Effect of chemical fixatives on accurate preservation of Escherichia coli and Bacillus subtilis structure in cells prepared by freeze-substitution.

Five chemical fixatives were evaluated for their ability to accurately preserve bacterial ultrastructure during freeze-substitution of select Escherichia coli and Bacillus subtilis strains. Radioisotopes were specifically incorporated into the peptidoglycan, lipopolysaccharide, and nucleic acids of E. coli SFK11 and W7 and into the peptidoglycan and RNA of B. subtilis 168 and W23. The ease of extraction of radiolabels, as assessed by liquid scintillation counting during all stages of processing for freeze-substitution, was used as an indicator of cell structural integrity and retention of cellular chemical composition. Subsequent visual examination by electron microscopy was used to confirm ultrastructural conformation. The fixatives used were: 2% (wt/vol) osmium tetroxide and 2% (wt/vol) uranyl acetate; 2% (vol/vol) glutaraldehyde and 2% (wt/vol) uranyl acetate; 2% (vol/vol) acrolein and 2% (wt/vol) uranyl acetate; 2% (wt/vol) gallic acid; and 2% (wt/vol) uranyl acetate. All fixatives were prepared in a substitution solvent of anhydrous acetone. Extraction of cellular constituents depended on the chemical fixative used. A combination of 2% osmium tetroxide-2% uranyl acetate or 2% gallic acid alone resulted in optimum fixation as ascertained by least extraction of radiolabels. In both gram-positive and gram-negative organisms, high levels of radiolabel were detected in the processing fluids in which 2% acrolein-2% uranyl acetate, 2% glutaraldehyde-2% uranyl acetate, or 2% uranyl acetate alone were used as fixatives. Ultrastructural variations were observed in cells freeze-substituted in the presence of different chemical fixatives. We recommend the use of osmium tetroxide and uranyl acetate in acetone for routine freeze-substitution of eubacteria, while gallic acid is recommended for use when microanalytical processing necessitates the omission of osmium.     

The L17 ribosomal protein of Bacillus subtilis binds preferentially to curved DNA

We searched in Bacillus subtilis for proteins that bind preferentially to curved DNA. Two proteins of 9 and 15 kDa displaying this property were purified from exponentially growing cells of B. subtilis strain 168. Sequencing of N-terminal amino acids identified them as the proteins HBsu and L17 respectively. The overproduction of L17 from B. subtilis in Escherichia coli was shown to have a strong effect on nucleoid morphology and segregation.     

Expression of Escherichia coli Min system in Bacillus subtilis and its effect on cell division

In both rod-shaped Bacillus subtilis and Escherichia coli cells, Min proteins are involved in the regulation of division septa formation. In E. coli , dynamic oscillation of MinCD inhibitory complex and MinE, a topological specificity protein, prevents improper polar septation. However, in B. subtilis no MinE is present and no oscillation of Min proteins can be observed. The function of MinE is substituted by that of an unrelated DivIVA protein, which targets MinCD to division sites and retains them at the cell poles. We inspected cell division when the E. coli Min system was introduced into B. subtilis cells. Expression of these heterologous Min proteins resulted in cell elongation. We demonstrate here that E. coli MinD can partially substitute for the function of its B. subtilis protein counterpart. Moreover, E. coli MinD was observed to have similar helical localization as B. subtilis MinD.     

Oxidative stress and growth temperature in Bacillus subtilis.

Pretreatment of Bacillus subtilis with low concentrations of hydrogen peroxide protected the cells against the lethal effects of higher levels of oxidative stress. During the period of adaptation, eight proteins were induced, as detected by one-dimensional gel electrophoresis. Four of these proteins were the same size as four of the proteins induced by the temperature upshift. The range of proteins synthesized in response to an elevation in temperature depended both on the starting (lower) temperature and on the temperature to which the cells were shifted. Both catalase and superoxide dismutase were present at high levels in B. subtilis, but neither was induced by oxidative stress or temperature upshift. In fact, catalase activity was reduced after the temperature upshift.     

Cloning and expression of the Escherichia coli recA gene in Bacillus subtilis

By means of homopolymer dG-dC tailing, using PstI linearized pBR327 as vector, we constructed small plasmids containing the entire Escherichia coli recA gene. The 1.8-kb inserts were recloned in the Bacillus subtilis expression vector pPL608 in a B. subtilis recE4 strain. Analysis of plasmid-coded proteins showed expression of the E. coli recA gene both in minicells and whole cells of B. subtilis. Expression was under control of the bacteriophage SP02 promoter, which is part of pPL608. A recA-expressing plasmid completely abolished the transformation deficiency of the recE4 mutant as well as its sensitivity to mitomycin C (MC). The expressed recA gene also restored recombination in other B. subtilis strains lacking the recE gene product. These results indicate a high similarity between the functions of the E. coli RecA and B. subtilis RecE proteins.     

Sulphation by cultured cells. Cysteine, cysteinesulphinic acid and sulphite as sources for proteoglycan sulphate.

The tryptophan-auxotrophic Bacillus subtilis LC33 mutant strain utilizes either tryptophan or 4-fluorotryptophan for growth. Proteins therefore could be isolated from these cells in either tryptophan-containing or 4-fluorotryptophan-containing forms. Since 4-fluorotryptophan is non-fluorescent, tryptophan fluorescence would be suppressed in the 4-fluorotryptophan-containing proteins, facilitating the investigation of other chromophores either on the proteins or interacting with the proteins. This approach, potentially applicable to any protein endogenous to or clonable into B. subtilis, was illustrated by an examination of the fluorescence of B. subtilis ribosomal proteins.     

Nucleotide sequence and characterization of a Bacillus subtilis gene encoding a flagellar switch protein.

The nucleotide sequence of the Bacillus subtilis fliM gene has been determined. This gene encodes a 38-kDa protein that is homologous to the FliM flagellar switch proteins of Escherichia coli and Salmonella typhimurium. Expression of this gene in Che+ cells of E. coli and B. subtilis interferes with normal chemotaxis. The nature of the chemotaxis defect is dependent upon the host used. In B. subtilis, overproduction of FliM generates mostly nonmotile cells. Those cells that are motile switch less frequently. Expression of B. subtilis FliM in E. coli also generates nonmotile cells. However, those cells that are motile have a tumble bias. The B. subtilis fliM gene cannot complement an E. coli fliM mutant. A frameshift mutation was constructed in the fliM gene, and the mutation was transferred onto the B. subtilis chromosome. The mutant has a Fla- phenotype. This phenotype is consistent with the hypothesis that the FliM protein encodes a component of the flagellar switch in B. subtilis. Additional characterization of the fliM mutant suggests that the hag and mot loci are not expressed. These loci are regulated by the SigD form of RNA polymerase. We also did not observe any methyl-accepting chemotaxis proteins in an in vivo methylation experiment. The expression of these proteins is also dependent upon SigD. It is possible that a functional basal body-hook complex may be required for the expression of SigD-regulated chemotaxis and motility genes.     

Bacteriophage SPO1-induced macromolecular synthesis in minicells of Bacillus subtilis.

SPO1 bacteriophage injects its DNA into minicells produced by Bacillus subtilis CU403 divIVB1. The injected DNA is partially degraded to small trichloracetic acid-precipitable material and trichloroacetic acid-soluble material. The injected DNA is not replicated; however, it serves as a template for RNA and protein synthesis. The RNA produced specifically hybridizes to SPO1 DNA, and the amount of RNA hybridized can be reduced by competition with RNA isolated at all stages of the phage cycle from infected nucleate cells of the B. subtilis CU403 divIVB1. An unrelated phage, SPP1, also induces phage-specific RNA in infected minicells. Translation occurs in SPO1-infected minicells resulting in at least eight proteins which have been separated by gel electrophoresis, and two of these proteins have mobilities similar to proteins found only in infected B. subtilis CU403 divIVB1 nucleate cells. A large proportion of the polypeptide material synthesized in infected minicells is very small and heterogeneous in size.     

Essential nature of the mreC determinant of Bacillus subtilis

The mre genes of Escherichia coli and Bacillus subtilis are cell shape determination genes. Mutants affected in mre function are spheres instead of the normal rods. Although the mre determinants are not required for viability in E. coli, the mreB determinant is an essential gene in B. subtilis. Conflicting results have been reported as to whether the two membrane-associated proteins MreC and MreD are essential proteins. Furthermore, although the MreB protein has been studied in some detail, the roles of the MreC and MreD proteins in cell shape determination are unknown. We constructed a strain of B. subtilis in which expression of the mreC determinant is dependent upon the addition of isopropyl-beta-D-thiogalactopyranoside to the culture medium. Utilizing this conditional strain, it was shown that mreC is an essential gene in B. subtilis. Furthermore, it was shown that cells lacking sufficient quantities of MreC undergo morphological changes, namely, swelling and twisting of the cells, which is followed by cell lysis. Electron microscopy was utilized to demonstrate that a polymeric material accumulated at one side of the division septum of the cells and that the presence of this material correlated with the bending of the cell. The best explanation for the results is that the MreC protein is involved in the control of septal versus long-axis peptidoglycan synthesis, that cells lacking MreC perform aberrant septal peptidoglycan synthesis, and that lysis results from a deficiency in long-axis peptidoglycan synthesis.     

Heterospecific expression of the Bacillus subtilis cell shape determination genes mreBCD in Escherichia coli

The divIVB operon of Bacillus subtilis includes the cell shape-associated mre genes, including the membrane-associated proteins MreC and MreD. TnphoA mutagenesis was utilized to analyze a topological model for MreC. MreC has a short cytoplasmic amino terminus, a single membrane-spanning domain, and a large carboxy terminal domain which lies externally to the outer leaflet of the cell membrane. Expression of the B. subtilis MreB protein, or the Mre C and D proteins, results in a morphological conversion of the Escherichia coli host cells from a rod to a roughly spherical cell, morphologically similar to mre-negative mutants of E. coli. Immunolocalization of the MreC protein in B. subtilis revealed that this protein is found at the midcell division site of the bacterial cells, consistent with the postulated role of the Mre proteins in the regulation of septum-specific peptidoglycan synthesis.     

Extreme C terminus of bacterial cytoskeletal protein FtsZ plays fundamental role in assembly independent of modulatory proteins

Bacterial cell division typically requires assembly of the cytoskeletal protein FtsZ into a ring (Z-ring) at the nascent division site that serves as a foundation for assembly of the division apparatus. High resolution imaging suggests that the Z-ring consists of short, single-stranded polymers held together by lateral interactions. Several proteins implicated in stabilizing the Z-ring enhance lateral interactions between FtsZ polymers in vitro. Here we report that residues at the C terminus of Bacillus subtilis FtsZ (C-terminal variable region (CTV)) are both necessary and sufficient for stimulating lateral interactions in vitro in the absence of modulatory proteins. Swapping the 6-residue CTV from B. subtilis FtsZ with the 4-residue CTV from Escherichia coli FtsZ completely abolished lateral interactions between chimeric B. subtilis FtsZ polymers. The E. coli FtsZ chimera readily formed higher order structures normally seen only in the presence of molecular crowding agents. CTV-mediated lateral interactions are important for the integrity of the Z-ring because B. subtilis cells expressing the B. subtilis FtsZ chimera had a low frequency of FtsZ ring formation and a high degree of filamentation relative to wild-type cells. Site-directed mutagenesis of the B. subtilis CTV suggests that electrostatic forces are an important determinant of lateral interaction potential.     

Immobilized hemin affinity chromatography as a probe for proteins having potentiality to bind with heme

After Sepharose 4B polymer beads were activated by using epichlorohydrin, hemin was binded with them to prepare an immobilized hemin affinity chromatography column. The coupling rate of this column was very high, more than 0.25mg hemin could be fixed by 1g of wet Sepharose 4B beads. The column equilibrated with deionized water and eluated with pH 3.0 NaAc-HAc buffer was applied to capture the proteins in human serum, earthworm body and Bacillus subtilis cells. Three polypeptides in human serum were captured, one of which was verified as serum albumin after comparison to the control. At least one polypeptide in earthworm body, two in Bacillus subtilis cells displayed the powerful binding specificity to hemin. Our experiments demonstrated that the immobilized hemin affinity chromatography was available as a probe for some proteins having potentiality to bind with heme.     

Characterization of a chimeric proU operon in a subtilin-producing mutant of Bacillus subtilis 168.

The ability to respond to osmotic stress by osmoregulation is common to virtually all living cells. Gram-negative bacteria such as Escherichia coli and Salmonella typhimurium can achieve osmotolerance by import of osmoprotectants such as proline and glycine betaine by an import system encoded in an operon called proU with genes for proteins ProV, ProW, and ProX. In this report, we describe the discovery of a proU-type locus in the gram-positive bacterium Bacillus subtilis. It contains four open reading frames (ProV, ProW, ProX, and ProZ) with homology to the gram-negative ProU proteins, with the B. subtilis ProV, ProW, and ProX proteins having sequence homologies of 35, 29, and 17%, respectively, to the E. coli proteins. The B. subtilis ProZ protein is similar to the ProW protein but is smaller and, accordingly, may fulfill a novel role in osmoprotection. The B. subtilis proU locus was discovered while exploring the chromosomal sequence upstream from the spa operon in B. subtilis LH45, which is a subtilin-producing mutant of B. subtilis 168. B. subtilis LH45 had been previously constructed by transformation of strain 168 with linear DNA from B. subtilis ATCC 6633 (W. Liu and J. N. Hansen, J. Bacteriol. 173:7387-7390, 1991). Hybridization experiments showed that LH45 resulted from recombination in a region of homology in the proV gene, so that the proU locus in LH45 is a chimera between strains 168 and 6633. Despite being a chimera, this proU locus was fully functional in its ability to confer osmotolerance when glycine betaine was available in the medium. Conversely, a mutant (LH45 deltaproU) in which most of the proU locus had been deleted grew poorly at high osmolarity in the presence of glycine betaine. We conclude that the proU-like locus in B. subtilis LH45 is a gram-positive counterpart of the proU locus in gram-negative bacteria and probably evolved prior to the evolutionary split of prokaryotes into gram-positive and gram-negative forms.     

Interaction of the Bacillus subtilis DnaA-like protein with the Escherichia coli DnaA protein.

Plasmids carrying the intact Bacillus subtilis dnaA-like gene and two reciprocal hybrids between the B. subtilis and Escherichia coli dnaA genes were constructed. None of the plasmids could transform wild-type E. coli cells unless the cells contained surplus E. coli DnaA protein (DnaAEc). A dnaA (Ts) strain integratively suppressed by the plasmid R1 origin could be transformed by plasmids carrying either the B. subtilis gene (dnaABs) or a hybrid gene containing the amino terminus of the E. coli gene and the carboxyl terminus of the B. subtilis gene (dnaAEc/Bs). In cells with surplus E. coli DnaA protein, expression of the E. coli dnaA gene was derepressed by the B. subtilis DnaA protein and by the hybrid DnaAEc/Bs protein, whereas it was strongly repressed by the reciprocal hybrid protein DnaABs/Ec. The plasmids carrying the different dnaA genes probably all interfere with initiation of chromosome replication in E. coli by decreasing the E. coli DnaA protein concentration to a limiting level. The DnaABs and the DnaAEc/Bs proteins effect this decrease possibly by forming inactive oligomeric proteins, while the DnaABs/Ec protein may decrease dnaAEc gene expression.     

Bacillus subtilis strain deficient for the protein-tyrosine kinase PtkA exhibits impaired DNA replication

Bacillus subtilis has recently come into the focus of research on bacterial protein-tyrosine phosphorylation, with several proteins kinases, phosphatases and their substrates identified in this Gram-positive model organism. B. subtilis protein-tyrosine phosphorylation system PtkA/PtpZ was previously shown to regulate the phosphorylation state of UDP-glucose dehydrogenases and single-stranded DNA-binding proteins. This promiscuity towards substrates is reminiscent of eukaryal kinases and has prompted us to investigate possible physiological effects of ptkA and ptpZ gene inactivations in this study. We were unable to identify any striking phenotypes related to control of UDP-glucose dehydrogenases, natural competence and DNA lesion repair; however, a very strong phenotype of DeltaptkA emerged with respect to DNA replication and cell cycle control, as revealed by flow cytometry and fluorescent microscopy. B. subtilis cells lacking the kinase PtkA accumulated extra chromosome equivalents, exhibited aberrant initiation mass for DNA replication and an unusually long D period.     

Transglutaminase in sporulating cells of Bacillus subtilis

We screened various Bacillus species producing transglutaminase (TGase), measured as labeled putrescine incorporated into N,N-dimethylcasein. As a result, we detected TGase activity in sporulating cells of B. subtilis, B. cereus, B. alvei and B. aneurinolyticus, and found TGase activity related to sporulation. TGase activity of Bacillus subtilis was detected in lysozyme-treated sporulating cells during late sporulation, but not in cells without lysozyme treatment or the supernatant of the culture broth. TGase was found to be localized on spores. TGase was preliminarily purified by gel filtration chromatography for characterization. Its activity was eluted in the fractions indicating a molecular weight of approximately 23 kDa. TGase could cross-link and polymerize a certain protein. The enzyme was strongly suggested to form epsilon-(gamma-glutamyl)lysine bonds, which were detected in the spore coat proteins of B. subtilis. The activity was Ca(2+)-independent like the TGases derived from Streptoverticillium or some plants. It is suggested that TGase is expressed during sporulation and plays a role in the assembly of the spore coat proteins of the genus Bacillus.     

Expression of functional Bacillus SpoIISAB toxin-antitoxin modules in Escherichia coli.

SpoIISA and SpoIISB proteins from Bacillus subtilis belong to a recently described bacterial programmed-cell death system. The current work demonstrates that the toxin-antitoxin module is also functional in Escherichia coli cells, where the expression of SpoIISA toxin leads to transient growth arrest coupled with cell lysis, and SpoIISA-induced death can be prevented by coexpression of its cognate antitoxin, SpoIISB. Escherichia coli cells appear to be able to escape the SpoIISA killing by activation of a specific, as yet unidentified protease that cleaves out the cytosolic part of the protein. Analysis of the toxic effects of the transmembrane and cytosolic portions of SpoIISA showed that neither of them separately can function as a toxin; therefore, both parts of the protein have to act in concert to exert the killing. This work also identifies genes encoding putative homologues of SpoIISA and SpoIISB proteins on chromosomes of other Bacilli species. The SpoIISA-like proteins from Bacillus anthracis and Bacillus cereus were shown to manifest the same effect on the viability of E. coli as their homologue from B. subtilis. Moreover, expression of the proposed spoIISB-like gene rescues E. coli cells from death induced by the SpoIISA homologue.     

Expression of functional Bacillus SpoIISAB toxin–antitoxin modules in Escherichia coli

SpoIISA and SpoIISB proteins from Bacillus subtilis belong to a recently described bacterial programmed-cell death system. The current work demonstrates that the toxin–antitoxin module is also functional in Escherichia coli cells, where the expression of SpoIISA toxin leads to transient growth arrest coupled with cell lysis, and SpoIISA-induced death can be prevented by coexpression of its cognate antitoxin, SpoIISB. Escherichia coli cells appear to be able to escape the SpoIISA killing by activation of a specific, as yet unidentified protease that cleaves out the cytosolic part of the protein. Analysis of the toxic effects of the transmembrane and cytosolic portions of SpoIISA showed that neither of them separately can function as a toxin; therefore, both parts of the protein have to act in concert to exert the killing. This work also identifies genes encoding putative homologues of SpoIISA and SpoIISB proteins on chromosomes of other Bacilli species. The SpoIISA-like proteins from Bacillus anthracis and Bacillus cereus were shown to manifest the same effect on the viability of E. coli as their homologue from B. subtilis . Moreover, expression of the proposed spoIISB -like gene rescues E. coli cells from death induced by the SpoIISA homologue.