The MerR metalloregulatory protein binds mercuric ion as a tricoordinate, metal-bridged dimer.

Bacterial MerR proteins are dimeric DNA-binding proteins that mediate the Hg(II)-dependent induction of mercury resistance operons. Site-directed mutagenesis of the Bacillus sp. RC607 MerR protein reveals that three of four Cys residues per monomer are required for Hg(II) binding at the single high-affinity binding site. Inactive mutant homodimers can exchange subunits to form heterodimers active for Hg(II) binding. Studies of a heterodimer retaining only three of eight cysteine residues per dimer reveal that Cys79 in one subunit and Cys114 and Cys123 in the second subunit are necessary and sufficient for high-affinity Hg(II) binding in an asymmetric, subunit bridging coordination complex.

A 1,2,3,4-tetrahydroxy pentane-substituted pentacyclic triterpene from Bacillus acidocaldarius.

A long-chained polyalcohol (polyol) was isolated from the glycolipid fraction of the extreme thermoacidophile Bacillus acidocaldarius. The polyol and its Smith degradation products, as well as the alkanes derived from these compounds were characterized by mobility on thin-layer and gas-liquid chromatography, and by infrared and mass spectrometry. The polyol is proposed to be a fully saturated pentacyclic tetrol (C35H62O4, Mr 546) containing a 1,2,3,4-tetrahydroxy pentane substituted to a hopane-derived pentacyclic triterpene nucleus.

Identification of surface proteins of a bacterial membrane using thiolactone-activated polyacrylamide beads.

A new method for the determination of protein accessibility in membranes and membrane fractions using a resin, 'Enzacryl' polythiolactone, is described. Enzacryl polythiolactone is a hydrophilic polymer of acrylamide and acrylamide derivatives with thiolactone ring substituents. The binding of enzymes and proteins to this resin is accomplished very simply by mixing them together in a simple aqueous buffer. Groups which react with the polymer in the pH range 5--9 include aliphatic and phenolic hydroxyls and aliphatic amino groups. Surface proteins of Bacillus licheniformis membrane and solubilised membrane fractions are bound irreversibly to this resin. Inaccessible proteins remaining in the fractions are solubilised with sodium dodecyl sulphate and examined by sodium dodecyl sulphate polyacrylamide gel electrophoresis.

Liposomal membranes. XI. A suggestion to structural characteristics of acido-thermophilic bacterial membranes.

To understand the role of omega-cyclohexyl fatty acid residue of lipids in acido-thermophilic bacterial membranes, three unusual phosphatidylcholines, 1, 2-di-11-cyclohexylundecanoyl-L-alpha-phosphatidylcholine (11CYPC), 1,2-di-13-cyclohexyltridecanoyl-L-alpha-phosphatidylcholine (13CYPc), and 1-13-cyclohexyltridecanoyl-2-11-cyclohexylundecanoyl-L-alpha- phosphatidylcholine (1-13CY-2-11CYPC) were prepared and the steady-state fluorescence anisotropy of 1, 6-diphenylhexatriene (DPH) in the hydrophobic domain of these liposomal bilayers was determined. Compared with the case of dipalmitoyl (DPPC) or dimyristoyl phosphatidylcholine (DMPC), introducing the omega-cyclohexyl moiety onto lecithins makes the bilayers fluid below the phase transition temperature, while immobilizes them above the phase transition temperatures. The properties of the unusual phosphatidylcholine liposomes suggested by the steady-state fluorescence anisotropy investigation were in good agreement with those obtained from the thermotropic and permeability investigations. Results obtained are discussed from the view point of the role and function of lipid membranes of acido-thermophilic bacteria which contain unusual fatty acids.

Purification and characterization of two soluble cytochromes from the alkalophile Bacillus firmus RAB.

A soluble cytochrome c and soluble cytochrome b were purified from the alkalophilic Bacillus firmus RAB. The cytochrome c, with an alpha band at 552 nm, had an apparent molecular weight of 16,500 and was acidic, with a pI of 3.4. At both pH 7.0 and 8.3, the midpoint potential of c-552 was +66 mV. Above pH 8.3, the cytochrome exhibited a pH-dependent decrease in midpoint potential. This property, among others, distinguished the cytochrome c-552 from other membrane-associated c-type cytochromes. The soluble cytochrome b, with an alpha band maximum at 558 nm, had a molecular weight of approx. 15,500 and was also an acidic protein, with a pI of 3.07. It exhibited a pH-independent midpoint potential of +28 mV.

Cardiolipin, a major phospholipid of Gram-positive bacteria that is not readily extractable.

Extraction of phospholipids from stationary phase grown cells of the Gram+ bacteria, Bacillus megaterium, Bacillus subtilis, Bacillus cereus and Micrococcus lysodeikticus was found to be incomplete with various commonly used extraction procedures. Phosphatidylglycerol and phosphatidylethanolamine were readily extracted but up to 95% of the cardiolipin appeared to be retained within the cell residue. Extraction of the cardiolipin could be slightly enhanced by increasing the temperature or the acidity of the extraction solutions but complete extraction was obtained only after lysozyme treatment of intact cells or cell residues remaining after extraction. In addition complete extraction could be observed in the case of cells harvested in the early logarithmic phase. Freeze-fracture electron microscopy was carried out on the cell residue remaining after extraction of all phospholipids except cardiolipin. A fracture plane through the plasma membrane could not be observed anymore. Instead fracture planes through lipid vesicles were observed. These vesicles reside within the remnants of the cytoplasm and consist most likely of the non-extracted cardiolipin.

On the relationship between glycerophosphoglycolipids and lipoteichoic acids in Gram-positive bacteria. II. Structures of glycerophosphoglycolipids.

1. Eight glycerophosphoglycolipids were isolated from six Gram-positive bacteria. Besides sn-glycero-1-phospho-beta-gentiobiosyldiacylglycerol (i) and sn-glycero-1-phospho-alpha-kojibiosyldiacylglycerol (ii), three novel structures have been established: 1,2-di-O-acyl-3-O-[6-(sn-glycero-1-phospho)-alpha-D-glucopyranosyl-(1 leads to 2)-(6-O-acyl-alpha-D-glucopyranosyl)]glycerol (iii), 1,2-di-O-acyl-3-O-[6-(sn-glycero-1-phospho)-beta-D-glucopyranosyl-(1 leads to 6)-alpha-D-galactopyranosyl-(1 leads to 2)-alpha-D-glucopyranosyl]glycerol (iv), and 1,2-di-O-acyl-3-O-[6-(sn-glycero-1-phospho)-beta-D-glucopyranosyl-(1 leads to 6)-alpha-D-galactopyranosyl-(1 leads to 2)-(6-O-acyl-alpha-D-glucopyranosyl)]glycerol (v). 2. Compound i was isolated from Bacillus licheniformis, Bacillus subtilis and Staphylococcus aureus, compound ii from a group B Streptococcus, compounds ii and iii from Streptococcus lactis, compounds iv and v from Lactobacillus casei. Lactobacillus plantarum contained besides compounds iv and v a glycerophosphate derivative of 1,2-di-O-acyl-3-O-[alpha-D-galactopyranosyl (1 leads to 2)-alpha-D-glucopyranosyl]glycerol. 3. Identical structural features of the described glycerophosphoglycolipids and the corresponding lipoteichoic acids are discussed.

An X-ray study of the cytoplasmic membranes of two gram-positive bacteria.

X-ray diffraction diagrams of partially disordered one-dimensional lattices of isolated bacterial cytoplasmic membranes are described and they provide a basis for suggesting possible molecular structures of bacterial membranes. Biochemical and electron microscope evidence points towards a lipid bilayer with a high degree of fluidity. The protein molecules are in a disordered configuration in the membrane.

On the nature of the cytosine-methylated sequence in DNA of Bacillus brevis var. G.-B.

On growing the cells of Bacillus brevis S methionine-auxotroph mutant in the presence of [Me-3H]methionine, practically all the radioactivity incorporated into DNA is found to exist in 5-methylcytosine and N6-methyladenine. The analysis of pyrimidine isopliths isolated from DNA shows that radioactivity only exists in mono- and dinucleotides and the content of 5-methylcytosine in R-m5 C-R and R-m5 C-T-R oligonucleotides is equal. The analysis of dinucleotides isolated from DNA by means of pancreatic DNAase hydrolysis allows the nature of purine residues neighbouring 5-methylcytosine to be identified and shows that 5-methylcytosine localizes in G-m5 C-A and G-m5 C-Tr fragments. B. brevis S DNA methylase modifying cytosine residues recognizes the GCA/TGC degenerate nucleotide sequence which is a part of the following complementary structure with a two-fold rotational axis of symmetry: (5')...N'-G-C-T-G-C-N... (3') (3')...N-C-G-A-C-G-N'... (5') (Methylated cytosine residues are askerisked). Cytosine-modifying DNA methylase activity is isolated from B. brevis cells; it is capable of methylating in vitro homologous and heterologous DNA. Hence DNA in bacterial cells can be undermethylated. This enzyme methylates cytosine residues in native and denatured DNA in the same nucleotide sequences. Specificity of methylation of cytosine residues in vitro and in vivo does not depend on the nature of substrate DNA. DNA methylases of different variants of B. brevis (R, S, P+, P-)) methylate cytosine residues in the same nucleotide sequences. It means that specificity or methylation of DNA cytosine residues in the cells of different variants of B. brevis is the same.

A sulfonolipid and novel glucosamidyl glycolipids from the extreme thermoacidophile Bacillus acidocaldarius.

The total lipid content of the extreme thermoacidophile Bacillus acidocaldarius comprises about 8.1% of the cell dry weight. Total lipid had a distribution of 15.7% neutral linique component initially characterized as an N-acylglucosamine beta-linked to the primary hydroxyl of an unusual fully saturated pentacyclic triterpene derived tetrol(C35H62O4, Mr 546), which appears to be a derivative of the pentacylcic triterpene hopane substituted at C-29 with a 1,2,3,4-tetrahydroxy pentane. Other major glycolipids present were partially characterized as O-beta-D-glucopyranosyl-(1 leads to 4)-O-2-acylamido-2-deoxy-beta D-glucopyranosyldiacylglycerol and O-beta-D-glucopyranosyl-(1 leads to 4)-O-2-acylamido-2-deoxy-beta-D-glucopyranosylmonoacylglycerol. Minor components of the glycolipid fraction included O-beta-D-glucopyranosyl-(1 leads to 4)-O-2-acylamido-2-deoxy-beta-D-glucopyranosylglycerol, O-2-amino-2-deoxy-beta-D-glucopyranosyl pentacyclic tetrol and free pentacyclic tetrol. The distributions of esterified and amide-linked fatty acids were similar, being comprised primarily of branched heptadecanoic, 11-cyclohexyundecanoic and 13-cyclohexyltridecanoic acids. The acid lipids were composed of a sulfonoglycosyldiacylglycerol (43.2%), diphosphatidylglycerol (32.3%), lysodiphosphatidylglycerol (5.3%), phosphatidic acid (5.8%) and phosphatidylglycerol (13.4%).

On the relationship between glycerophosphoglycolipids and lipoteichoic acids in Gram-positive bacteria. I. The occurrence of phosphoglycolipids.

1. Gram-positive bacteria out of the families of Streptococcaceae, Lactobacillaceae, Micrococcaceae and Bacillaceae were investigated with respect to the occurrence and the concentration of phosphoglycolipids. 2. Phosphatidylglycolipids occur exclusively in group D Streptococci and in Streptococcus hemolyticus D-58. Phosphatidyl-alpha-kojibiosyldiacylglycerol, the prevalent species, accounts for up to 28% of the polar lipids. The related glycerophospho-phosphatidyl-alpha-kojibiosyldiacylglycerol is restricted to Streptococcus faecalis. 3. Glycerophosphoglycolipids, usually minor components, comprise thirteen compounds most of which have so far not been described. Except Micrococcus lysodeikticus all examined bacteria contained one or more glycerophosphoglycolipids. Their occurrence parallels, therefore, that of lipoteichoic acids, which supports the hypothesis of a metabolic relationship between these two membrane components.

Membrane associated phospholipoproteins of Bacillus lichenformis 749.

The membrane-bound penicillinase of Bacillus licheniformis 749/C is a phospholipoprotein that differs from the hydrophilic exoenzyme in that its polypeptide chain carries an additional 25 residues (mostly hydrophilic) with phosphatidylserine as the NH2-terminus. To determine if other phospholipoproteins are present in the plasma membrane, the penicillinase-inducible strain 749 was grown without inducer in the presence of [2-(3)H] glycerol. Electrophoretic separation of the membrane proteins (after removal of free lipids) showed an association of 3H-activity with certain of the proteins which could not be broken by lipid solvents and strongly denaturing conditions. Pronase digestion of the membrane proteins (after solvent extraction) released phosphatidylserine, thus indicating the covalent linkage of protein and phospholipid. Treatment of the isolated membranes with trypsin solubilized the protein portion of some of the phospholipoproteins (as with penicillinase), but not the 3H-labelled fragment. Penicillinase should be considered as the first observed example of a group of phosphatidylserine-containing proteins present in the plasma membrane of B. licheniformis 749 and 749/C.

Tertiary structure of Bacillus thermoproteolyticus [4Fe-4S] ferredoxin. Evolutionary implications for bacterial ferredoxins.

The structure of a low-potential [4Fe-4S] ferredoxin from Bacillus thermoproteolyticus has been solved using anomalous scattering data from iron atoms in the diffraction data of native crystals and refined partially to a crystallographic R-factor of 0.33, with 2.3 A (1 A = 0.1 nm) resolution data. The least-squares refinement based on the Bijvoet differences has determined that the four iron atoms in the cluster are an equal distance, approximately 2.8 A, apart. The NH ... S hydrogen bonds between polypeptide nitrogen atoms, and both cysteine and inorganic sulfur atoms, are present, as in ferrodoxin from Peptococcus aerogenes. The polypeptide chain of the B. thermoproteolyticus ferredoxin has a fold closely similar to that of 2[4Fe-4S] ferredoxin from P. aerogenes. The structural correspondence indicates strongly that both types of ferredoxin evolved from a common ancestor. The second cluster-binding region in P. aerogenes ferredoxin corresponds to the alpha-helix in B. thermoproteolyticus ferredoxin. The secondary-structure predictions strongly suggest that the alpha-helix is generally present in the monocluster-type ferredoxins. The conformational change to alpha-helix, insertions of a loop and a protrusion, as well as the absence of the second cluster in B. thermoproteolyticus ferredoxin, result in the lack of 2-fold symmetry present in P. aerogenes ferredoxin. So, the track of gene duplication is no longer detectable in the tertiary structure alone. The evolutionary events that may have occurred in the ferredoxins with the [4Fe-4S] cluster are discussed.

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

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

[Purification of an extracellular thiol-dependent hemolysin from Bacillus alvei]. / Purification de l'hémolysine thiol-dépendante extracellulaire de Bacillus alvei.

Alveolysin a sulfhydryl-dependent cytolytic extracellular protein released by Bacillus alvei has been purified by salting-out by ammonium sulfate, gel filtration, isoelectric focusing on pH gradient and chromatography on DEAE-cellulose. The purified protein after reduction by thiols (active hemolytic form) proved homogeneous by disc polyacrylamide gel electrophoresis and by gel immunodiffusion. The molecular weight was 60,000 daltons, Two molecular forms of pI 5.1 and 7.0 were detected by gel isoelectrofocusing. The toxin was lethal to the mouse. Lytic activity was inhibited by cholesterol and antistreptolysin O anstisera. Immunological cross-reaction was observed between alveolysin and streptolysin O.

1H-2-Benzopyran-1-one derivatives, microbial products with pharmacological activity. Conversion into orally active derivatives with antiinflammatory and antiulcer activities.

A novel gastroprotective substance, 6-[[1(S)-[3(S),4-dihydro-8-hydroxy-1-oxo-1H-2-benzopyran-3-yl] -3-methylbutyl]amino]-4(S),5(S)-dihydroxy-6-oxo-3(S)-ammoniohexanoate (AI-77-B, 1), isolated from a culture broth of Bacillus pumilus AI-77, was chemically modified to prodrugs that are active by oral dosing. Compound 1 was lactonized and then monoalkylated at the primary amine position. Six N-alkylated gamma-lactone derivatives of 1 (with alkyl chains being methyl 5a, ethyl 5b, n-propyl 5c, n-butyl 5d, n-pentyl 5e, or n-hexyl 5f) were synthesized and eight compounds including 1 and gamma-lactone derivative 2 were compared for their gastroprotective activities and blood levels after oral administration in rats. Further, chloroform-water partition coefficients of 5a-f were also compared as a measure of lipid solubility. The protective effects of these compounds on stress ulcers were mutually related to blood levels of dealkylated compounds (1 and 2). Parent compound 1 was detected in blood at 1 h after each of 5a-d was administered. When 5b or 5c was administered, high activity and high blood levels of 1 were observed in comparison with those levels obtained with 5a or 5d. Neither 5e nor 5f were detected in any amount in blood by oral administration without special formulation due to extremely low solubilities and agglutinative properties in intestinal fluid. Interestingly, 5b and 5c were found to have antiinflammatory activities in addition to potent antiulcerogenicity action.

Bis(monoacylglycero)phosphate in alkalophilic bacteria.

Bis(monoacylglycero)phosphate (BMP) was found in an alkalophilic bacterium, Bacillus sp. A-007. This lipid comprised 4% of the phospholipid of this organism. The very low rate and extent of triphenylmethylation of BMP indicated that the two fatty acid residues occupied the C-1 and C-3' positions of the two glycerol moieties. The stereochemical configuration of the backbone of BMP was identified as sn-glycero-3-phosphoryl-1'-sn-glycerol by sn-glycerol 3-phosphate dehydrogenase analysis after acetic acid hydrolysis and mild alkaline methanolysis. This configuration was the same as that of phosphatidylglycerol, but differed from that of BMP from mammalian cells. Finally, the structure of BMP in Bacillus sp. A-007 was concluded to be 1-acyl-sn-glycero-3-phosphoryl-1' (3'-acyl)-sn-glycerol. The fatty acids of BMP were those common in Bacillus species. The fatty acid composition of BMP was very similar to that of the major lipids in this organism. The fatty acid distribution between the C-1 and C-3' positions of BMP was determined by a newly developed method which included successive steps of acetic acid hydrolysis, chemical acylation and phospholipase A2 treatment. Fatty acids with longer chain length were concentrated in the C-1 position, and the shorter fatty acids were the major ones esterified at the C-3' position.

Bacteriophage phiNS11: a lipid-containing phage of acidophilic thermophilic bacteria. III. Characterization of viral components.

Components of a lipid-containing phage phiNS11 were characterized. The phage had five protein components, the molecular weights of which were 59,000, 44,000, 33,000, 23,000, and 18,000. Viral lipid consisted of six components, which were also found in the host bacterial lipid. The relative amounts of these viral lipid components were very similar to those of the bacterial lipid. The phage contained omega-cyclohexyl fatty acids characteristic of Bacillus acidocaldarius as the main fatty acids. The phage nucleic acid was a linear double-stranded DNA, the molecular weight of which was 9.3--9.4 X 10(6) daltons. The guanine plus cytosine content of the DNA was determined to be about 52% from chemical analysis, buoyant density (1.711 g/cm3 in CsCl) and melting temperature (90.6 degrees C in 0.15 M NaCl plus 0.015 M sodium citrate). The phage contained two kinds of polyamine; spermidine and spermine.

Structural studies on thiocillins I, II and III (studies on antibiotics from the genus Bacillus XXIX).

Thiocillins I, II and III were compared with micrococcin P1 by analysis of acid hydrolyzates of the native and the reduced antibiotics as well as by means of 1H and 13CNMR spectroscopies. As a result of these studies, the differences of these antibiotics were clarified in their structural units, and the structures of thiocillins I, II and III were assigned on the basis of the proposed structure of micrococcin P1.

Glysperin, a new antibiotic complex of bacterial origin. II. Structures of glysperins A, B and C.

Structures of glysperins A, B and C were determined on the basis of chemical degradation studies in conjunction with spectroscopic analyses. Glysperin A consisted of L-alanine, p-hydroxybenzoic acid, a C11-alkyl tetramine and four sugar moieties, three of which were identified as D-ribose, D-galactose and 2,4-diamino-2,4,6-trideoxy-D-galactose. The fourth sugar was a novel exoenose, 6-deoxy-D-xylo-hex-5-enose. Structural difference between glysperins A and B resided solely in the terminal polyamine moiety which was spermidine in glysperin B. Glysperin C contained D-glucose in place of the exoenohexose moiety of glysperin A. Glysperins A, B and C are, in some respects, structurally related to the glycocinnamoylspermidine antibiotics, LL-BM 123 beta, gamma 1 and gamma 2.