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Surfactin is a lipopeptide produced by certain strains of Bacillus subtilis and has potent surface activity. Here, we present the first results showing that ion-conducting pores can be formed by surfactin in artificial lipid membranes. With a low aqueous concentration of surfactin (1 microM) and a restricted membrane area (5.10(-5) cm2) we observed conductance jumps that indicate the formation of individual ionic channels in the presence of K+, Rb+, Cs+, Na+ or Li+ chlorides. Although for every salt concentration (Ci), the distribution in amplitude of the conductance steps (lambda i) may be rather broad, there is always a step amplitude which is more frequent than the others. In addition, the channels corresponding to this most frequent step amplitude are the longest in duration. For Ci = 1 M, the cationic selectivity sequence deduced from these most frequent events is K+ greater than Rb+ greater than Na+ greater than Cs+ = Li+ with respective values for lambda Mi: 130, 110, 80 and 30 pS. In KCl solutions lambda MKCl increases as a function of Ci for low Ci, and shows a plateau for Ci greater than 0.5 M. When measured on larger area membranes (10(-2)cm2) with 1 M solutions of the monovalent salts KCl, NaCl, RbCl and CsCl or the divalent salt CaCl2, the macroscopic low voltage conductance (G0) increases with a slope of 2 on a log-log plot as a function of surfactin concentration. These results demonstrate that surfactin produces selective cationic channels in lipid bilayer membranes and suggest that at higher salt concentration, a dimer is involved in this functional channel-forming process.

Gas-liquid chromatography of cellular fatty acids is a useful tool for the identification of bacteria. Derivatization of bacterial fatty acids to methyl esters by conventional techniques is usually time-consuming and complicated. A new one-step technique using trimethyl-sulfonium hydroxide allows the direct formation of fatty acid methyl esters within 1-2 min. Some random examples of profiles demonstrate that straight, branched, saturated, unsaturated, hydroxy and cyclopropyl fatty acids match conventional preparations well. The method is a very sensitive one, since only a few colonies are sufficient for preparation of fatty acid methyl esters.

The primary and tertiary structures of DNA-binding protein HU from Bacillus stearothermophilus are already known. The primary structure has been previously determined for HU from the closely related B. globigii and the determinations of the sequences from B. caldolyticus and B. subtilis are described here. These bacteria have optimum growth temperatures of greater than 70 degrees C (B. caldolyticus), 65 degrees C (B. stearothermophilus), 37 degrees C (B. subtilis) and 30 degrees C (B. globigii). In vitro measurements from circular dichroic spectra described here give Tm values reflecting these growth temperatures, of 68, 64, 43 and 41 degrees C respectively. We discuss here the relative thermostability of the four proteins in terms of the amino acid differences between the sequences and the three-dimensional model of the B. stearothermophilus HU. The current model for the interaction of the protein with DNA is only discussed in terms of its relevance with regard to thermostability.

By use of a T7 expression system, large amounts of active Bacillus subtilis RNA polymerase sigma A factor were produced in Escherichia coli cells. This overproduced protein was found in the form of inclusion bodies and constituted 40% of the total cellular protein. Because of the ease of isolation of the inclusion bodies and the acidic properties of sigma A, the protein was purified to more than 99% purity and the yield was about 90 mg/liter of culture. Gel mobility, antigenicity, specificity of promoter recognition, and N-terminal amino acid sequence of the overproduced sigma were found to be the same as those of native sigma A. Partial proteolysis analysis of sigma A protein suggested the presence of a protease-sensitive surface region in the C-terminal part of the sigma A protein. The promoter -10 binding region of sigma A was less sensitive to proteases and was probably involved in a hydrophobic, tightly folded domain of sigma A protein.

High-performance liquid chromatography (HPLC) on newly developed spherical beads of hydroxyapatite was applied to the analysis of purified E. coli tRNAs (Val, Met, Tyr and Phe). tRNAs were eluted from the column separately with appreciable differences in retention time by a 45-min gradient of phosphate buffer (pH 6.8) of concentration from 64 to 123 mM; both the retention times and peak areas of respective tRNAs were highly reproducible. Total tRNA (tRNA(Total)) preparations obtained from E. coli and B. subtilis were also analysed on the column. It is possible even to elute tRNA(Total) which may, in general, contain 60 or more tRNA species with a relatively shallow gradient such as 75-132 mM. The recovery of tRNA from the column was as high as 90%. Owing to the complicated composition, the elution profile of tRNA(Total) had a wide spread irregular shape but, with a 1-h gradient more than ten peaks were easily detected. When the amino acid-accepting activity of tRNA in the eluate of tRNA(Total) was determined, for ten specific tRNAs, each activity peak was eluted sharply from the column. In addition, several tRNA activities were eluted in different fractions. This indicates that isoacceptors were separated by the column. The results show that HPLC on hydroxyapatite beads is useful for the purification and characterization of tRNA.

A method for enhancing the precision in the calculation of isotope enrichment for 15N-labeled amino acids is presented. The method utilizes multiple line pairs for the calculation of isotope enrichment. Using multiple line pairs allows the evaluation of calibration curves for nonlinear behavior and permits differentiation among sites containing more than one labeled nitrogen. The increase in precision is related to the number of isotopically shifted line pairs used in calculating the isotopic enrichment and varies with the amino acid of interest.

Genetic and biochemical studies have shown that the product of the Escherichia coli secY gene is an integral membrane protein with a central role in protein secretion. We found the Bacillus subtilis secY homologue within the spc-alpha ribosomal protein operon at the same position occupied by E. coli secY. B. subtilis secY coded for a hypothetical product 41% identical to E. coli SecY, a protein thought to contain 10 membrane-spanning segments and 11 hydrophilic regions, six of which are exposed to the cytoplasm and five to the periplasm. We predicted similar segments in B. subtilis SecY, and the primary sequences of the second and third cytoplasmic regions and the first, second, fourth, fifth, seventh, and tenth membrane segments were particularly conserved, sharing greater than 50% identity with E. coli SecY. We propose that the conserved cytoplasmic regions interact with similar cytoplasmic secretion factors in both organisms and that the conserved membrane-spanning segments actively participate in protein export. Our results suggest that despite the evolutionary differences reflected in cell wall architecture, Gram-negative and Gram-positive bacteria possess a similar protein export apparatus.

SPR, a temperate Bacillus subtilis phage, codes for a DNA methyltransferase that can methylate the sequences GGCC (or GGCC) and CCGG at the cytosines indicated. We show here that it can also methylate the sequence CC(A/T)GG and protect it from cleavage with EcoRII and ApyI. This methylation can be seen in vivo as well as in vitro with purified SPR methyltransferase. SPR19 and SPR83 are two mutant phages, defective in GGCC or CCGG methylation, respectively. These mutants have not lost their ability to methylate CC(A/T)GG sites. Mutation SPR26 has lost the ability to methylate all three sites. Thus the SPR methyltransferase codes for three genetically distinguishable methylation abilities.

Some bacterial penicillin-binding proteins (PBPs) are reported to exist as multiple isoelectric forms. Other PBPs were examined by two-dimensional electrophoresis to establish whether multiple isoelectric forms are widespread amongst PBPs. Bacillus subtilis PBPs 3 and 5 and Escherichia coli PBPs 1b alpha and 1b gamma focussed as discrete spots with no evidence of multiple isoforms. However, E. coli PBPs 1b beta and 4 displayed isoelectric variants. The results are discussed in relation to current models of bacterial peptidoglycan structure.

DnaA protein interacts with cAMP with a KD of 1 microM. This interaction stimulates DnaA protein binding to the chromosome replication origin (oriC) and the mioC promoter region, protects DnaA protein from thermal inactivation, releases ADP but not ATP bound to DnaA protein, and restores normal DNA replication activity and ATPase activity in inactive ADP-DnaA protein preparations. A model is proposed in which cellular cAMP levels govern the replication activity of DnaA protein by promoting the recycling of the inactive ADP-DnaA protein form into the active ATP form.

After heat-induction of the defective phage PBSX in a xhi-1479 mutant of Bacillus subtilis 168, the culture lysed rapidly even if the lyt-2 mutation was present (which greatly reduces the amount of the bacterial autolysins). Two lytic enzymes, an N-acetylmuramoyl-L-alanine amidase and an endo-N-acetylmuramidase, were purified from the culture supernatant. The amidase was readily distinguished from the bacterial amidase by its low molecular weight. In addition, it was not inhibited by antibody directed against the bacterial enzyme. These results indicate that PBSX does not rely on the bacterial autolysins to accomplish lysis.

Six thionucleosides found in Bacillus subtilis transfer ribonucleic acids were investigated: N6-(delta 2-isopentenyl)-2-methylthioadenosine, 5-carboxymethylaminomethyl-2-thiouridine, 4-thiouridine, 2-methylthioadenosine, N-[(9-beta-D-ribofuranosyl-2-methylthiopurin-6-yl)carbamoyl]threonine, and one unknown (X1). The presence of N-[(9-beta-D-ribofuranosyl-2-methylthiopurin-6-yl)carbamoyl]threonine was demonstrated based on the affinity of the transfer ribonucleic acid containing it for an immunoadsorbent made with the antibody directed toward N-[9-(beta-D-ribofuranosyl)purin-6-ylcarbamoyl]-L-threonine. The existance of N-[(9-beta-D-ribofuranosyl-2-methylthiopurin-6-yl)carbamoyl]threonine in two species of lysine transfer ribonucleic acids was also confirmed by high-resolution mass spectrometry. Four of these thionucleosides--N6-(delta 2-isopenenyl)-2-methylthioadenosine, 2-methylthioadenosine, 5-carboxymethylaminomethyl-2-thiouridine, and the unknown designated X1--occurred only in specific areas in the elution profile of an RPC-5 column and probably affect the chromatographic properties of the transfer ribonucleic acids containing them. In contrast with Escherichia coli, where 4-thiouridine is the most frequent type of sulfur-containing modification, approximately one-third of the sulfur groups in B. subtilis transfer ribonucleic acid are present as thiomethyl groups on the 2 position of an adenosine or modified adenosine residue.

Bacillus subtilis Ni15 is deficient in cell wall turnover. The deficiency is removed if the medium contains 0.2 M NaCl, which does not affect growth. The levels of amidase and glucosaminidase, the most likely enzymes involved in turnover, were, in stationary phase Ni15 cells, similar to those in late-exponential phase cells of a standard strain. The Ni15 enzymes were not salt sensitive. However, the Ni15 walls contained 4.7-fold less phosphorus than the walls of the standard strain. Since the phosphorus content of B. subtilis walls reflects the level of teichoic acid, it is proposed that the turnover deficiency of this strain is due to a decrease in wall teichoic acid.

A flow cytometer was used to measure the fluorescence intensity of Bacillus anthracis spores, B. subtilis spores and Escherichia coli stained in suspension with specific rabbit fluorescein-conjugated antibody. The effect of normal sera and a number of other additives on the binding of conjugate to the surface of the homologous bacteria was assessed by measuring the median fluorescence intensity of the bacterial population in the reaction mixture. Non-ionic detergent depressed binding of one conjugate (anti-E. coli) by up to 22%. Bovine serum albumin, gelatin, foetal calf serum and normal rabbit serum did not affect the median fluorescence value for these 3 bacterial species by more than 14%. Normal serum from 5 goats reduced the specific staining of B. anthracis by up to two-thirds. Anti-B. anthracis antibodies were detected in goat serum by indirect immunofluorescence microscopy, and it is inferred that these goat antibodies were in competition with fluorescein conjugate for the bacterial antigens. Normal goat and sheep serum stimulated the specific staining of B. subtilis and E. coli measured by the cytometer; in the case of goat serum previous heating of the serum to 56 degrees C resulted in repression of staining of E. coli. Since anti-E. coli antibody was detected in this normal sera by indirect immunofluorescence assays, it is proposed that repression was caused by anti-bacterial antibodies and stimulation by a separate factor, heat-labile in the case of goat serum. The stimulatory factor was also apparently inactivated by increasing the NaCl concentration, suggesting that stimulation depends heavily on charge interactions. Preliminary evidence is presented that the stimulatory factor may be anti-antibody, possibly of the IgA or IgG class.

Bacillus subtilis strains 168 met ile leu and 23 thy contain folates which differ from one another in the number of glutamyl residues. The folate species were identified by reductive cleavage to the corresponding p-aminobenzoylglutamyl poly-gamma-glutamates and chromatography on diethylaminoethyl-cellulose. Pteroyltriglutamate is the predominant folate type, accounting for 86 to 88% of the total. Pteroyltetraglutamate is the only other type present in appreciable quantities, accounting for 5 to 6% of the total folates. Pteroyldiglutamate and pteroylpentaglutamate are present in small amounts, accounting for 1 to 3% and 1% of the total folates, respectively. Strain 168 met ile leu contains a very small amount of pteroylmonoglutamate (less than 0.5% of the total folates), but the other strain contains none.

The DNA-membrane complexes were isolated from the membranes of Bacillus subtilis, disrupted by ultrasonication and subjected to chromatography on Sepharose 4B. The membrane-bound fraction of native 3H-DNA was found only in the freshly isolated material. The phospholipid and protein composition of this fraction was determined. It was assumed that the newly synthesized membrane-associated 3H-DNA from Bacillus subtilis is bound to the lipoprotein complexes enriched with phospholipids.

The ribosomal subunit proteins(30S and 50 S) from vegetative and sporulating cells of Bacillus subtilis 168M were analyzed by two dimensional acrylamide gel electrophoresis. Twenty two proteins were identified in the 30S subunits and 28 proteins are detectable in the 50S subunits. The number of proteins and their electrophoretic mobility seem to remain unaltered during the sporualtion process. The ribosomal proteins of a thermosensitive sporulation mutant (ts-4), isolated from stationary phase cultures, under permissive (for sporulation) and non-permissive conditions, did not show any qualitative difference in either of the subunits. The 21S precursor particles derived from log phase cell ribosomes show two different proteins, in addition to those present in the 30 S subunit. It is suggested that these two proteins either disappear or are modified during the maturation process.

Several physicochemical properties of the teichoic acid of Bacillus subtilis 168 have been determined. The teichoic acid partial specific volume was found to be 0.57 ml/g. The apparent weight-average molecular weight of the polymer was 24,800. Sedimentation was strongly dependent on solvent. The sedimentation coefficient of the teichoic acid was found to have a value of s(20.w) (0) = 1.90S. In dilute buffers and distilled water, the teichoic acid possessed a rigid rod or extended conformation. Salts induced a loss of secondary structure in the polymer, resulting in a random coil configuration. Salt-induced structural changes in the teichoic acid were determined by viscosities, ultraviolet difference spectra, and inhibition of precipitation with concanavalin A. Divalent cations such as Mg(2+) had little effect on the teichoic acid structure. The salt-induced structural changes were reversible, as evidenced by return of the original properties upon dialysis of the teichoic acid against water. Sodium chloride inhibited the adsorption of bacteriophage ø25 to B. subtilis cell walls. Teichoic acid conformation may have a significant influence on the physiology of bacteria.

We have developed a rapid method to quantitate specific bacterial RNA species. The method measures the steady-state level of RNA, produces a linear response over more than a 16-fold range of RNA concentration, and can be used for Staphylococcus aureus, Escherichia coli and Bacillus subtilis. In this method, a sheared whole-cell lysate of approx. 7 x 10(8) organisms, prepared as for plasmid screening, is separated on agarose, blotted to a nitrocellulose filter, hybridized with a radiolabeled DNA probe, and autoradiographed. The RNA species are quantitated by counting the radioactive bands on the filter. We have applied the method to the measurement of mRNA induction of the genes encoding beta-lactamase, ermC rRNA methylase, and the alpha-complementing fragment of beta-galactosidase. Upon induction, a ten-fold increase in the mRNA for each gene was observed. The peak mRNA level occurred after 30 min for beta-lactamase, 20 min for beta-galactosidase, and 5 min for the ermC rRNA methylase.

The sequences of the anticodon of tRNAAla, tRNAThr, and tRNAVal from Bacillus subtilis W 168 were N-G-C, N-G-U, and N-A-C, respectively. A new minor constituent, N, occupied the first position of the anticodon of each tRNA. N was indentified as 5-methoxyuridine (mo5U, Figure 1) by comparison of its UV absorption spectra, Rf values in thin-layer chromatography using several solvent systems and mass spectra with those of chemically synthesized specimen.

Competent cells of Bacillus subtilis are able to bind but not to take up DNA in magnesium-free medium. Wall-membrane complexes, but not cell walls, bind homologous DNA. Binding of DNA to membrane vesicles suggests that DNA-receptor sites are located at the membrane level. On the basis of competition experiments with homologous and heterologous DNAs, in cells and in membrane vesicles, different types of DNA receptors are detected.

Purified flagellins derived from 16 strains of Bacillus subtilis were classified into at least five distinct groups on the basis of their reaction with antiflagellar filament antibody and antiflagellin antibody. This classification was in good accord with that derived independently on the basis of amino acid analyses of the flagellins. Flagellar antigenicity appears to provide a useful typological character in classifying B. subtilis strains.

Phosphorylserine was isolated from the spores of Bacillus subtilis NRRL B 558. This phosphorylated amino acid was only found in the spore coat protein and was not detectable in vegetative cells. The compound was characteristically synthesized at a late stage of sporulation and its turnover did not occur until the completion of mature spore formation. The protein which contains phosphorylserine was not solubilized from the spore coat by digestion with pronase or treatment with any detergents.

A 75,000-dalton protein complex involved in DNA binding during transformation was purified from membranes of competent Bacillus subtilis cells. Previous results (Smith et al., J. Bacteriol. 156:101-108, 1983) showed that the complex contained two polypeptides, polypeptide a (molecular weight, 18,000; isoelectric point, 5.0) and polypeptide b (molecular weight, 17,000; isoelectric point, 4.7) in approximately equal amounts. In the present experiments the two polypeptides were extracted from two-dimensional gels and studied separately and in combination with respect to DNA binding and nuclease activities. For DNA binding the interaction of both polypeptides was required. DNA binding occurred efficiently in the presence of EDTA. Nuclease activity was restricted to polypeptide b. The nucleolytic properties of b were identical to those of the native 75,000-dalton complex. Polypeptide a affected b by reducing its nuclease activity. Analysis of the nuclease subunit b on DNA-containing polyacrylamide gels revealed nuclease activities at four different molecular weight positions. These activities were identical to the major competence-specific nuclease activities which were previously implicated in the entry of donor DNA during transformation (Mulder and Venema, J. Bacteriol. 152:166-174, 1982). These results indicate that the 75,000-dalton protein complex is composed of two different competence-specific polypeptides involved in both binding and entry of donor DNA. The possible roles of the two polypeptides in the transformation of B. subtilis are discussed.

In order to circumvent problems associated with direct chemical analysis of the phase-1 flagellar filament protein (flagellin) of Salmonella typhimurium, the covalent structure was determined by recombinant DNA procedures. The corresponding structural gene (H-1i) was cloned into plasmid pBR322 in a 4.3-kilobase fragment produced by EcoRI digestion of chromosomal DNA, and the nucleotide sequence of the region specifying the flagellar protein was determined. Comparison of the data obtained with the limited information available for other salmonellar flagellins supported the concept that both ends of the molecule are conserved in this genus. Additionally, a conservation of base sequence in the region of H-1 genes coding for the N-terminal end of flagellins was apparent, suggesting that this area may have an additional regulatory role. The i flagellin was found to be unrelated to proteins in the NBRF data base with the exception of other flagellins. The three flagellins which have been sequenced to date (those produced by Bacillus subtilis, Caulobacter crescentis, and phase-1 S. typhimurium) show homologies in amino acid sequence at both the N-terminal and C-terminal ends despite large differences in their total molecular weight, and comparison suggests that B. subtilis and Salmonella are more closely related to each other than either is to Caulobacter.

The distribution of mannosamine, mannosaminuronic acid, and the enzymes responsible for the formation of these saccharides was studied in nine species (18 strains) of Bacillus. Whereas UDP-N-acetylglucosamine 2-epimerase activity was detected in all of the strains examined, UDP-N-acetylmannosamine dehydrogenase, as well as the activity incorporating N-acetylmannosaminuronic acid residues from UDP-N-acetylmannosaminuronic acid into polymer, was found only in four strains of B. megaterium and one strain each of B. subtilis and B. polymyxa. The cell walls prepared from the six above-named strains were shown to contain mannosaminuronic acid in amounts of 135 to 245 nmol/mg. In contrast, mannosamine had a wide distribution. The cell walls from two strains of B. cereus and one strain each of B. circulans, B. polymyxa, B. sphaericus, and B. cereus subsp. mycoides contained mannosamine in amounts of 370 to 470 nmol/mg. In addition, the cell walls from five strains of B. subtilis, two strains of B. megaterium, and one strain each of B. cereus. B. coagulans, and B. licheniformis also contained this amino sugar in amounts as small as 10 to 35 nmol/mg. On the basis of analytical data, it is suggested that the mannosamine present in small amounts may be a common constituent of linkage units between peptidoglycan and other cell wall components such as glycerol teichoic acid.

Inhibition of d-alanine carboxypeptidase in Bacillus subtilis by 95% caused no measurable change in the degree of cross-linking of the peptidoglycan.

The available comparative data on procaryotic 5S rRNA was extended through sequencing studies of eight gram positive procaryotes. Complete nucleotide sequences were presented for 5S rRNA from Bacillus subtilis, B. firmus, B. pasteurii, B. brevis, Lactobacillus brevis and Streptococcus faecalis. In addition, 5S rRNA oligonucleotide catalogs and partial sequence data were provided for B. cereus and Sporosarcina ureae. These sequences and catalogs were discussed in terms of known features of procaryotic 5S rRNA architecture.

Hot and cold, 80% aqueous phenol extraction procedures together with an aqueous extraction technique have been evaluated for the isolation of lipoteichoic acids from the cytoplasmic membrane of Gram-positive bacteria. Lipoteichoic acids of Staphlococcus aureus H, Micrococcus 2102, Baccillus subtilis 168, and Bacillus subtilis W-23 were examined as each of them emphasises a different problem of contamination. The purity of the lipoteichoic acids with respect to cell-wall material, nucleic acid, and protein is discussed together with the criteria of purity which enables critical structural analysis of lipoteichoic acids to be carried out.

Intrastrand self-complementary sequences have been isolated from the DNA of Bacillus subtilis by hydroxyapatite (HA) chromatography following thermal renaturation of strands separated by chromatography on methylated albumin kieselguhr (MAK). The instrastrand structures derived from the MAK H strand (HA HII) were biologically active showing transforming activity for a wide variety of markers, as well as hybridization to both pulse-labelled and ribosomal RNA. Removal of regions of single-strand DNA with S1 nuclease did not significantly alter the biological activity of the self-annealed molecules. The overall efficiency of transformation and hybridization of the intrastrand self-annealing DNA was low suggesting that many sequences in the population are neither active in transformation to prototrophy nor transcribed into RNA.

Experiments are described in which the tensile strength, the extensibility and the initial Young's modulus of bacterial cell wall have been determined as functions of relative humidity in the range 11-98%. Data on stress relaxation and recovery are also given. Standard fibre-measuring technique has been used on 'bacterial thread', made from a cell-separation-suppressed mutant of Bacillus subtilis. The data show that peptidoglycan, the load bearing polymer in the cell wall, behaves very much like other viscoelastic polymers. Its mechanical behaviour when dry is that of a glassy polymer with tensile strength about 300 MPa and modulus about 20 GPa. When wet, it is weaker and much less stiff with tensile strength about 3 M Pa and modulus 10 M Pa. The relaxation data indicate a wide spectrum of relaxation times. The results are discussed in terms of the structure of peptidoglycan and its orientation in the bacterial cell wall. The way in which mechanical behaviour depends strongly on humidity is compared with that of other biopolymers in terms of possible hydrogen-bond density and the ordering of water molecules. The possibility of a well-defined glass transition is briefly examined.

The protoplasts of a mutant of Bacillus subtilis 168 (B. subtilis CMK33) are osmotically fragile when compared to protoplasts of the parent organism and contain an active, membrane-associated phospholipase A1. A protein found in the parent organism specifically inhibits the phospholipase A1 (Kent, C., and Lennarz, W.J. (1972) Proc. Nat. Acad. Sci. U.S.A. 69, 2793-2797). The inhibitor exists in both a soluble and particulate form. The soluble inhibitor is not found in the cytoplasm, but rather in a "periplasmic" fraction released from the cell during incubation with lysozyme. The soluble inhibitor has been purified to homogeneity by diethylaminoethyl-cellulose and hydroxylapatite chromatography. Its molecular weight is 28,000 to 32,000 as determined by gel filtration chromatography and 36,000 to 37,000 as determined by sodium dodecyl sulfate-urea gel electrophoresis. The inhibitor appears to inactivate the membrane bound phospholipase A1 by an enzymatic process that is dependent on time and protein concentration. Binding of the inhbitor to the membrane-associated phospholipase cannot be detected. When purified inhibitor is added to cells of B. subtilis CMK33 during treatment with lysozyme, the osmotic stability of the resultant protoplasts is similar to that of protoplasts of the wild type of organism.

About 13% of the deoxyribonucleic acid (DNA) of various strains of Bacillus subtilis, independent of the stage of growth or competence for transformation, was rendered acid soluble by endonuclease S1. In a pH 11.2 CsCl gradient, 4% of the untreated DNA banded at the density typical for single-stranded molecules, whereas 9% of the remaining DNA (main band) was sensitive to endonuclease S1. Selective inhibition of DNA polymerase III, or of DNA-dependent ribonucleic acid polymerase, did not increase or abolish single-strandedness. The DNA purification procedure did affect the level of single-stranded DNA, indicating its binding to cell constituents containing ribonucleic acid, protein, and membranous material. The molecular weight of the single-stranded fraction resembled that of total denatured DNA, and its buoyant density in an alkaline CsCl gradient was centered partially at a density of 1.772 g/cm3 and partially at a density of 7.759 g/cm3. Incubation of DNA under conditions leading to renaturation of its single-stranded fraction led to an increase in transforming activity for the purA16+ marker (close to the origin of replication) relative to leu-8+ and metC3+ markers (located in the middle of the chromosome), indicating this region is the main source of the single-stranded fraction.

Minicells produced by Bacillus subtilis strains carrying the div IV-B1 mutation, (CU 403 div IV-B1 and CU 403 div IV-B1, tag-1), were purified by a procedure which destroys parental cells with ultrasound, but spares minicells. Such preparations generally contain 10(9) or more minicells/ml and less than 10(4) colony-forming units/ml. Purified minicells are resistant to autolysis in tris(hydroxymethyl)aminomethane buffer, pH 7.5, at 30 C, conditions which result in total lysis of parental cells. Minicells are not completely devoid of autolytic activity, however. The medium in which minicells are produced, the temperature at which purified minicells are incubated, and the genotype of cells from which the minicells are derived all influence the rate of autolysis of purified minicells. These parameters are demonstrated by using minicells obtained from div IV-B1 and div IV-B1, tag-1 strains. Ultrastructural differences have been observed in the products of autolysis of these two minicell strains. Minicells are sensitive to low levels of lysozyme and yield miniprotoplasts when the wall is removed in an osmotically protective environment. Although minicells are unable to grow, they can maintain their integrity over long periods of time, which suggests functional energy metabolism in minicells. Direct measurements of adenosine 5'-triphosphate (ATP) levels by the luciferase assay indicated that minicells can produce ATP. Oxygen consumption, measured by standard respirometry techniques, also indicates functional metabolism in minicells. These findings demonstrate that minicells purified by ultrasound are suitable material for study of physiological processes in anucleate cells.

A recombinant cosmid clone was isolated from a library created from cosmid pQB79-1 and Bacillus subtilis DNA, and a 15 kb BamHI fragment derived from the cloned insert was transferred to the vector pHV33. The recombinant clone, pRC12, was capable of complementing eight auxotrophic markers in the spoIIA-tyrA region of the B. subtilis chromosome (map positions 205-210). It also complemented eight of nine markers in the spoIIA locus. The exception, spoIIA176, is the most distal marker from lysine. Although pRC12 failed to complement sporulation defects in spoVA or spoIVA (spoIIA+) strains, subclones of pRC12, lacking a functional spoIIA gene, did complement these mutations. pRC12 inhibited sporulation in a spo+ recE strain, possibly due to the presence of multiple functional spoIIA genes. Both the original cosmid and pRC12 were unstable in Escherichia coli and B. subtilis. Antibiotic selection of the vector resulted in extensive deletion of the insert, while selection for insert function in B. subtilis invariably led to loss of the chloramphenicol resistance vector function.

Three tRNAs specific for methionine, phenylalanine and tyrosine were isolated from the total tRNA of Bacillus subtilis by chromatographic procedures using BD-cellulose and reversed-phase (5) chromatography. The acceptor activities of the purified tRNAs are 1160, 1260 and 1320 pmoles per A260nm unit for tRNAMetf, tRNAPhe and tRNATyr2 respectively. In tRNAMetf and tRNAPhe ribothymidine, pseudouridine and dihydrouridine are present, in addition, in tRNAPhe 7-methyguanosine and a 2'-O-methylated nucleoside were found. The modified nucleosides of tRNATyr2 are ribothymidine, pseudouridine, dihydrouridine, 4-thiouridine and 1-methyladenosine. The results suggest the presence of 2-methylthio-N6(delta 2-isopentenyl)adenosine in tRNAPhe and tRNATyr2. The thermal denaturation profiles of the three tRAN species are presented.

Although it is generally admitted that Gram-positive bacteria cannot induce interferon, we were able to show that organisms and cell walls of Bacillis subtilis as well as components obtained through the extraction of endotoxins from Gram-negative bacteria can induce type II interferon. Moreover we have ascertained that those components have the same biological activity as the endotoxins, espcially with regard to their behaviour with B cells of lymphocytes, the results of pyrogen test, the incidence of cycloheximide and their function as inhibitors of bacteriophages. However, it has appeared that chemically they are quite different from the lipopolysaccharide of the Gram-negative bacteria: no lipid A could be detected.