- Volume 133, Issue 3, 1987
Volume 133, Issue 3, 1987
- Biochemistry
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Investigation of Escherichia coli Fumarate Reductase Subunit Function Using Transposon Tn5
More LessSeventy two Tn5 transposon insertions were isolated in the frd operon carried on the multicopy plasmid pFRD79. The polar nature of these mutations permitted examination of the expression and localization of the frd polypeptides in novel subunit combinations. The minimal catalytic unit is the FRDA plus B dimer. A transposon within frdB (frdB::Tn5) produces inactive, soluble FRDA polypeptide which has covalently attached 8α(N3-histidyl)flavin adenine dinucleotide cofactor. A transposon mutation within frdC (frdC::Tn5) produces soluble, catalytically active dimer. An insertion in frdD (frdD::Tn5) produces both a soluble trimer composed of FRDABC, and a tetramer of FRDABC and truncated FRDD bound to the inner membrane. Eighty percent of the activity is in the soluble form. Using this mutant, the requirement for FRDD both for optimal activity of the catalytic domain and for proper anchorage in the cytoplasmic membrane was demonstrated.
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Metabolism of Both Stereoisomers of Phenylglycine by Different Routes in Flavobacterium F24
More LessFlavobacterium F24 metabolized both stereoisomers of phenylglycine and enzyme studies revealed that l-phenylglycine was transaminated by a constitutive enzyme while the d-stereoisomer was oxidized by a phenazine-methosulphate-dependent d-amino-acid dehydrogenase. This latter enzyme was not induced during growth on l-phenylglycine. Phenylglyoxylate formed in the reactions was decarboxylated by an inducible enzyme to benzaldehyde, which was oxidized mainly by an inducible phenazine-methosulphate-dependent benzaldehyde dehydrogenase not described earlier. Benzoate was further metabolized via 3-hydroxybenzoate to gentisate, which in turn was further degraded through a glutathione-dependent pathway.
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The Purification and Properties of Two Isofunctional 2-Hydroxy-6-oxohepta-2,4-dienoate Hydrolases from Alcaligenes eutrophus Strain 345
More LessTwo 2-hydroxy-6-oxohepta-2,4-dienoate (HOD) hydrolases have been purified to homogeneity from Alcaligenes eutrophus ATCC 17707. One, HODHII, is encoded by the TOL-like plasmid, pRA1000, and the other, HODHI, is chromosomally-encoded. HODHI and HODHII have an M r of 104 × 103 and 116 × 103, respectively, and both are dissociated by SDS into three subunits of equal size which are not linked by disulphide bonds. Both hydrolases are active against a similar range of substrates formed by the action of catechol 2,3-oxygenases, generally being more active against substrates formed from 3-alkyl- rather than 4-alkyl-substituted catechols. HODHI and HODHII had similar K m values for HOD, but HODHI had a significantly lower K m value for, and a higher activity against, 2-hydoxymuconic semialdehyde than HODHII. It was shown by immunodiffusion studies that the two hydrolases were immunologically distinct and that HODHI had no common antigenic determinants with HOD hydrolases from the Pseudomonas putida strains NCIB 9865, NCIB 10015 and mt-2; HODHII gave a line of identity with antiserum to the hydrolase from P. putida NCIB 9865. A comparison of the physico-chemical properties of HODHI and HODHII with the same properties of the HOD hydrolases from the three P. putida strains demonstrated several similarities between the five HOD hydrolases which have been purified.
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- Development And Structure
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Flagellation of Salmonella typhimurium Treated with Nalidixic Acid
More LessFilamentous cells of Salmonella typhimurium, obtained after treatment with nalidixic acid in the exponential phase of growth, elongated up to 10 μm, corresponding to 4 unit cell lengths, per nucleoid. During elongation, division of nucleoids and septum formation did not occur, but de novo formation of flagella continued. Most of the filamentous cells were motile, and flagella were evenly distributed on their surface.
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- Genetics And Molecular Biology
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Structure and Function in a Bacillus subtilis Sporulation-specific Sigma Factor: Molecular Nature of Mutations in spoIIAC
More LessSUMMARY: The spoIIAC gene was cloned from chromosomal DNA of seven spoIIAC mutants of Bacillus subtilis, and the complete sequence of the gene was determined for each mutant. Three of the mutants proved to have chain-terminating mutations (one of which, previously shown to be suppressible by sup-3, was identified as amber); these led in every case to complete failure either to manufacture spores or to synthesize two enzymes normally associated with stage II of sporulation. The four remaining mutations were missense, and these corresponded to a phenotype in which a few spores are formed and about half the wild-type quantities of the two enzymes are made. Of the four missense mutations, two were near the promoter-distal end of the gene, in a region believed to correspond to the DNA-binding domain of the sigma factor that spoIIAC encodes. The remaining two mutations were in the region of the gene that is thought to correspond to the domain of the protein that interacts with core RNA polymerase.
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Construction of Improved Bacteriophage ɸ105 Vectors for Cloning by Transfection in Bacillus subtilis
D. Jones and J. ErringtonA series of improved phage vectors have been constructed, based on Bacillus subtilis bacteriophage ø105, which can be used to clone genes in B. subtilis by direct transfection of protoplasts. The new vectors, designated ø105J23, ø105J24, ø105J27 and ø105J28, show frequencies of plaque formation that are equal to those of wild-type ø105. This represents at least a 10-fold improvement over ø105J9, the vector used in previous cloning experiments. Two of the new vectors ø105J27 and ø105J28 incorporate a mutation, cts-52, that renders the prophage temperature inducible. This has made it possible to devise a rapid small-scale procedure for screening progeny phage for the presence of inserted DNA. The usefulness of the new vectors is illustrated in the accompanying paper by cloning more than 20 B. subtilis sporulation genes.
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Cloning in Bacillus subtilis by Transfection with Bacteriophage Vector ɸ105J27: Isolation and Preliminary Characterization of Transducing Phages for 23 Sporulation Loci
J. Errington and D. JonesSUMMARY: Bacteriophage cloning vector ɸ105J27, the construction of which is described in an accompanying paper, has been used for shotgun cloning of sporulation genes in Bacillus subtilis. Various genomic libraries have been constructed and screened for the presence of recombinant phages capable of transducing strains containing sporulation (spo) mutations to Spo+. Of a total of 30 spo loci tested, transducing phages have been isolated for 23, more than half of the known spo loci. Included are nine loci (spo0D, spo0J, spoIIA, spoIIIE, spoIIIF, spoIVF, spoVB, spoVH and spoVJ) that do not appear to have been cloned previously. Preliminary genetic characterization of some of the new clones by a rapid screening procedure has enabled the status of various sporulation loci to be clarified.
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l-Cysteine Biosynthesis in Escherichia coli: Nucleotide Sequence and Expression of the Serine Acetyltransferase (cysE) Gene from the Wild-type and a Cysteine-excreting Mutant
More LessSUMMARY: Serine acetyltransferase (SAT) from Escherichia coli is subject to feedback inhibition by l-cysteine. A mutant was isolated which excretes l-cysteine because of a lesion in cysE, the structural gene for SAT, rendering the enzyme less feedback sensitive. To analyse the structural basis for this mutation the cysE genes both from wild-type E. coli and the mutant strain were cloned and their nucleotide sequences determined. The cysE gene contained an open reading frame consisting of 819 bp, equivalent to a protein of 273 amino acids. The mutant gene showed a single base change in position 767 resulting in a methionine to isoleucine substitution. A causal connection between this SAT sequence alteration, feedback insensitivity and l-cysteine excretion was demonstrated. The SAT from the wild-type strain was purified. It was composed of a single polypeptide chain migrating in SDS gels according to an M r of 34000. As in Salmonella typhimurium, the enzyme was associated in a bifunctional complex with O-acetylserine (thiol)-lyase.
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Nucleotide Sequence of bglC, the Gene Specifying EnzymeIIbg1 of the PEP:Sugar Phosphotransferase System in Escherichia coli K12, and Overexpression of the Gene Product
More LessThe EnzymeIIbg1 of the phosphoenolpyruvate- (PEP-) dependent phosphotransferase system catalyses the uptake and concomitant phosphorylation of β-glucosides by Escherichia coli; it is specified by the gene bglC. The nucleotide sequence of a 3·6 kb HindIII restriction fragment spanning bglC, cloned on a plasmid, was determined. DNA analysis strongly suggests that the published order of this and other genes involved in β-glucoside utilization, bgl C, S, B, is incorrect, and that the regulatory gene bglS may be located upstream of the structural genes bglC and bglB. From the deduced amino acid sequence it is predicted that the membrane protein specified by bglC consists of 625 amino acid residues (66·48 kDa). The protein has the hydropathic profile expected of an integral membrane protein (average hydropathy = 0·62). Comparisons between the amino acid sequences deduced for the EnzymeIIbgl and for the mannitol-specific EnzymeIImtl show that these proteins are related, and a little direct homology is apparent. A 2·3 kb AluI fragment spanning bglC was subcloned into an expression vector which carries the λpL promoter and then transformed into a host strain which produces thermolabile c1857 repressor and the anti-terminator N; thermoinduction resulted in the overproduction of a membrane protein and the appearance of Bgl activity.
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Cloning and Expression of Treponema pallidum Common Antigen (Tp-4) in Escherichia coli K12
More LessA library of Treponema pallidum DNA was constructed using a cosmid cloning system. Sixteen hundred Escherichia coli recombinant clones were generated covering the T. pallidum genome with a probability of 99%. Three hundred of the clones were screened for expression of T. pallidum antigens by a modified rocket immunoelectrophoresis technique using a polyspecific antiserum to T. pallidum. One clone was identified which produced the ‘common antigen’ (CA) of T. pallidum (Tp-4). CA shares epitopes with antigens present in more than 50 different bacterial species, but nothing is known about its structure, function and localization. The recombinant E. coli clone will be of value for a structural analysis of the CA gene.
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Characterization of a Pseudomonas aeruginosa Transposon Insertion Mutant with Defective Release of Exoenzymes
More LessA Pseudomonas aeruginosa transposon insertion mutant with defective release of several exoenzymes has been characterized. The Tn5-751 insertion mutation was located in the previously described xcp-1 locus at 0 min on the chromosomal map and caused several exoenzymes to remain in cell-bound form. At least one of the exoenzymes, elastase, was accumulated in the periplasmic space. The periplasmic elastase had the same M r as the extracellular enzyme produced by the wild-type strain. The virulence of the mutant was comparable to that of wild-type strains in experimental burn infection in mice. The presence of an easily selectable antibiotic resistance marker in the xcp-1 locus offers the possibility of cloning the gene(s) involved in exoprotein secretion.
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Isolation and Characterization of Pseudomonas putida R-Prime Plasmids
More LessA number of enhanced chromosome mobilizing (ECM) plasmids derived from the wide host range plasmid R68 have been used to construct R-prime plasmids carrying a maximum of two map minutes of the Pseudomonas putida PPN chromosome, using Pseudomonas aeruginosa PAO as the recipient. For one ECM plasmid, pMO61, the ability to form R-primes did not correlate with the ability to mobilize chromosomes in intrastrain crosses, suggesting that different mechanisms are involved. Physical analysis of one R-prime showed that 3·5 kb of chromosomal DNA had been inserted between the tandem IS21 sequences carried by the parent ECM plasmid.
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Cloning of the Galactokinase Gene (galK) from Streptomyces coelicolor A3(2)
More LessStreptomyces coelicolor A3(2) and Streptomyces lividans 66 strains were shown to be sensitive to the galactose analogue 2-deoxy-d-galactose. Spontaneous resistant mutants were isolated that were Gal- and lacked the enzyme galactokinase. The galK gene (structural gene for galactokinase) from S. coelicolor was cloned into S. lividans using the low copy number vector pIJ922. The resulting plasmid (pMT650), which contained a 14 kb insert, complemented gal mutations in both species. The presence of the galK gene on a 28 kb EcoRI fragment was confirmed by expressing it in Escherichia coli where it complemented a well characterized galK mutation.
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Use of Mu-lac Insertions to Study the Secretion of Pectate Lyases by Erwinia chrysanthemi
More LessMembers of the species Erwinia chrysanthemi are phytopathogenic enterobacteria causing soft-rot disease due, in part, to the secretion of pectolytic enzymes. We studied the secretion of pectate lyases by using Mu-lac insertion mutagenesis in E. chrysanthemi strain 3937. Analysis of β-galactosidase expression of the out—lac fusions, in different growth conditions, showed that the expression of the out gene is constitutive. Compartmentation of pectate lyases during growth suggests that these enzymes are first exported to the periplasm with formation of an intracellular pool of active pectate lyases and then released into the extracellular medium. In the out mutants, pectate lyases are retained in the periplasm. Analysis by electrofocusing showed that the pectate lyases are not modified during the transfer via the outer membrane. A 65 kDa protein is absent from the periplasm of the out mutants; this protein could have a role in the secretory system of E. chrysanthemi.
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Correlation between the Rate of Exoprotein Synthesis and the Amount of the Multiprotein Complex on Membrane-bound Ribosomes (MBRP-Complex) in Staphylococcus aureus
More LessThe membrane-bound ribosome protein (MBRP)-complex of Staphylococcus aureus was studied using antibodies to its individual components. The four polypeptides of the complex were firmly held together, and none were present in large excess. The membrane-bound fraction of the MBRP-complex was accessible to trypsin only after removal of the membrane-bound ribosomes; it also remained associated with the membrane-bound ribosomes even after solubilization of the membranes with Triton X-100. Furthermore, the amount of MBRP-complex in the membrane was proportional to the rate of exoprotein synthesis. These results strongly suggest a role for the MBRP-complex in protein secretion.
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- Pathogenicity And Medical Microbiology
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Characterization of Binding of Human Fibrinogen to the Surface of Germ-tubes and Mycelium of Candida albicans
More LessThe binding of human fibrinogen to germ-tubes and mycelium of Candida albicans, forms usually found in infected tissues, was studied in vitro by an immunofluorescence assay. Binding was quantified by using 125I-labelled fibrinogen. The degree of binding differed according to the morphological form of the fungus. Binding relative to that of the yeast form was greater for mycelium (12-fold) than for germ-tube (7·7-fold). Pretreatment of yeasts with fragments D and E (terminal degradation products of fibrinogen) before fibrinogen binding showed that fragment D possessed a higher affinity for C. albicans than fragment E. Binding of fibrinogen was diminished when C. albicans was pretreated with 2-mercaptoethanol alone or in combination with pronase, or pretreated with α-mannosidase or trypsin. Binding was not decreased by pretreatment with pronase alone or chitinase. Inhibition experiments using C. albicans dialysed culture filtrate, C. albicans mannan, chitin, sugars or amino sugars were done by preabsorbing the fibrinogen with each of the above mentioned compounds. C. albicans dialysed culture filtrate inhibited the binding more strongly than C. albicans mannan. However, fibrinogen binding to C. albicans was not significantly reduced by mannose, several other sugars or chitin. These studies demonstrate the existence of a fibrinogen-binding factor (FBF) strongly associated with the surface of germ-tube and filamentous forms of C. albicans, and indicate a possible role for FBF in the pathogenicity of C. albicans.
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Genetic Analysis of Variant Pilin Genes from Neisseria gonorrhoeae P9 Cloned in Escherichia coli: Physical and Immunological Properties of Encoded Pilins
More LessA series of genomic DNA fragments that encode gonococcal pilins from four well-characterized pilus variants of Neisseria gonorrhoeae strain P9 have been cloned in Escherichia coli K12. At least nine classes of cloned P9 pilin genes have been identified on the basis of restriction mapping of cloned pilin-encoding DNA and physical and immunological analysis of expressed pilin proteins. Each antigenic variant of strain P9 possesses many genomic segments of pilin gene information, although our results suggest that strain P9 contains only a single pilin-expressing (pilE) locus.
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Isolation and Partial Characterization of an Adhesin from Candida albicans
More LessCandida albicans produces extracellular polymeric material (EP) which contains a mannoprotein adhesin. EP isolated from culture supernatants of C. albicans GDH 2346 consisted of a mixture of glycoprotein components and inhibited yeast adhesion to buccal epithelial cells by up to 60 %. Partial purification of the adhesin was achieved by a two-step procedure involving chromatography of EP on concanavalin A-Sepharose and DEAE-cellulose. The purified adhesin inhibited adhesion to buccal cells 30 times more efficiently (on a weight basis) than unfractionated EP. Pretreatment of EP with heat, dithiothreitol or proteolytic enzymes either partially or completely destroyed its ability to inhibit adhesion, whereas pretreatment with sodium periodate or α-mannosidase had little or no effect. These results suggest that the protein portion of the mannoprotein adhesin is more important than the carbohydrate moiety in mediating yeast attachment to buccal epithelial cells.
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Role of Glycosides as Epithelial Cell Receptors for Candida albicans
More LessThe effect of various lectins and sugars on adhesion of five strains of Candida albicans to buccal and vaginal epithelial cells in vitro was investigated. Adhesion of C. albicans GDH 2346 was inhibited primarily by l-fucose and winged-pea lectin, whereas adhesion of strain GDH 2023 was inhibited by N-acetyl-d-glucosamine, or d-glucosamine, and wheat-germ agglutinin. Three other strains of C. albicans (MRL 3153, GRI 681 and GRI 682) gave results similar to those obtained with strain GDH 2346. Extracellular polymeric material (EP) isolated from strain GDH 2346 inhibited adhesion of strains MRL 3153, GRI 681 and GRI 682 by more than 50%, but that of strain GDH 2023 by only 30%. EP from strain GDH 2023 had little or no effect on the adhesion of any other yeast strain. Lectin-like proteins with affinities for l-fucose, N-acetyl-d-glucosamine and d-mannose were detected in EP from all five strains in different amounts. These results indicate that there are at least two types of adhesion mechanism and that glycosides containing l-fucose or N-acetyl-d-glucosamine can function as epithelial cell receptors for C. albicans.
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Animal Models in Q Fever: Pathological Responses of Inbred Mice to Phase I Coxiella burnetii
More LessThe susceptibility of inbred strains of mice to infection by phase I Coxiella burnetii, the aetiological agent of Q fever, was investigated by evaluating morbidity, mortality, antibody production and in vitro proliferative responses of splenic lymphocytes. Among the 47 strains of mice tested for morbidity and mortality to C. burnetii infection, 33 were resistant, 10 were of intermediate sensitivity, and four were sensitive. A/J mice exhibited the highest mortality, and surviving mice of this strain yielded high concentrations of viable rickettsiae from essentially all organs for more than 3 weeks after inoculation. However, A/J mice developed a protective immune response after vaccination with inactivated C. burnetii cells. Induction of gross pathological responses and antibody production were similar in sensitive mice (strain A/J) and resistant mice (strain C57BL/6J). The LD50 of phase I C. burnetii for A/J mice was about 1000-fold lower than that for the more resistant C57BL/6J mice. Mice of both strains developed antibody titres against phase I cells, phase II cells, and phase I lipopolysaccharide after the injection of one or more viable phase I organisms of C. burnetii; five or more rickettsiae caused splenomegaly that was almost proportional to the infecting dose. Suppression of in vitro proliferative responses of splenic lymphocytes to concanavalin A, a T-cell mitogen, was apparent after infection of sensitive A/J mice with as few as one to five phase I micro-organisms. However, suppression of proliferation of splenic lymphocytes from resistant C57BL/6J mice required 107 phase I C. burnetii.
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β-Lactamase Production by Intestinal Spirochaetes
More Lessβ-Lactamase production was demonstrated in four of nineteen strains of intestinal spirochaetes isolated from human subjects. The enzyme was preferentially active against penicillins and was inhibited by clavulanic acid; it was membrane bound and non-inducible. No plasmids were detected in the intestinal spirochaetes and the β-lactamase-production characteristic was not transferable to non-producing strains.
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Isolation from Urine of Two Serratia marcescens Strains Excreting a Diffusible Yellow Pigment
More LessTwo bacterial strains excreting a yellow pigment were isolated from human urine and identified as Serratia marcescens. The pigment was produced in the late exponential and early stationary phases of growth. Minimal media supplemented with tyrosine, phenylalanine, 3,4-dihydroxyphenylacetate or tryptophan, as well as complex media, induced pigment production. UV-visible spectra of the extracted pigment had peaks characteristic of 2-hydroxy-5-carboxymethyl-muconate semialdehyde, produced from meta-cleavage of 3,4-dihydroxyphenylacetate by the enzyme 3,4-dihydroxyphenylacetate 2,3-dioxygenase (EC 1.13.11.15). This enzyme was active when the bacteria were grown under conditions promoting pigment production. The kinetics and factors affecting pigment production are also reported.
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Human Choriogonadotropin-like Material in Bacteria of Different Species: Electron Microscopy and Immunocytochemical Studies with Monoclonal and Polyclonal Antibodies
More LessImmunocytochemical studies using antisera to whole human choriogonadotropin (hCG), to its α- and β-subunits and to the COOH-terminal peptide of hCGβ, and two monoclonal antibodies to hCGβ, demonstrated expression of hCG-like material, its individual subunits and/or fragments in nine bacterial strains. Seven of these were isolated from patients with cancer and were definitely identified as Streptococcus faecalis (three strains), Staphylococcus haemolyticus (two strains) and Staphylococcus epidermidis and Escherichia coli (single strains). The other two strains were cell-wall-deficient (CWD) variants, one identified as Streptococcus bovis, isolated from the blood of a patient with a fever of unknown origin and a possible brain abscess. The other was a Gram-negative diphtheroid isolated from the urine of a pregnant woman, which during the period of study reverted to a Gram-positive Corynebacterium identified as a ‘C. ulcerans’ strain and expressed the hCG-like factor only during its phase as Gram-negative diphtheroid. Electron microscopy of these nine strains (including negative controls of strains of the same species subjected to the same immunocytochemical analyses and under identical cultural conditions) revealed morphological alterations in the bacterial cell walls and cytoplasmic material and/or bizarre forms of reproduction in six of the nine strains expressing hCG-like material including the two CWD variants. Collectively, these results provided evidence that (1) hCG-producing bacteria isolated from patients with overt cancer are not a new and unique species as claimed by others, and (2) there is a close resemblance between the bacterial protein and the human trophoblastic hormone, based on immunochemical recognition of different parts of the hCG molecule. The morphological changes observed by electron microscopy may indicate that some of the bacteria expressing hCG-like material are revertants of CWD variants.
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- Physiology And Growth
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Comparison of Methods for Protoplast Formation in Bacillus thuringiensis
More LessSUMMARY: In preparation of Bacillus thuringiensis protoplasts or cell-free lysates, hydrolysis of cell walls was due to endogenous autolytic activity and was largely independent of lysozyme. Purified cell walls of B. thuringiensis were resistant to lysozyme, lysostaphin, chitinase, trypsin and Bacillus subtilis proteinase, but were hydrolysed by B. thuringiensis autolytic enzymes or an N-acetylmuramidase (mutanolysin) from Streptomyces globisporus. Mutanolysin was effective in preparation of protoplasts or lysates of B. thuringiensis, and, unlike lysozyme, did not interfere with electrophoretic analysis of DNA.
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Influence of Divalent Cations on the Growth and Morphology of Bacillus stearothermophilus
More LessSUMMARY: Bacillus stearothermophilus grows within the temperature range 40–70 °C in a complex medium that contains 115 μm-Ca2+ and 95 μm-Mg2+. The addition of Ca2+ to a final concentration ranging from 2·5 to 10 mm stimulated growth at suboptimal and supraoptimal temperatures, extending growth above 70°C, but had no effect on growth within the optimal temperature range. Mg2+ (2·5 mm) also stimulated growth although to a lesser extent. Furthermore, 10 mm-Mg2+ inhibited growth at temperatures higher than 65°C. This inhibitory effect was relieved by the addition of 2·5 mm-Ca2+. Sr2+ (10 mm), which often behaves as a Ca2+ analogue in biological systems, strongly inhibited growth and produced gross morphological alterations in the cells. The inhibitory effect of Sr2+ could also be relieved by addition of Ca2+.
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Morphology, Growth and Reversion in a Stable L-form of Escherichia coli K12
T. Onoda, A. Oshima, S. Nakano and A. MatsunoSUMMARY: An L-form isolated from Escherichia coli K12 by sequential treatment with N-methyl-N′-nitro-N-nitrosoguanidine and lysozyme was adapted to grow in hyperosmolar liquid cultures. It was stable in the absence of antibiotic when cultured in brain heart infusion (BHI) broth containing NaCl and CaCl2, the optimal concentrations being 0·34 m and 1 mm, respectively. No growth of the L-form was observed when CaCl2 was not added to BHI medium containing 0·34 m-NaCl. On the other hand, when KCl replaced NaCl as the osmotic stabilizer, growth of the L-form was repressed in the presence of CaCl2. Electron microscopy of the L-form confirmed the absence of a cell wall. A revertant strain derived from the L-form grew as a stable bacillary form in BHI medium without osmotic stabilizer. The growth characteristics of the revertant strain resembled those of the parent strain. The revertant strain produced L-forms in the presence of NaCl.
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External K+ Affects the Internal Acidification Caused by the Addition of Glucose to Yeast Cells
More LessSUMMARY: In glucose-grown cells of Saccharomyces cerevisiae, collected during the stationary phase of growth, the addition of K+ to the external medium reversed glucose-induced internal acidification in 2 min. However, in ethanol-grown cells external K+ did not reverse the effect of glucose even after 20 min. The presence or absence of external K+ did not alter the modification of trehalase and fructose-1,6-bisphosphatase induced by glucose. It is concluded that transient acidification may be sufficient to cause the associated transient increase in cAMP.
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Cadmium Accumulation by a Citrobacter sp.: the Chemical Nature of the Accumulated Metal Precipitate and its Location on the Bacterial Cells
Cells of a strain of a Citrobacter sp., pre-grown in cadmium-free continuous culture, accumulated cadmium extensively when resuspended in a buffer that contained Cd2+ and glycerol 2-phosphate. The accumulated compound was identified by X-ray microanalysis and magic angle spinning NMR analysis as cell-bound cadmium phosphate, probably CdHPO4. Its accumulation is consistent with the activity of a phosphatase, induced during pre-growth, that continues to function in the resuspended cells to liberate HPO2− 4 from glycerol 2-phosphate. This anion then combines with Cd2+ to form insoluble cell-bound CdHPO4.
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Topography of Peptidoglycan Synthesis during Elongation and Polar Cap Formation in a Cell Division Mutant of Escherichia coli MC4100
More LessA cell division mutant of Escherichia coli K12 lysA, the temperature sensitive ftsZ strain, was pulse-labelled with [3H]diaminopimelic acid (DAP) during growth in minimal salts medium both at the permissive (28 °C) and restrictive (42 °C) temperature. In contrast to other known cell division mutants, ftsZ filaments obtained during growth at 42 °C show no sign of persisting or newly initiated constrictions. The location of the incorporated DAP in dividing cells and in filaments was analysed with an improved autoradiographic method in which preparations of well-spread sacculi are covered with a dry emulsion. From the populations of sacculi complete distributions were obtained, which compared well with those of the intact cells. The grain-density distributions of cells dividing at 28 °C showed that the rate of surface synthesis was strongly increased at the site of constriction at the expense of the activity in the lateral wall, suggesting a redistribution of surface synthesis activity. In individual filaments elongating at 42 °C no indication for the existence of narrow or broad growth zones was found, suggesting a dispersed mode of lateral wall synthesis. These observations are in accordance with theoretical predictions on the rate of surface synthesis during the constriction period in cells which elongate at a constant diameter.
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Exo-1,3-βglucanase Activity in Candida albicans: Effect of the Yeast-to-mycelium Transition
More LessYeast cells of Candida albicans1001 produced glucan-hydrolysing activity, most of which was due to an exo-1,3-β-glucanase. The enzyme was periplasmically located; it could be found in culture medium samples, and was secreted by protoplasts when cultured under regeneration conditions. In contrast to most yeast exoglucanases, this enzyme was practically inactive against p-nitrophenyl-β-d-glucoside, hydrolysis of this substrate being carried out by a β-glucosidase located inside the cytoplasmic membrane and not secreted to the external medium. Supernatant fluids from cell-free extracts reached their maximum glucanase level after several days at 0 °C, suggesting that the active enzyme was formed from an inactive precursor. Glucanase activity substantially decreased and sometimes disappeared from the cells when the yeast-to-mycelium transition was induced, but a significant (though lesser) reduction was also observed in yeast cells incubated in the same medium under conditions (temperature, cell concentration) that did not lead to formation of hyphae. It is suggested that C. albicansexo-1,3-β-glucanase may not be necessary for mycelial growth.
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Production and Secretion of Saccharomyces cerevisiae β-Glucanases: Differences between Protoplast and Periplasmic Enzymes
More LessCell-free extracts and culture medium from Saccharomyces cerevisiae S288C contained the two glucan hydrolases that are known to be periplasmically located in vegetative cells of this yeast. These were an endo-1,3-β-glucanase and a much more abundant exo-1,3-β-glucanase. Cell-free extracts of strains carrying two different mutant alleles of the EXB1 gene were totally deficient in the latter enzyme, whereas the former appeared in multiple heterogeneous forms, probably due to incomplete glycosylation. In contrast, protoplast lysates of wild-type and exb1 mutant strains were identical in their complement of glucanases, which consisted of two enzymes clearly distinguishable from the periplasmic glucanases. One was an exo-1,3-β-glucanase, which was active on pustulan, p-nitrophenyl β-d-glucoside, salicin and cellobiose, and was of higher M r than periplasmic exoglucanase. The other was a hydrolase acting on 1,3-β-glucan and 1,6-β-glucan but not the simple glucosides, which was not retained by DEAE-Biogel. Wild-type and exb1 mutant protoplasts secreted portions of the glucanases detected in protoplast lysates, when cultured in osmotically stabilized regeneration medium; the former also secreted the periplasmic exo-1,3-β-glucanase but the latter consistently failed to secrete it. It is concluded that the classically known glucanases of S. cerevisiae, located in the periplasmic space, must be formed as active enzymes, upon secretion, from inactive cytoplasmic precursors. On the other hand, the two new protoplast glucanases could be secreted by an alternative route that assures their incorporation into the wall structure, thus making their release into the culture medium more difficult.
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Isolation and Characterization of Escherichia coli Mutants Lacking Inducible Cyanase
More LessTo determine the physiological role of cyanate aminohydrolase (cyanase, EC 3.5.5.3) in bacteria, mutants of Escherichia coli K12 devoid of this inducible activity were isolated and their properties investigated. Five independent mutations were localized next to lac; three of them lay beween lacY and codA. Thus cyanase activity could depend on the integrity of one gene or set of clustered genes; we propose for this locus the symbol cnt. Growth of the mutant strains was more sensitive to cyanate than growth of wild-type strains. This difference was noticeable in synthetic medium in the presence of low concentrations of cyanate (⩽ 1 mm). Higher concentrations inhibited growth of both wild-type and mutant strains. Urea in aqueous solutions dissociates slowly into ammonium cyanate. Accordingly wild-type strains were able to grow on a synthetic medium containing 0·5 m-urea whereas mutants lacking cyanase were not. We conclude that cyanase could play a role in destroying exogenous cyanate originating from the dissociation of carbamoyl compounds such as urea; alternatively cyanate might constitute a convenient nitrogen source for bacteria able to synthesize cyanase in an inducible way.
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Cyanate Specifically Inhibits Arginine Biosynthesis in Escherichia coli K12: a Case of By-product Inhibition?
More LessGrowth of Escherichia coli K12 cultivated in minimal medium was strongly inhibited by 2 mm-cyanate. This inhibition could be specifically reversed by arginine. Citrulline (but not ornithine, N-α-acetylornithine or N-acetylglutamate) could also restore a normal growth rate. Since growth inhibition by cyanate was followed by an accumulation of ornithine within the cell it was concluded that cyanate specifically inhibits the formation of citrulline from ornithine. The effect of cyanate on the growth of defined strains was consistent with a specific inhibition of carbamoylphosphate synthase. A kinetic study of carbamoylphosphate synthase and ornithine carbamoyltransferase in vitro supported this conclusion. Since carbamoylphosphate is probably the only source of endogenous cyanate it is postulated that carbamoylphosphate synthase activity can be regulated by cyanate resulting from the dissociation of carbamoylphosphate in metabolic circumstances leading to its overproduction.
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Effects of Moenomycin on Escherichia coli
More LessThe antibiotic moenomycin is a valuable biochemical tool for studying the metabolism of peptidoglycan and the autolytic system in Escherichia coli, since as a specific inhibitor of peptidoglycan polymerases it can efficiently promote cell lysis. In liquid media the bacteriolytic effect on E. coli K12 was dependent on the concentration of moenomycin, on growth phase and on growth rate. Before lysis cells underwent major morphological alterations. In sucrose-containing medium complete transformation to osmotically sensitive spheroplasts was easily achieved by addition of moenomycin. The minimum inhibitory concentration of the antibiotic varied with the strain of E. coli and was highly dependent on the growth medium. A tritiated derivative of moenomycin, [3H]decahydromoenomycin A, was prepared and found to have the same inhibiting efficiency. Its binding to E. coli membranes and membrane proteins was investigated. The absence of irreversible binding suggested that moenomycin might be a competitive inhibitor of the peptidoglycan polymerases. Spontaneous moenomycin resistant variants were isolated at a frequency of about 10−9.
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Regulation of Chlamydia psittaci (Strain Guinea Pig Inclusion Conjunctivitis) Growth in McCoy Cells by Amino Acid Antagonism
More LessChlamydiae have amino acid requirements for growth in tissue culture as defined by those amino acids whose individual omission from the growth medium prevents chlamydial multiplication. We have tested the hypothesis that this inhibition of growth arises as a result of antagonism between particular amino acids such that inhibition occurs when the concentration of one amino acid is reduced in the presence of the antagonist amino acid at high concentration. Using the Chlamydia psittaci strain guinea pig inclusion conjunctivitis (GPIC), in the presence of cycloheximide, the requirement for valine was abrogated by the simultaneous omission of isoleucine, that for phenylalanine by simultaneous omission of tryptophan and that for leucine by simultaneous omission of isoleucine plus valine. The antagonism shown between leucine and isoleucine plus valine appears to be unique among bacteria. In the absence of cycloheximide, GPIC had an additional need for tryptophan, tyrosine and isoleucine; these amino acid requirements were shown for both infected McCoy, HeLa and BHK cells. The results are consistent with a mechanism for regulation of parasite growth which depends on the balance of amino acid concentrations in the extracellular environment.
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The Uptake and Cellular Distribution of Zinc in Saccharomyces cerevisiae
More LessZn2+ uptake by Saccharomyces cerevisiae was biphasic. The first phase was independent of metabolic energy, consisting of adsorption to the cell surface, and followed a Freundlich isotherm. The second phase was dependent on metabolic energy, ATPase activity and the transmembrane proton gradient, and consisted of uptake into the cell. Energy-dependent uptake showed Michaelis-Menten kinetics with a K m of 3·7μm-Zn2+ and a V max of 1·6 nmol min-1 per 107 cells at Zn2+ concentrations below 80 μm but deviated at higher concentrations. K+ and Mg2+ inhibited energy-dependent Zn2+ uptake while Na+ and Ca2+ did not. The effect of heavy metals was complex and included both inhibition and stimulation of Zn2+ uptake. K+ efflux accompanied Zn2+ uptake at all Zn2+ concentrations but there was no simple stoichiometric relationship between the two. Toxic effects of Zn2+ such as inhibition of H+ efflux and K+ uptake and reduction of viability were observed at all Zn2+ concentrations and toxicity appeared to be a major factor in K+ efflux. Toxicity also affected the kinetics of Zn2+ uptake, being a major cause of deviation from Michaelis-Menten kinetics. Zn2+ was compartmented within the cell: 56 % of the total intracellular pool was in the soluble vacuolar fraction, 39 % was bound to insoluble components and only 5 % was found in the cytosol. Isolated yeast vacuoles possessed an ATP-dependent Zn2+ uptake system whose properties were consistent with a Zn2+/H+ antiport.
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Interaction of Non-lytic β-Lactams with Penicillin-binding Proteins in Streptococcus pneumoniae
More LessThe monobactam aztreonam and the cephalosporin ceftazidime, β-lactam antibiotics that possess the same side chain R1, showed unusual effects on exponentially growing pneumococci compared to other β-lactams. Both antibiotics did not induce lysis even at concentrations up to 2 mg ml−1, values well above the respective MICs. However, morphological alterations and growth inhibition of the cells were observed at much lower concentrations. Binding to penicillin-binding proteins (PBPs) in vitro could be monitored directly by using anti-aztreonam antiserum and the Western blot technique. Both antibiotics showed high affinity for PBP 3, but had an extremely low affinity for PBP 2b. It is suggested that the failure to bind to PBP 2b is responsible for the failure to induce lysis in pneumococci.
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Influence of the Culture Medium on the Production of Iturin A by Bacillus subtilis
More LessThe production of iturin A by Bacillus subtilis was studied with respect to the composition of the culture medium. Increasing phosphate concentrations did not modify the antibiotic yield. Fructose, sucrose and mannitol were better carbon sources than glucose for antibiotic production. The nature of the nitrogen source was an important factor in the production of antibiotic. Among the amino acids which are components of iturin A, l-asparagine was the best substrate for the biosynthesis of iturin A; l-glutamine and l-serine were rather poor substrates while l-proline and d-tyrosine gave no antibiotic. Ammonium salts permitted good synthesis of antibiotic but the addition of calcium ions to the culture medium inhibited the excretion of antibiotic from the cells.
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- Systematics
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DNA:DNA Hybridization Studies on the Pink-pigmented Facultative Methylotrophs
More LessThe genomic relatedness among 36 strains of pink-pigmented facultatively methylotrophic bacteria (PPFMs) was estimated by determination of DNA base composition and by DNA:DNA hybridization studies. A reproducible hybridization system was developed for the rapid analysis of multiple DNA samples. Results indicated that the PPFMs comprise four major and several minor homology groups, and that they should remain grouped in a single genus, Methylobacterium.
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Biochemical Characteristics and Fatty Acid Compositions of Some Armadillo-derived Mycobacteria and Their Relation to Mycobacterium gordonae
More LessThe long-chain components of 75 strains of mycobacteria, cultivated from Mycobacterium leprae-infected or non-infected armadillos, and of eight clinical and 15 environmental isolates of M. gordonae, were compared. Four major groups could be distinguished based on the presence of 10-methyloctadecanoic (tuberculostearic) and 2-methyl 3-hydroxyeicosanoic acids and secondary alcohols (2-octadecanol and 2-eicosanol). Some heterogeneity was found in strains assigned to M. gordonae: the characteristic absence of tuberculostearic acid and secondary alcohols and the presence of the branched C14 and the hydroxylated C20 acids were seen in only 34 of the 49 strains studied. Three strains were identified as M. malmoense, one as M. kansasii, ten as belonging to the M. avium–M. intracellulare–M. scrofulaceum complex and eight as belonging to new groups of armadillo-derived mycobacteria (ADM 1, ADM 2 and ADM 3) by conventional bacteriological tests and fatty acid compositions, though M. malmoense was heterogeneous in its fatty acids composition. Four strains, identified as M. avium by conventional tests, differed from this species by their fatty acid compositions. Thirteen strains showed some similarity to M. simiae and ten strains differed from all other known mycobacteria.
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- Corrigendum
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