ComEA is a DNA receptor for transformation of competent Bacillus subtilis

Competent cells of Bacillus subtilis efficiently bind and internalize DNA. ComEA and the seven proteins encoded by the comG operon are required in vivo for the binding step. We show here that ComEA, a bitopic membrane protein, is itself capable of high-affinity DNA binding. A domain necessary for DNA binding is located at the C-terminus of ComEA. Proteins with similar 60-80 amino acid residue domains are widespread among bacteria and higher organisms. ComEA shows a marked preference for double-stranded DNA and can bind to oligomers as small as 22 bp in length. DNA binding by ComEA exhibits no apparent base sequence specificity. Using a membrane vesicle DNA-binding assay system we show that in the absence of cell wall, ComEA is still required for DNA binding, whereas the requirement for the ComG proteins is bypassed. We conclude that the ComG proteins are needed in vivo to provide access of the binding domain of ComEA to exogenous DNA. Possible specific roles for the ComG proteins are discussed.     

Homology in natural cryptic plasmids of Bacillus subtilis

Peculiarities of DNA homology in a number of cryptic plasmids isolated from soil bacillary strains and related or identical to Bacillus subtilis were studied. Fragments generated after digestion of one of these plasmids, p1414, were employed as a probe for blot hybridization with identical fragments of other plasmids. The data obtained suggest that nearly all the studied plasmids possess a common homology site that occupies a significant plasmid portion. Regions of detectable and weak homology were located throughout this site. Moreover, plasmid p1414 was shown to carry a large site that lacks homology with plasmids belonging to two groups. Eventually, one of these plasmids demonstrates complete lack of homology with the remaining plasmids.     

Cell surface localization and processing of the ComG proteins, required for DNA binding during transformation of Bacillus subtilis

The comG operon of Bacillus subtilis encodes seven proteins essential for the binding of transforming DNA to the competent cell surface. We have explored the processing of the ComG proteins and the cellular localization of six of them. All of the proteins were found to be membrane associated. The four proteins with N-terminal sequence motifs typical of type 4 pre-pilins (ComGC, GD, GE and GG) are processed by a pathway that requires the product of comC, also an essential competence gene. The unprocessed forms of ComGC and GD behave like integral membrane proteins. Pre-ComGG differs from pre-ComGC and pre-ComGD, in that it is accessible to proteolysis only from the cytoplasmic face of the membrane and at least a portion of it behaves like a peripheral membrane protein. The mature forms of these proteins are translocated to the outer face of the membrane and are liberated when peptidoglycan is hydrolysed by lysozyme or mutanolysin. ComGG exists in part as a disulphide-cross-linked homodimer in vivo. ComGC was found to possess an intramolecular disulphide bond. The previously identified homodimer form of this protein is not stabilized by disulphide bond formation. ComGF behaves as an integral membrane protein, while ComGA, a putative ATPase, is located on the inner face of the membrane as a peripheral membrane protein. Possible roles of the ComG proteins in DNA binding to the competent cell surface are discussed in the light of these and other results.     

The minimal sequence needed to define a functional DNA terminator in Bacillus subtilis

BACKGROUND: We wished to determine whether transcutaneous oximetry or laser Doppler flowmetry (LDF) could identify patients at risk for wound failure after conservative, limb-sparing surgery for extremity sarcomas. METHODS: Studies were performed on postoperative days (PODs) 1, 4/5, 7, and 9. Measurements of transcutaneous oxygen pressure (tcPO2) were taken at breathing room air (BL) and 100% oxygen (rate tcPO2). LDF measurements were taken at multiple sites along the wound, and a perfusion index was calculated. RESULTS: Twenty-four patients were studied. Four (17%) had nonhealing wounds. There was no difference in tcPO2 (BL) values between healed and nonhealing wounds. Measurement of rate tcPO2 on POD 1 was significantly lower in the nonhealing wounds than in those with normal healing (28.5 +/- 12.1 mm Hg vs 14.3 +/- 16.2 mm Hg, mean +/- SD, p = 0.03). Rate tcPO2 values increased significantly in healing wounds from POD 1 to PODs 7 and 9 (p = 0.006, p = 0.009). This increase was absent in nonhealing wounds. A clear separation was noted in rate tcPO2 values between groups, with a minimum rate tcPO2 value recorded in a healed wound of 9 mm Hg/min, compared with the maximum value in a nonhealing wound of 7 mm Hg/min. The LDF perfusion index failed to predict wound healing at any of the measured time points. CONCLUSIONS: This study showed that measurement of tcPO2 during oxygen inhalation can accurately predict wound healing in patients after excision of an extremity sarcoma.     

Unstable association in vitro between donor and recipient DNA in Bacillus subtilis

In addition to stable donor-recipient DNA complexes, unstable complexes between donor and recipient DNA were formed in vitro with Bacillus subtilis. Whereas the stable complexes survived CsCl gradient centrifugation at pH 11.2 and phenol plus sodium p-aminosalicylate extraction with 0.17 M NaCl, the unstable complexes dissociated during these manipulations. The donor moiety from the unstable complexes remained associated with the recipient DNA during phenol plus sodium p-aminosalicylate treatment at 0.85 M NaCl. The unstable complexes could be stabilized artificially by cross-linking with 4,5',8-trimethylpsoralen. Dissociation of the complexes during CsCl gradient centrifugation could be prevented by centrifuging at pH 10. Heterologous DNA fragments derived from phage H1 DNA appeared to be unable to form complexes with the recipient B. subtilis DNA. Unstable complexes were also formed with Escherichia coli DNA, although under all conditions tested, more complex was detectable by using homologous B. subtilis DNA.     

comF, a Bacillus subtilis late competence locus, encodes a protein similar to ATP-dependent RNA/DNA helicases

The family has increasingly been recognized as an important component in the development, maintenance, and treatment of alcoholism. Few empirical studies, however, have examined alcoholism within a family context. Questionnaire and interview data were collected from women whose husbands received inpatient treatment for alcoholism. Since wives now typically work outside the home, this article focuses on the 60 employed wives. Employment was examined as a source of stress as well as social support. The majority of working wives reported minimal negative impact of their husbands' drinking on all areas of their work functioning, with a small subset indicating impairment attributable to the drinking. These wives were very satisfied with their current positions and described work as a positive experience. However, unobtrusive measures that alcoholism in a family member intrudes into the workplace were apparent, including changing jobs, absenteeism, and discussing husbands' drinking at work. Further, these women scored closer to a sample of depressed women than a community sample on measures of physical and mental health, depressed mood, and smoking symptoms. Possible reasons for the discrepancy between subjective reports and objective indicators are discussed.     

Sequence and analysis of the genetic locus responsible for surfactin synthesis in Bacillus subtilis

The chromosomal region of Bacillus subtilis comprising the entire srfA operon, sfp and about four kilobases in between have been completely sequenced and functionally characterized. The srfA gene codes for three large subunits of surfactin synthetase, 402, 401 and 144 kDa, respectively, arranged in a series of seven amino acid activating domains which, as shown in the accompanying communication, recognize and bind the seven amino acids of the surfactin peptide. The srfA amino acid activating domains share homologies with similar domains of other peptide synthetases; in particular, regions can be identified which are more homologous in domains activating the same amino acid. A fourth gene in srfA encodes a polypeptide homologous to grsT. Four genes are positioned between srfA and sfp, the disruption of which does not affect surfactin biosynthesis.     

Subcellular localization of Bacillus subtilis SMC, a protein involved in chromosome condensation and segregation

We have investigated the subcellular localization of the SMC protein in the gram-positive bacterium Bacillus subtilis. Recent work has shown that SMC is required for chromosome condensation and faithful chromosome segregation during the B. subtilis cell cycle. Using antibodies against SMC and fluorescence microscopy, we have shown that SMC is associated with the chromosome but is also present in discrete foci near the poles of the cell. DNase treatment of permeabilized cells disrupted the association of SMC with the chromosome but not with the polar foci. The use of a truncated smc gene demonstrated that the C-terminal domain of the protein is required for chromosomal binding but not for the formation of polar foci. Regular arrays of SMC-containing foci were still present between nucleoids along the length of aseptate filaments generated by depleting cells of the cell division protein FtsZ, indicating that the formation of polar foci does not require the formation of septal structures. In slowly growing cells, which have only one or two chromosomes, SMC foci were principally observed early in the cell cycle, prior to or coincident with chromosome segregation. Cell cycle-dependent release of stored SMC from polar foci may mediate segregation by condensation of chromosomes.     

Bacillus subtilis RNase III gene: cloning, function of the gene in Escherichia coli, and construction of Bacillus subtilis strains with altered rnc loci

The rnc gene of Bacillus subtilis, which has 36% amino acid identity with the gene that encodes Escherichia coli RNase III endonuclease, was cloned in E. coli and shown by functional assays to encode B. subtilis RNase III (Bs-RNase III). The cloned B. subtilis rnc gene could complement an E. coli rnc strain that is deficient in rRNA processing, suggesting that Bs-RNase III is involved in rRNA processing in B. subtilis. Attempts to construct a B. subtilis rnc null mutant were unsuccessful, but a strain was constructed in which only a carboxy-terminal truncated version of Bs-RNase III was expressed. The truncated Bs-RNase III showed virtually no activity in vitro but was active in vivo. Analysis of expression of a copy of the rnc gene integrated at the amy locus and transcribed from a p(spac) promoter suggested that expression of the B. subtilis rnc is under regulatory control.     

Identification of a zinc-specific metalloregulatory protein, Zur, controlling zinc transport operons in Bacillus subtilis

Zinc is an essential nutrient for all cells, but remarkably little is known regarding bacterial zinc transport and its regulation. We have identified three of the key components acting to maintain zinc homeostasis in Bacillus subtilis. Zur is a metalloregulatory protein related to the ferric uptake repressor (Fur) family of regulators and is required for the zinc-specific repression of two operons implicated in zinc uptake, yciC and ycdHIyceA. A zur mutant overexpresses the 45-kDa YciC membrane protein, and purified Zur binds specifically, and in a zinc-responsive manner, to an operator site overlapping the yciC control region. A similar operator precedes the ycdH-containing operon, which encodes an ABC transporter. Two lines of evidence suggest that the ycdH operon encodes a high-affinity zinc transporter whereas YciC may function as part of a lower-affinity pathway. First, a ycdH mutant is impaired in growth in low-zinc medium, and this growth defect is exacerbated by the additional presence of a yciC mutation. Second, mutation of ycdH, but not yciC, alters the regulation of both the yciC and ycdH operons such that much higher levels of exogenous zinc are required for repression. We conclude that Zur is a Fur-like repressor that controls the expression of two zinc homeostasis operons in response to zinc. Thus, Fur-like regulators control zinc homeostasis in addition to their previously characterized roles in regulating iron homeostasis, acid tolerance responses, and oxidative stress functions.     

Isolation, characterization, molecular gene cloning, and sequencing of a novel phytase from Bacillus subtilis

The Bacillus subtilis strain VTT E-68013 was chosen for purification and characterization of its excreted phytase. Purified enzyme had maximal phytase activity at pH 7 and 55 degrees C. Isolated enzyme required calcium for its activity and/or stability and was readily inhibited by EDTA. The enzyme proved to be highly specific since, of the substrates tested, only phytate, ADP, and ATP were hydrolyzed (100, 75, and 50% of the relative activity, respectively). The phytase gene (phyC) was cloned from the B. subtilis VTT E-68013 genomic library. The deduced amino acid sequence (383 residues) showed no homology to the sequences of other phytases nor to those of any known phosphatases. PhyC did not have the conserved RHGXRXP sequence found in the active site of known phytases, and therefore PhyC appears not to be a member of the phytase subfamily of histidine acid phosphatases but a novel enzyme having phytase activity. Due to its pH profile and optimum, it could be an interesting candidate for feed applications.     

Isolation of the Bacillus subtilis cdd downstream region and analysis of genetic structure around the cdd vicinity

A 40-year experience consisting of 91 cases of acute slipped capital femoral epiphysis (SCFE) was reviewed to assess the safety of manipulative reduction and to determine whether urgent reduction has an effect on the development of avascular necrosis (AVN) of the capital femoral epiphysis. All patients had a history of sudden onset of severe hip pain and were documented to have an unstable (acute) slipped epiphysis. Treatment modalities included manipulative reduction under general anesthesia followed by internal fixation (41 hips), epiphysiodesis and internal fixation (15 hips), epiphysiodesis and cast immobilization (31 hips), and cast immobilization alone (three hips). One case was treated with cast immobilization after reduction by skeletal traction. Patient follow-up averaged 44 months, and ranged from 12 to 216 months. Radiographic review identified 13 (14%) cases of AVN in the series of 91 hips. Of 42 hips reduced in <24 h from presentation, AVN developed in three (7%). Of 49 hips reduced in >24 h from presentation, AVN developed in 10 (20%). Manipulative reduction of the acute SCFE may be accomplished without increased risk of AVN. Time to reduction may be an important risk factor for development of AVN after acute SCFE.     

Alkyl hydroperoxide reductase, catalase, MrgA, and superoxide dismutase are not involved in resistance of Bacillus subtilis spores to heat or oxidizing agents

Only a single superoxide dismutase (SodA) was detected in Bacillus subtilis, and growing cells of a sodA mutant exhibited paraquat sensitivity as well as a growth defect and reduced survival at an elevated temperature. However, the sodA mutation had no effect on the heat or hydrogen peroxide resistance of wild-type spores or spores lacking the two major DNA protective alpha/beta-type small, acid-soluble, spore proteins (termed alpha(-)beta(-) spores). Spores also had only a single catalase (KatX), as the two catalases found in growing cells (KatA and KatB) were absent. While a katA mutation greatly decreased the hydrogen peroxide resistance of growing cells, as found previously, katA, katB, and katX mutations had no effect on the heat or hydrogen peroxide resistance of wild-type or alpha(-)beta(-) spores. Inactivation of the mrgA gene, which codes for a DNA-binding protein that can protect growing cells against hydrogen peroxide, also had no effect on spore hydrogen peroxide resistance. Inactivation of genes coding for alkyl hydroperoxide reductase, which has been shown to decrease growing cell resistance to alkyl hydroperoxides, had no effect on spore resistance to such compounds or on spore resistance to heat and hydrogen peroxide. However, Western blot analysis showed that at least one alkyl hydroperoxide reductase subunit was present in spores. Together these results indicate that proteins that play a role in the resistance of growing cells to oxidizing agents play no role in spore resistance. A likely reason for this lack of a protective role for spore enzymes is the inactivity of enzymes within the dormant spore.     

Induction of surfactin production in Bacillus subtilis by gsp, a gene located upstream of the gramicidin S operon in Bacillus brevis

The deduced amino acid sequence of the gsp gene, located upstream of the 5' end of the gramicidin S operon (grs operon) in Bacillus brevis, showed a high degree of similarity to the sfp gene product, which is located downstream of the srfA operon in B. subtilis. The gsp gene complemented in trans a defect in the sfp gene (sfpO) and promoted production of the lipopeptide antibiotic surfactin. The functional homology of Gsp and Sfp and the sequence similarity of these two proteins to EntD suggest that the three proteins represent a new class of proteins involved in peptide secretion, in support of a hypothesis published previously (T. H. Grossman, M. Tuckman, S. Ellestad, and M. S. Osburne, J. Bacteriol. 175:6203-6211, 1993).