillus subtilis↗

PubMed1999

Septal localization of penicillin-binding protein 1 in Bacillus subtilis.

Previous studies have shown that Bacillus subtilis cells lacking penicillin-binding protein 1 (PBP1), encoded by ponA, have a reduced growth rate in a variety of growth media and are longer, thinner, and more bent than wild-type cells. It was also recently shown that cells lacking PBP1 require increased levels of divalent cations for growth and are either unable to grow or grow as filaments in media low in Mg2+, suggesting a possible involvement of PBP1 in septum formation under these conditions. Using epitope-tagging and immunofluorescence microscopy, we have now shown that PBP1 is localized at division sites in vegetative cells of B. subtilis. In addition, we have used fluorescence and electron microscopy to show that growing ponA mutant cells display a significant septation defect, and finally by immunofluorescence microscopy we have found that while FtsZ localizes normally in most ponA mutant cells, a significant proportion of ponA mutant cells display FtsZ rings with aberrant structure or improper localization, suggesting that lack of PBP1 affects FtsZ ring stability or assembly. These results provide strong evidence that PBP1 is localized to and has an important function in the division septum in B. subtilis. This is the first example of a high-molecular-weight class A PBP that is localized to the bacterial division septum.

Bacillus subtilis↗

PubMed1999

Isolation and characterization of mutations in Bacillus subtilis that allow spore germination in the novel germinant D-alanine.

Bacillus subtilis spores break their metabolic dormancy through a process called germination. Spore germination is triggered by specific molecules called germinants, which are thought to act by binding to and stimulating spore receptors. Three homologous operons, gerA, gerB, and gerK, were previously proposed to encode germinant receptors because inactivating mutations in those genes confer a germinant-specific defect in germination. To more definitely identify genes that encode germinant receptors, we isolated mutants whose spores germinated in the novel germinant D-alanine, because such mutants would likely contain gain-of-function mutations in genes that encoded preexisting germinant receptors. Three independent mutants were isolated, and in each case the mutant phenotype was shown to result from a single dominant mutation in the gerB operon. Two of the mutations altered the gerBA gene, whereas the third affected the gerBB gene. These results suggest that gerBA and gerBB encode components of the germinant receptor. Furthermore, genetic interactions between the wild-type gerB and the mutant gerBA and gerBB alleles suggested that the germinant receptor might be a complex containing GerBA, GerBB, and probably other proteins. Thus, we propose that the gerB operon encodes at least two components of a multicomponent germinant receptor.

Alanine↗

PubMed1999

Spore peptidoglycan structure in a cwlD dacB double mutant of Bacillus subtilis.

Bacillus subtilis cwlD and dacB mutants produce spore peptidoglycan (PG) with increased cross-linking but with little change in spore core hydration compared to the wild type. A cwlD dacB double mutant produced spores with a two- to fourfold greater increase in PG cross-linking and novel muropeptides containing glycine residues but no significant changes in spore resistance or core hydration.

Amino Acids↗

PubMed1998

Identification of protein-protein contacts between alpha/beta-type small, acid-soluble spore proteins of Bacillus species bound to DNA.

Small, acid-soluble spore proteins (SASP) of the alpha/beta-type from several Bacillus species were cross-linked into homodimers, heterodimers and homooligomers with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) in the presence of linear plasmid DNA. Significant protein cross-linking was not detected in the absence of DNA. In all four alpha/beta-type SASP examined, the amino donor in the EDC induced amide cross-links was the alpha-amino group of the protein. However, the carboxylate containing amino acid residues involved in cross-linking varied. In SASP-A and SASP-C of Bacillus megaterium two conserved glutamate residues, which form part of the germination protease recognition sequence, were involved in cross-link formation. In SspC from Bacillus subtilis and Bce1 from Bacillus cereus the acidic residues involved in cross-link formation were not in the protease recognition sequence, but at a site closer to the N terminus of the proteins. These data indicate that, although there are likely to be subtle structural differences between different alpha/beta-type SASP, the N-terminal regions of these proteins are involved in protein-protein interactions while in the DNA bound state.

Amino Acid Sequence↗

PubMed1998

Characterization of yhcN, a new forespore-specific gene of Bacillus subtilis.

A new Bacillus subtilis sporulation-specific gene, yhcN, has been identified, the expression of which is dependent on the forespore-specific sigma factor sigmaG and to a much lesser extent on sigmaF. A translational yhcN-lacZ fusion is expressed at a very high level in the forespore, and the protein encoded by yhcN was detected in the inner spore membrane. A yhcN mutant sporulates normally and yhcN spores have identical resistance properties to wild-type spores. However, the outgrowth of yhcN spores is slower than that of wild-type spores.

Amino Acid Sequence↗

PubMed1998

Small, Acid-Soluble Spore Proteins of the alpha/beta Type Do Not Protect the DNA in Bacillus subtilis Spores against Base Alkylation

Ethyl methanesulfonate (EMS) killed wild-type Bacillus subtilis spores as rapidly as spores lacking small, acid-soluble proteins (SASP) of the alpha/beta type (alpha-beta- spores), and 20% of the survivors had obvious mutations. A recA mutation increased the EMS sensitivity of wild-type and alpha-beta- spores similarly but reduced their mutagenesis; EMS treatment of dormant spores also resulted in the induction of RecA synthesis during spore germination. EMS generated similar levels of alkylated bases in wild-type and alpha-beta- spore DNAs, in purified DNA, or in DNA saturated with alpha/beta-type SASP. Ethylene oxide (EtO) also generated similar levels of base alkylation in wild-type and alpha-beta- spore DNAs. These data indicate that EMS and EtO kill spores at least in part by DNA damage but that alpha/beta-type SASP, which protect DNA against many types of damage, do not protect spore DNA from base alkylation.

Journal Article↗

PubMed1998

Nucleotide sequence of the sspE genes coding for gamma-type small, acid-soluble spore proteins from the round-spore-forming bacteria Bacillus aminovorans, Sporosarcina halophila and S. ureae.

The single sspE genes coding for gamma-type small, acid-soluble spore proteins (SASP) of three round-spore-forming bacteria, Bacillus aminovorans, Sporosarcina halophila and S. ureae, have been cloned and sequenced. While the deduced amino acid sequences of these three gamma-type SASP show clear homology to those from six Bacillus species that do not form round spores, there are no residues conserved completely among the 9 sequences known. In addition, the 139 residue B. aminovorans protein is 35 residues larger than any other while the 60 residue S. halophila protein is one of the smallest. These data suggest that the sspE genes have been under little selective pressure in recent evolutionary time.

Amino Acid Sequence↗

PubMed1998

Bacillus subtilis contains multiple Fur homologues: identification of the iron uptake (Fur) and peroxide regulon (PerR) repressors.

Fur (ferric uptake regulator) proteins control iron uptake in many Gram-negative bacteria. Although Fur homologues have been identified in Gram-positive bacteria, their roles in gene regulation are unknown. Genome sequencing has revealed three fur homologues in Bacillus subtilis: yqkL, yqfV and ygaG. We demonstrate that yqkL encodes an iron uptake repressor: both siderophore biosynthesis and transcription of ferri-siderophore uptake genes is constitutive in the yqkL mutant. Thus, yqkL encodes a repressor that is functionally as well as structurally related to Fur. B. subtilis peroxide stress genes are induced by either H2O2 or by metal ion limitation. Previous genetic studies defined a regulatory locus, perR, postulated to encode the peroxide regulon repressor. We demonstrate that a ygaG mutant has the perR phenotype: It is highly resistant to peroxides and overexpresses catalase, alkyl hydroperoxide reductase and the DNA binding protein MrgA. Nine spontaneous perR mutations, isolated by virtue of their ability to derepress mrgA transcription in the presence of managanous ion, all contain sequence changes in the ygaG locus and can be complemented by the cloned ygaG gene. Thus, ygaG encodes the peroxide regulon repressor and is allelic with perR.

Amino Acid Sequence↗

PubMed1998

Heat killing of Bacillus subtilis spores in water is not due to oxidative damage.

The heat resistance of wild-type spores of Bacillus subtilis or spores (termed alpha-beta-) lacking DNA protective alpha/beta-type small, acid-soluble spore proteins was not altered by anaerobiosis or high concentrations of the free radical scavenging agents ethanethiol and ethanedithiol. Heat-killed wild-type and alpha-beta- spores exhibited no increase in either protein carbonyl content or oxidized bases in DNA. These data strongly suggest that oxidative damage to spore macromolecules does not contribute significantly to spore killing by heat.

Aerobiosis↗

PubMed1998

In vitro and in vivo oxidation of methionine residues in small, acid-soluble spore proteins from Bacillus species.

Methionine residues in alpha/beta-type small, acid-soluble spore proteins (SASP) of Bacillus species were readily oxidized to methionine sulfoxide in vitro by t-butyl hydroperoxide (tBHP) or hydrogen peroxide (H2O2). These oxidized alpha/beta-type SASP no longer bound to DNA effectively, but DNA binding protected alpha/beta-type SASP against methionine oxidation by peroxides in vitro. Incubation of an oxidized alpha/beta-type SASP with peptidyl methionine sulfoxide reductase (MsrA), which can reduce methionine sulfoxide residues back to methionine, restored the alpha/beta-type SASP's ability to bind to DNA. Both tBHP and H2O2 caused some oxidation of the two methionine residues of an alpha/beta-type SASP (SspC) in spores of Bacillus subtilis, although one methionine which is highly conserved in alpha/beta-type SASP was only oxidized to a small degree. However, much more methionine sulfoxide was generated by peroxide treatment of spores carrying a mutant form of SspC which has a lower affinity for DNA. MsrA activity was present in wild-type B. subtilis spores. However, msrA mutant spores were no more sensitive to H2O2 than were wild-type spores. The major mechanism operating for dealing with oxidative damage to alpha/beta-type SASP in spores is DNA binding, which protects the protein's methionine residues from oxidation both in vitro and in vivo. This may be important in vivo since alpha/beta-type SASP containing oxidized methionine residues no longer bind DNA well and alpha/beta-type SASP-DNA binding is essential for long-term spore survival.

Bacillus↗

PubMed1998

Bacillus subtilis cells lacking penicillin-binding protein 1 require increased levels of divalent cations for growth.

Bacillus subtilis strains lacking penicillin-binding protein 1 (PBP1), encoded by ponA, required greater amounts of Mg2+ or Ca2+ for vegetative growth or spore outgrowth than the wild-type strain and strains lacking other high-molecular-weight (HMW) PBPs. Growth of ponA cells in a medium low in Mg2+ also resulted in greatly increased cell bending compared to wild-type cells or cells lacking other HMW PBPs. The addition of high levels of Mg2+ to growth media eliminated these phenotypes of a ponA mutant. In contrast to the effects of divalent cations, NaCl did not restore ponA cell growth in a divalent-cation-deficient medium. Surprisingly, wild-type cells swelled and then lysed during both vegetative growth and spore outgrowth when 500 mM NaCl was included in a divalent-cation-deficient medium. Again, Mg2+ addition was sufficient to allow normal vegetative growth and spore outgrowth of both wild-type and ponA cells in a medium with 500 mM NaCl. These studies demonstrate that (i) while HMW PBPs possess largely redundant functions in rich medium, when divalent cations are limiting, PBP1 is required for cell growth and spore outgrowth; and (ii) high levels of NaCl induce cell lysis in media deficient in divalent cations during both vegetative growth and spore outgrowth.

Bacillus subtilis↗

PubMed1998

Characterization of dacC, which encodes a new low-molecular-weight penicillin-binding protein in Bacillus subtilis.

The pbp gene (renamed dacC), identified by the Bacillus subtilis genome sequencing project, encodes a putative 491-residue protein with sequence homology to low-molecular-weight penicillin-binding proteins. Use of a transcriptional dacC-lacZ fusion revealed that dacC expression (i) is initiated at the end of stationary phase; (ii) depends strongly on transcription factor sigmaH; and (iii) appears to be initiated from a promoter located immediately upstream of yoxA, a gene of unknown function located upstream of dacC on the B. subtilis chromosome. A B. subtilis dacC insertional mutant grew and sporulated identically to wild-type cells, and dacC and wild-type spores had the same heat resistance, cortex structure, and germination and outgrowth kinetics. Expression of dacC in Escherichia coli showed that this gene encodes an approximately 59-kDa membrane-associated penicillin-binding protein which is highly toxic when overexpressed.

Bacillus subtilis↗

PubMed1998

Structure and mechanism of action of the protease that degrades small, acid-soluble spore proteins during germination of spores of Bacillus species.

The germination protease (GPR) of Bacillus megaterium initiates the degradation of small, acid-soluble proteins during spore germination. Trypsin treatment of the 46-kDa GPR zymogen (termed P46) removes an approximately 15-kDa C-terminal domain generating a 30-kDa species (P30) which is stable against further digestion. While P30 is not active, it does autoprocess to a smaller form by cleavage of the same bond cleaved in conversion of P46 to the active 41-kDa form of GPR (P41). Trypsin treatment of P41 cleaves the same bond in the C-terminal part of the protein as is cleaved in the P46-->P30 conversion. While the approximately 29-kDa species generated by trypsin treatment of P41 is active, it is rapidly degraded further by trypsin to small inactive fragments. These results, as well as a thermal melting temperature for P41 which is 13 degreesC lower than that for P46 and the unfolding of P41 at significantly lower concentrations of guanidine hydrochloride than for P46, are further evidence for a difference in tertiary structure between P46 and P41, with P46 presumably having a more compact stable structure. However, circular dichroism spectroscopy revealed no significant difference in the secondary structure content of P46 and P41. The removal of approximately 30% of P46 or P41 without significant loss in enzyme activity localized GPR's catalytic residues to the N-terminal two-thirds of the molecule. This finding, as well as comparison of the amino acid sequences of GPR from three different species, analysis of several site-directed GPR mutants, determination of the metal ion content of purified GPR, and lack of inhibition of P41 by a number of protease inhibitors, suggests that GPR is not a member of a previously described class of protease.

Amino Acid Sequence↗