- Volume 135, Issue 11, 1989
Volume 135, Issue 11, 1989
- Biochemistry
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Purification and Properties of the ATPase of the Halophilic and Alkaliphilic Phototrophic Bacterium Ectothiorhodospira halochloris
More LessThe membrane-bound ATPase of the halophilic and alkaliphilic phototrophic bacterium Ectothiorhodospira halochloris was solubilized by washing membranes with buffer of low ionic strength and purified by ion-exchange chromatography, ultrafiltration and gel filtration. The M r of the trypsin-activated enzyme as determined by gel chromatography was approximately 400000. Four subunits were found by SDS electrophoresis with apparent Mr values of 58000, 53700, 46800 and 36900. The purified enzyme was cold labile, but was more stable at room-temperature and in the presence of p-aminobenzamidine. Both the membrane-bound and the solubilized enzyme were inhibited by DCCD after preincubation, but not by oligomycin. The ATPase was activated by bicarbonate and sulphite. NaCl was inhibitory at low concentrations. The results are discussed with respect to optimum growth conditions for E. halochloris and its cytoplasmic solute concentrations. Responses of the enzyme to salts are compared to those of ATPases from two other halophilic bacteria.
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Occurrence and Chemistry of Cell Wall Teichoic Acids in the Genus Brevibacterium
F. Fiedler and A. BudeThe cell walls of Brevibacterium casei NCDO 2048, B. epidermidis NCDO 2286 and 14 B. linens isolates from cheese were shown to contain teichoic acids as the dominant non-peptidoglycan polymers. The variation in teichoic acid composition was similar to that described earlier for B. linens strains and for B. iodinum. In B. casei NCDO 2048 a glycerol teichoic acid was found, whereas in B. epidermidis NCDO 2286 glycerol teichoic acid existed together with mannitol teichoic acid. The occurrence of teichoic acids in the cell walls of all Brevibacterium strains investigated constitutes a biochemical characteristic of the genus and is of chemotaxonomic relevance. The co-existence of glycerol and mannitol within the cell walls of the B. linens strains AC480 and AC577 was investigated in detail. The polyols belonged to separate polymers: a glycerol teichoic acid and a mannitol teichoic acid.
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Glutathione Transferase in Bacteria: Subunit Composition and Antigenic Characterization
The presence of glutathione transferase (GST; EC 2.5.1.18) in Escherichia coli ATCC 25922, E. coli ATCC 25422, Proteus vulgaris ATCC 8427, Pseudomonas aeruginosa ATCC 27853, Klebsiella oxytoca CIP 666, K. oxytoca AF 101, Enterobacter cloacae CIP 6085, Serratia marcescens CIP 6755, and Proteus mirabilis AF 2924 was investigated. Using 1-chloro-2,4-dinitrobenzene as substrate, GST activity was found in the glutathione- (GSH-) affinity-purified fraction of all strains tested. SDS-PAGE analysis of GSH-affinity-purified enzyme indicated that the GSTs of all these bacteria are dimers of two identical subunits of M r about 22500. Rabbit antiserum directed against the major isoenzyme present in Proteus mirabilis AF 2924, Pm-GST-6·0, was used to investigate the antigenic properties of bacterial GSTs. Western blot analysis indicated that a GST antigenically identical to Pm-GST-6·0 is present in Enterobacter cloacae CIP 6085, Escherichia coli ATCC 25422 and Proteus vulgaris ATCC 8427, but absent in Escherichia coli ATCC 25922, Klebsiella oxytoca CIP 666, K. oxytoca AF 101 and Serratia marcescens CIP 6755. The presence of Pm-GST-6·0, but not mammalian GST, increased the MIC values of amikacin, ampicillin, cefotaxime, cephalothin and nalidixic acid for E. coli ATCC 25922. It is suggested that bacterial GST may represent a defence against the effects of antibiotics.
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Isolation and Characterization of Lectins from Rhizoctonia crocorum and Athelia rolfsii
More LessTwo new lectins were isolated from mycelium of Rhizoctonia crocorum and Athelia rolfsii by affinity chromatography on mucin-Sepharose. The Rhizoctonia crocorum lectin (RCL) was a tetramer of 11 kDa subunits whereas the Athelia rolfsii lectin (ARL) was a dimer of two 17 kDa subunits. Both lectins were rich in asparagine/aspartic acid, glutamine/glutamic acid, glycine, lysine and serine. In contrast to the previously described Rhizoctonia solani agglutinin (RSA), which exhibits specificity towards N-acetylgalactosamine, RCL and ARL had a complex specificity. Despite obvious differences between the three lectins, they are serologically related to each other.
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- Biotechnology
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The Segregation of the 2μ-based Yeast Plasmid pJDB248 Breaks Down under Conditions of Slow, Glucose-limited Growth
More LessThe stability of the 2μ-based yeast plasmid pJDB248 in Saccharomyces cerevisiae S150-2B(cir0) was investigated in glucose-limited chemostat culture. Plasmid-free cells were detected by loss of (plasmid-encoded) leucine prototrophy and confirmed by colony hybridization. The plasmid was considerably more stable at a high dilution rate (0·12 h−1) than at a lower dilution rate (0·05 h−1). The average plasmid copy number in the cells retaining the plasmid remained constant at approximately 50 in the high dilution rate culture whereas it rose to almost 600 in the slow dilution rate culture. However, in both cultures the overall plasmid level in the total population remained constant, indicating that plasmid segregation breaks down at the low growth rate. Similar experiments on the native 2μ plasmid demonstrated high stability and no significant differences between the high and low growth rate cultures. It is postulated that the difference in behaviour between the native and chimeric plasmids is related to an interaction between the growth conditions and the loss of the D gene product.
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- Development And Structure
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Ultrastructure of the Infectious and Reproductive Forms of Holospora obtusa, a Bacterium Infecting the Macronucleus of Paramecium caudatum
More LessThe ultrastructure of the infectious and reproductive forms of Holospora obtusa, a bacterium colonizing the macronucleus of Paramecium caudatum, was analysed with the aid of ultrathin sections, freeze-fracture, freeze-etching and cytochemical electron microscopical techniques. The infectious form differed considerably from the reproductive form. The bacterial cytoplasm of the infectious form was confined to less than half of the cell, the rest being occupied by a voluminous periplasmic space. The periplasmic space contained a highly osmiophilic fine granular material. At the end of the cell distal from the cytoplasm, a tip containing less osmiophilic fine granular material was observed. Freeze-fracture and freeze-etching studies revealed differences in the patterns of intramembranous particles between the two forms. It is suggested that some of the structures characterizing the infectious form have a function in the infection process.
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Coat and Enterotoxin-related Proteins in Clostridium perfringens Spores
More LessCoat proteins from mature spores of two enterotoxin-positive (Ent+) and two enterotoxin-negative (Ent+) strains of Clostridium perfringens were solubilized using 50 mm-dithiothreitol and 1% sodium dodecyl sulphate at pH 9·7, and alkylated using 110 mm-iodoacetamide to prevent aggregation. The coat proteins and C. perfringens type A enterotoxin (CPE) were separated by SDS-PAGE and analysed by Western blotting using anti-CPE antibody. As previously reported, CPE aggregated in the presence of SDS, but no aggregation occurred at concentrations below 15 μg CPE ml−. Two CPE-related proteins (34 and 48 kDa) were found in the solubilized spore coat protein of Ent+ strains while only the 48 kDa CPE-related protein was found in the spore coat fraction of Ent+ strains. CPE-related proteins comprised 2·7% and 0·8% of the total solubilized coat protein of Ent+ and Ent+ strains respectively. CPE-related proteins could be extracted from the spores with 1 % SDS alone. They could also be released by disruption of whole spores, indicating that the CPE-related proteins may be in the spore core or trapped between the core and coat layers. The results suggest that CPE is not a major structural component of the coat fraction of C. perfringens spores.
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- Genetics And Molecular Biology
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A Plasmid Which Can Be Transferred Between Escherichia coli and Pasteurella haemolytica by Electroporation and Conjugation
More LessThree broad-host-range plasmids (pRK290, pSa4 and pKT230) and one native Pasteurella haemolytica plasmid (pPH33) were used in transformation experiments with P. haemolytica strains T179 (serotype Al), Y216 (serotype A2)and its capsular-deficient variant Y216/NS1. No transformants were detected with either heat-shock or freeze-thaw techniques. However, by electroporation, all P. haemolytica strains were transformed by pPH33 but not by pRK290 or pSa4. The highest frequency obtained was 91 × 104 transformants per µg of pPH33 DNA with P. haemolytica strain Y216/NS1. Although pPH33 itself was non-transmissible by conjugation, it could be mobilized from Escherichia coli, using the transfer function of the IncP plasmid pRK2013, into P. haemolytica at a frequency of 0·3–2·2 × 10−3 per recipient cell.
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Characterization of Pseudomonas Mercury-resistance Transposon Tn502, Which Has a Preferred Insertion Site in RP1
More LessTn502mer differs in size and restriction map from the well-characterized Tn501mer. It also differs in its preferential and high-frequency insertion into the 6 kb PstI-C region of RP1. The affinity for this region is perpetuated in pVS76, a clone of RP1 PstI-C in pBR322. Restriction mapping of independent pVS76::Tn502 derivatives revealed that Tn502 inserted at the same site (or small region) in PstI-C corresponding to the 35 kb coordinate in RP1. Insertion occurred in both orientations, but one was preferred. When PstI-C was deleted from RP1, acquisition of Tn502 was reduced and the sites of insertion randomized.
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Cloning and Genetic Analysis of Six Pyrroloquinoline Quinone Biosynthesis Genes in Methylobacterium organophilum DSM 760
More LessAfter EMS mutagenesis, mutants of Methylobacterium organophilum DSM 760 unable to synthesize pyrroloquinoline quinone (PQQ) were selected among mutants which did not utilize methanol but were still able to use methylamine as growth substrate. Six different pqq genes (pqqA to pqqF) were identified by complementation analysis. The genes pqqA to pqqD, cloned in a single R′ plasmid, were grouped in a 3·9 kb DNA fragment. The genes pqqA and pqqB belonged to a single transcription unit independent from the adjacent gene pqqC. The gene pqqD was contained in a short DNA segment of approximately 0·1 kb, separated from pqqC by a region with no apparent role in PQQ biosynthesis. Two other genes were identified:pqqE, which was closely linked to pqqD; and pqqF, located approximately 19 kb from the other genes. Directed mutagenesis by marker exchange provided chromosomal insertion mutations of these genes in M. organophilum. Attempts to express the pqq genes in two heterologous hosts, Escherichia coli and Pseudomonas testosteroni, were unsuccessful, and no plasmid containing all of the pqq genes was isolated.
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Chromosomal Location of the Bacillus subtilis Aspartokinase II Gene and Nucleotide Sequence of the Adjacent Genes Homologous to uvrC and trx of Escherichia coli
More LessThe aspartokinase II(ask) operon of Bacillus subtilis consists of two in-phase overlapping genes that encode the two subunits of the lysine-sensitive isoenzyme of aspartokinase (ATP:l-aspartate 4-phosphotransferase, EC 2.7.2.4). Transduction mapping of the ask operon, inactivated by recombinational insertion of a cat marker, indicates a chromosomal location (about 253°) between leuA and aroG. ask is thus remote from aecB, eliminating aecB as a possible locus for the structural gene of aspartokinase II, but close to aecA and uvrB. The nucleotide sequence of a 2 kb DNA fragment just upstream of the ask operon was determined and found to contain two open reading frames. The deduced amino acid sequence of the distal reading frame exhibits extensive homology with Escherichia coli thioredoxin and that of the proximal one, which overlaps with the ask promoter, is homologous to the deduced product of the E. coli uvrC gene. Insertional mutagenesis of the proximal open reading frame led to a mitomycin-sensitive phenotype, consistent with a role in DNA repair. In conjunction with the data of M. Petricek, L. Rutberg & L. Hederstedt [FEMS Microbiology Letters 61, 85–88] our results define the nucleotide sequence of an 8·8 kb segment of the B. subtilis chromosome near 253 ° and the following order of genes: trx-uvrB-ask-orfX-sdhC-sdhA-sdhB-orfY-gerE.
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A Novel Phage Genome Integrated into a Plasmid in Bacillus thuringiensis Strain AF101
More LessBacillus thuringiensis strain AF101 possesses a single plasmid (pAF101) with a molecular size of 42 MDa (69 kb). During plasmid curing experiments in strain AF101, we found that a phage (J7W-1) was induced by ethidium bromide treatment. Moreover, the phage genome (48 kb) hybridized only with pAF101 on a Southern blot of the DNA of a cleared lysate prepared from strain AF101. Comparison of the restriction patterns of pAF101 and J7W-1 phage DNA revealed that pAF101 contains not only the entire phage DNA but also a plasmid-specific DNA region. These results indicate that the J7W-1 genome has been stably integrated into pAF101 in strain AF101. Integration of the J7W-1 genome into a plasmid was also observed after phage infection of the type strain of B. thuringiensis subsp. israelensis.
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The entD Gene of the Escherichia coli K12 Enterobactin Gene Cluster
More LessThe Escherichia coli entD gene encodes a product necessary for the synthesis of the iron-chelating and transport molecule enterobactin (Ent); cells harbouring entD mutations fail to grow in iron-deficient environments. For unknown reasons, it has not been possible to identify the entD product. The nucleotide sequence of the entD region has now been determined. An open reading frame extending in the same direction as the adjacent fepA gene and capable of encoding an approximately 24 kDa polypeptide was found; it contained a high percentage of rare codons and two possible translational start sites. Complementation data suggested that EntD proteins truncated at the carboxy terminus retain some activity. Two REP sequences were present upstream of entD and an IS186 sequence was observed downstream. RNA dot-blot hybridizations demonstrated that entD is transcribed from the strand predicted by the sequencing results. An entD-lacZ recombinant plasmid was constructed and shown to express low amounts of a fusion protein of the anticipated size (approximately 125 kDa). The evidence suggests a number of possible explanations for difficulties in detecting the entD product. Sequence data indicate that if entD has its own promoter, it is weak; the REP sequences suggest that entD mRNA may be destabilized; and translation may be slow because of the frequency of rare codons and a possible unusual start codon (UUG). The data are also consistent with previous evidence that the entD product is unstable.
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The Location of a Temperature-sensitive trans-Dominant Mutation and Its Effect on Restriction and Modification in Escherichia coli K12
More LessAn Escherichia coli K12 chromosomal EcoRI-BamHI fragment containing a mutant hsdS locus was cloned into plasmid pBR322. The mcrB gene, closely linked to hsdS, was used for selection of clones with the inserted fragment using T4αgt57βgt14 and λvir. PvuII phages; the phage DNAs contain methylated cytosines and hence can be used to demonstrate McrB restriction. For the efficient expression of the hsdS gene, a BglII fragment of phage λ carrying the pR promoter was inserted into the BamHI site of the hybrid plasmid. Under these conditions a trans-dominant effect of the hsdX ts +d mutation on restriction and modification was detected. Inactivation of the hsdS gene by the insertion of the λ phage BglII fragment into the BglII site within this gene resulted in the disappearance of the trans-dominant effect. When the cloned BamHI-EcoRI fragment was shortened by HpaI and EcoRI restriction enzymes, the trans-dominant effect was fully expressed. The results indicate that the X ts +d mutation is located in the hsdS gene. The effect of gene dosage of the HsdS subunit on the expression of X ts +d mutation was studied. The results of complementation experiments, using F′-merodiploids or plasmid pBR322 with an inserted X ts +d mutation, support the idea that the HsdSts +d product competes with the wild-type HsdS product, and has a quantitatively different effect on restriction and modification.
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Transposon-916-like Elements in Clinical Isolates of Enterococcus faecium
More LessTetracycline (Tc) resistance was found in nine out of ten clinical isolates of Enterococcus faecium. Conjugative transposons, designated Tn5031, Tn5032 and Tn5033, were present in the chromosome of three isolates. The transposons were similar both structurally and functionally to Tn916 containing the tetM determinant. A large non-conjugative plasmid found in a fourth isolate contained an element homologous to Tn916. The four isolates containing the element showing homology to Tn916 exhibited a substantially higher level of Tc resistance than the remaining five Tc-resistant isolates. Tc-resistance genes which have not been identified are apparently responsible for the low-level Tc resistance in five clinical isolates.
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Cloning, Sequencing and Expression of a Sialidase Gene from Clostridium sordellii G12
More LessA 4·3 kb XbaI restriction fragment of DNA from Clostridium sordellii G12 hybridized with a synthetic oligonucleotide representing the N-terminus of the sialidase protein secreted by C. sordellii. This cloned fragment was shown to encode only part of the sialidase protein. The sialidase gene of C. sordellii was completed by a 0·7 kb RsaI restriction fragment overlapping one end of the XbaI fragment. After combining the two fragments and transformation of Escherichia coli, a clone that expressed sialidase was obtained. The nucleotide sequence of the sialidase gene of C. sordellii G12 was determined. The sequence of the 18 N-terminal amino acids of the purified extracellular enzyme perfectly matched the predicted amino acid sequence near the beginning of the structural gene. The amino acid sequence derived from the complete gene corresponds to a protein with a molecular mass of 44735 Da. Upstream from the putative ATG initiation codon, ribosomal-binding site and promoter-like consensus sequences were found. The encoded protein has a leader sequence of 27 amino acids. The enzyme expressed in E. coli has similar properties to the enzyme isolated from C. sordellii, except for small differences in size and isoelectric point. Significant homology (70%) was found with a sialidase gene from C. perfringens.
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Molecular Cloning and Expression in Escherichia coli of the recA Gene of Legionella pneumophila
More LessInterspecific complementation of an Escherichia coli recA mutant with a Legionella pneumophila genomic library was used to identify a recombinant plasmid encoding the L. pneumophila recA gene. Recombinant E. coli strains harbouring the L. pneumophila recA gene were isolated by replica-plating bacterial colonies on medium containing methyl methanesulphonate (MMS). MMS-resistant clones were identified as encoding the L. pneumophila recA analogue by their ability to protect E. coli HB101 from UV exposure and promote homologous recombination. Subcloning of selected restriction fragments and Tn5 mutagenesis localized the recA gene to a 1·7 kb BglII-EcoRI fragment. Analysis of minicell preparations harbouring a 1·9 kb EcoRI fragment containing the recA coding segment revealed a single 37·5 kDa protein. Insertional inactivation of the cloned recA gene by Tn5 resulted in the disappearance of the 37·5 kDa protein, concomitant with the loss of RecA function. The L. pneumophila recA gene product did not promote induction of a λ lysogen; instead, the presence of the heterologous recA gene caused a significant reduction in spontaneous and mitomycin-C-induced prophage induction in recA + and recA E. coli backgrounds. Despite the lack of significant genetic homology between the L. pneumophila recA gene and the E. coli counterpart, the L. pneumophila RecA protein was nearly identical to that of E. coli in molecular mass, and the two proteins showed antigenic cross-reactivity. Western blot analysis of UV-treated L. pneumophila revealed a significant increase in RecA antigen in irradiated versus control cells, suggesting that the L. pneumophila recA gene is regulated in a manner similar to that of E. coli recA.
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- Physiology And Growth
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D-Xylose Utilization by Saccharomyces cerevisiae
More LessSummary: Although it is generally accepted that Saccharomyces cerevisiae is unable to assimilate d-xylose, four strains were found to utilize xylose aerobically at different efficiencies in the presence of a mixture of substrates. The degree of d-xylose utilization by S. cerevisiae ATCC 26602 depended upon the presence of other substrates or yeast extract. The greatest amount of xylose (up to 69% over 7 d) was utilized when sugar substrates such as d-ribose were co-metabolized. Much lower degrees of utilization occurred with co-metabolism of organic acids, polyols or ethanol. A mixture of d-glucose, d-ribose, d-raffinose, glycerol and d-xylose resulted in greater xylose utilization than the presence of a single substrate and xylose. The absence of growth on a co-substrate alone did not prevent the utilization of xylose in its presence. Xylose was co-metabolized with ribose under anaerobic conditions but at a much slower rate than under aerobic conditions. When [14C]xylose was utilized in the presence of ribose under anaerobic conditions, the radioactive label was detected mainly in xylitol and not in the small amounts of ethanol produced. Under aerobic conditions the radioactive label was distributed between xylitol (91·3 α 0·8%), CO2 (2·6 α 2·3%) and biomass (1·7 α 0·6%). No other metabolic products were detected. Whereas most xylose was dissimilated rather than assimilated by S. cerevisiae, the organism apparently possesses a pathway which completely oxidizes xylose in the presence of another substrate.
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The Role of Hydrogen Peroxide in the Degradation of Crystalline Cellulose by Basidiomycete Fungi
More LessSummary: Extracellular hydrogen peroxide has in the past been implicated in the degradation of the crystalline cellulose component of plant cell walls, particularly by brown-rot fungi. Using assays sensitive to 1 μg peroxide ml−1 no evidence could be found of extracellular hydrogen peroxide in culture media of several basidiomycete fungi examined, although some free radicals may have been produced on the surface of the fungal hyphae. Analysis of the products of enzymic degradation of crystalline cellulose by gas chromatography/mass spectroscopy revealed a range of oxidized sugars which most probably resulted from the action of peroxide free radicals derived from hydrogen peroxide.
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Invertase Secretion by Phycomyces blakesleeanus: Regulation by Carbon Catabolite Repression and by the pH of the Growth Medium
More LessSummary: Carbon catabolite repression was studied in secreted (invertase) and mitochondrial (isocitrate dehydrogenase) enzymes of Phycomyces blakesleeanus. Both enzymes were subject to repression by glucose when the fungus was grown at pH 4·5. Repression of invertase but not of isocitrate dehydrogenase was overcome when xylose was added to the growth medium together with glucose. Derepression of invertase activity was obtained when the fungus was grown in a medium with glucose and at pH 7·3. This pH-dependent derepression was not due to enzyme redistribution or stimulation of activity. In contrast, isocitrate dehydrogenase activity was not derepressed at pH 7·3, but was significantly reduced either in the presence or absence of glucose.
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Sporulation of a Bacitracin-sensitive Mutant of Bacillus licheniformis Is Self-inhibited by Bacitracin
More LessA mutant of Bacillus licheniformis (BLU166) sensitive to its own antibiotic bacitracin was isolated and the mutation ber-1 was mapped close to the bacitracin synthetase genes. The sensitivity was shown to be specific for bacitracin. Two further bacitracin-sensitive strains were constructed, one (BLU171) with normal ability to synthesize bacitracin, and one (BLU170) a bacitracin non-producer. In addition to an increased sensitivity of growing cells to bacitracin, sporulation of the mutant strain BLU171 was self-inhibited by bacitracin. It is concluded that (1) there might exist at least two levels of resistance to bacitracin; (2) mutation ber-1 affects a ‘structural’ component, which may protect the sensitive reaction of cell-wall biosynthesis; (3) sporulation is affected to a greater extent by bacitracin than vegetative growth; and (4) synthesis of bacitracin is independent of the presence of this resistance mechanism since the sensitive mutant produces similar amounts of the antibiotic to the wild-type strain.
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Bacterial Ethylene Synthesis from 2-Oxo-4-thiobutyric Acid and from Methionine
More LessThe ability of selected bacterial cultures to synthesize ethylene during growth in nutrient broth supplemented with methionine or 2-oxo-4-methylthiobutyric acid (KMBA) was examined. Although most cultures transformed KMBA into ethylene, only those of Escherichia coli SPAO and Chromobacterium violaceum were able to convert exogenously added methionine to ethylene. In chemically defined media, E. coli SPAO produced the highest amounts of ethylene from methionine and KMBA. This capability was affected by the nature of the carbon source and the type and amount of nitrogen source used for growth. When glutamate was used as sole source of carbon and nitrogen for growth, the activity of the ethylenogenic enzymes was reduced to 25% of that observed with cultures grown with glucose and NH4Cl. Neither methionine nor KMBA significantly affected the ethylenogenic capacity of E. coli SPAO. Menadione and paraquat, compounds that generate superoxide radicals, stimulated ethylene synthesis by harvested cells, but not by cell-free extracts of E. coli SPAO. In addition, cells of Pseudomonas aeruginosa, which produced no ethylene in culture in the presence of exogenously added KMBA, yet possessed the necessary enzymes in an active form, were able to synthesize ethylene from KMBA when incubated with menadione or paraquat.
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1,3-β-Glucanase, 1,6-β-GIucanase and β-Glucosidase Activities of Sclerotium glucanicum: Synthesis and Properties
More LessSummary: The filamentous fungus Sclerotium glucanicum excreted significant amounts of 1,3-β-glucanase, 1,6-β-glucanase and --glucosidase activities when the culture medium was depleted of carbon sources. During starvation small amounts of intracellular 1,3-β- and 1,6-β-glucanase and β-glucosidase activities were also detected. Very low levels of -glucanase activity remained bound to mycelium and some activity was found loosely attached to the cells and/or to water-soluble 1,3-β-1,6-β-glucan adhering to the cell walls. During active growth intracellular 1,3-β-glucanase and mycelium-bound 1,3-β- and 1,6-β-glucanase activities were detected in small or trace amounts. During hyphal growth very low levels of 1,3-β- and 1,6-β-glucanase activities were also found to be weakly associated with the cells and/or with water-soluble -glucan covering the hyphae. Cycloheximide inhibited the increase in intra- and extracellular 1,3-β- and 1,6-β-glucanase and -glucosidase activities. This indicated that de novo protein synthesis was involved in the intra- and extracellular appearance of these three enzyme activities in derepressed cells. The formation of the extracellular 1,3-β-glucanase, 1,6-β-glucanase and -glucosidase activities was regulated by catabolite repression. 1,3-β- and 1,6-β-glucanase activities were uncompetitively inhibited and -glucosidase activity noncompetitively inhibited by glucose and glucono-δ-lactone. Optimum pH and temperature values as well as thermal stabilities of the three extracellular enzyme activities were determined. Almost all of the -glucosidase activity but only one-third of the extracellular 1,3-β- and 1,6-β-glucanase activities were found to bind to Con A-Sepharose. Under conditions of carbon limitation almost 90% of the extracellular 1,3-β-/1,6-β-glucan excreted during fungal growth was degraded by the extracellular -glucanases.
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The Regulation of Exopolysaccharide Production and of Enzymes Involved in C1 Assimilation in Methylophilus methylotrophus
More LessSummary: Methylophilus methylotrophus produced viscous and non-viscous exopolysaccharides (EPS) when grown in batch culture. Both types contained glucose, galactose, mannose and an unidentified 6-desoxyhexose, and were substituted with pyruvate and acetate residues. When the organism was grown in continuous culture only the non-viscous EPS was synthesized; the rate of production was 18·5 mg h−1 (g biomass)−1 in methanol-limited cultures and increased by approximately 3- and 4-fold when growth was limited by oxygen or nitrogen respectively. The specific activity of methanol dehydrogenase in cell extracts was relatively low when bacteria were grown under conditions of methanol excess and increased 2-fold in carbon-limited cells, reflecting the need to scavenge the small amounts of available methanol. In contrast, the specific activities of several key enzymes of the ribulose monophosphate (RuMP) pathway were greater in cells grown under conditions of nitrogen or oxygen limitation than when growth was Limited by the availability of carbon, indicating the potential for increased carbon flux round the cycle when excess methanol was present in the growth medium. When methylotrophs are grown under conditions of methanol excess it is important that there is a mechanism to prevent the overproduction of formaldehyde, and we suggest that these changes in EPS production and in the specific activities of the key enzymes of the RuMP cycle are necessary for the efficient removal of this toxic metabolite of methanol.
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Methanol Metabolism in Corynebacterium sp. XG, a Facultatively Methylotrophic Strain
More LessSummary: Corynebacterium sp. XG is a facultative methylotroph. Determination of the activities of several enzymes and use of mutants unable to grow on methanol indicated that in this Gram-positive strain methanol was metabolized through the serine pathway. Synthesis of the enzymes of this pathway was induced when cells were grown in the presence of methanol or its catabolic products.
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Control of Carbon Flux to Acetate Excretion During Growth of Escherichia coli in Batch and Continuous Cultures
More LessDuring growth of Escherichia coli ML308 on pyruvate in a continuous culture (turbidostat) or batch culture, flux of carbon into the cells exceeds the amphibolic capacity of the central pathways. This is balanced by diversion of carbon flux to acetate excretion which in turn diminishes the efficiency of carbon conversion to biomass [g] dry wt (mol substrate)−1]. However, restriction of carbon supply in a chemostat diminishes flux to acetate excretion and at a dilution rate (D = µ) of 0·35 h−1 or less, no flux to acetate excretion was sustained thus permitting perfect balance between carbon input on the one hand, and the output to biosynthesis and energy generation on the other. This, in turn, improves the efficiency of carbon conversion to biomass. Inclusion of 3-bromopyruvate (an inhibitor of pyruvate dehydrogenase) at a concentration which diminishes growth rate (µ) to 0·35 h−1 or less also prevented flux to acetate excretion. Furthermore, in a family of fluoroacetate-resistant strains, excessive flux of pyruvate was balanced by diversion of carbon flux to lactate excretion rather than acetate and a higher growth rate (µ = 0·63 h−1) was sustained.
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Binding-protein-dependent Sugar Transport by Agrobacterium radiobacter and A. tumefaciens Grown in Continuous Culture
More LessBinding-protein-dependent sugar transport has been investigated in Agrobacterium radiobacter and A. tumefaciens. A. radiobacter contained two high-affinity glucose-binding proteins (GBP1 and GBP2) that additionally bound d-galactose (K D 0·26 μm) and d-xylose (K D 0·04 μm) respectively and were involved in the transport of these sugars. Partial sequencing of GBP1 and GBP2 showed that GBP2 exhibited significant homology with both the arabinose-binding protein (ABP) and the galactose-binding protein (GalBP) from Escherichia coli, whereas GBP1 exhibited significant homology only with ABP. Antiserum raised against GBP1 cross-reacted with GBP1 but not with GBP2, and vice versa. Anti-GBP1 and anti-GBP2 also cross-reacted with proteins corresponding to GBP1 and GBP2 respectively in A. tumefaciens, but little or no cross-reaction was observed with selected members of the Enterobacteriaceae, Rhizobiaceae and Pseudomonadaceae families grown under glucose limitation. GBP1 was less strongly repressed than GBP2 following batch growth of A. radiobacter on various carbon sources. The growth of A. radiobacter for more than approximately 10 generations in continuous culture under galactose or xylose limitation (D 0·045 h−1) led to the emergence of new strains which exhibited increased rates of glucose/galactose or glucose/xylose uptake, and which respectively hyper-produced GBP1 (strain AR18a) or GBP2 (strain AR9a). Similarly, growth of A. tumefaciens for more than approximately 15 generations under glucose or galactose limitation produced new strains which exhibited increased rates of glucose/xylose or glucose/galactose uptake and which respectively hyperproduced proteins analogous to GBP2 (strain AT9) or GBP1 (strain AT18a). It is concluded that growth of Agrobacterium species under carbon-limited conditions leads to the predictable emergence of new strains which specifically hyperproduce the transport system for the limiting nutrient. The GBP1-dependent system of A. radiobacter is unique amongst these transport systems in that the mutations that lead to its hyperproduction under carbon limitation render it least susceptible to repression by excess glucose during ammonia limitation, with the result that succinoglucan exopolysaccharide is produced from glucose at an enhanced rate.
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Cross-linking and O-Acetylation of Newly Synthesized Peptidoglycan in Staphylococcus aureus H
More LessStaphylococcus aureus H growing exponentially was labelled with N-acetyl[14C]glucosamine, which became incorporated into the peptidoglycan. The portion of peptidoglycan not linked to teichoic acid (60–75% of the whole) was degraded with Chalaropsis muramidase to yield disaccharide-peptide monomers and dimers, trimers and oligomers formed by biosynthetic cross-linking of the monomers. The degree of O-acetylation of these fragments was also examined. Pulse-chase experiments showed that the proportion of label initially in the monomer fraction immediately after the 1 min pulse declined rapidly during a 3 min chase, while the oligomer fraction (fragments greater than trimer) gained the radioactivity proportionately. The radioactivity of the dimer and trimer fractions remained virtually unchanged. At 4 min after the commencement of labelling (i.e. approx. one-tenth of a generation time) final values had been reached. The O-acetylation of all fragments had achieved final values even at 1 min, except for the monomer fraction, which showed an increase from 40% to 60% during the first 3 min of chase. Although O-acetylation was clearly a very rapid process, no O-acetylated peptidoglycan lipid-intermediates could be detected.
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Early Cell Envelope Alterations by Tobramycin Associated with its Lethal Action on Pseudomonas aeruginosa
More LessThe immediate activities of the aminoglycoside antibiotic tobramycin were investigated in Pseudomonas aeruginosa PAO1. The lethal action of a low concentration of tobramycin (8 μgml−1) occurred rapidly (1–3 min) and was associated with leakage of certain cellular components into the supernatant. The presence of magnesium at the time of initial exposure protected cells by preventing uptake of tobramycin; however, magnesium addition following a brief exposure did not restore viability. Analyses of supernatant material revealed a rapid 2-fold increase in protein released following tobramycin treatment. A prominent 29 kDa protein, observed by SDS-PAGE in the released material was identified as the periplasmic β-lactamase. Brief exposure to tobramycin did not result in major morphological damage or cell lysis as observed by transmission electron microscopy, and release of LPS was not a primary event. Although activity at the ribosomal level was observed by 2–3 min, leakage was detected after only 1 min. These data indicate that leakage of cellular components, particularly β-lactamase, occurs simultaneously, if not prior to inhibition of protein synthesis by tobramycin.
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A Morphology Index for Characterization of Cell Shape in Candida albicans
More LessThe morphology of Candida albicans cells was determined from their maximum length, maximum diameter and septal diameter in a mathematical ratio, the morphology index (Mi), which usually ranged from approximately 1 for spherical yeast cells to approximately 4 for true hyphae, with elongated yeast cells and pseudohyphae giving intermediate values. Mi could be determined with high reproducibility for C. albicans grown in a variety of environments. The highest mean Mi was seen with cells grown in serum and Eagle's medium at 37°C, the lowest with cells grown in Sabouraud glucose broth at 26°C. Variant strains of C. albicans gave Mi values that remained constant in a variety of growth environments. The Mi facilitated detection of two variants that grew exclusively in the yeast form, one that grew as elongated yeasts but could be induced to form pseudohyphae in serum, and one consistently pseudohyphal variant. Cells with a mean Mi up to 2·5 could be easily separated at septal junctions by mild ultrasonication, whereas cells with a mean Mi greater than 3·5 tended not to separate under these conditions. The chitin content of C. albicans cells was almost twice as great in cells with a Mi approaching 4 as in cells with a Mi close to 1. The wide range of Mi distributions for a single C. albicans isolate in different environments demonstrates that the fungus does not undergo abrupt changes of morphological phase: rather there are continual changes in morphology between spherical yeasts and true hyphae at the extremes. The study shows that Mi can be used reliably in place of subjective descriptions of morphology to indicate the shape of a C. albicans cell. It should facilitate the detection of molecular and cellular markers specific for morphogenesis in the fungus.
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Physiology of Amidase Production by Methylophilus methylotrophus: Isolation of Hyperactive Strains Using Continuous Culture
More LessThe obligately methylotrophic bacterium Methylophilus methylotrophus hydrolyses aliphatic amides to ammonia and aliphatic acid using a cytoplasmic amidase. Physiological regulation of amidase activity was investigated by growing the organism under various conditions in batch, fed-batch and continuous culture. The results showed that synthesis of the enzyme was induced by various amides (acrylamide > acetamide) and repressed by ammonia. Growth of the wild-type organism in acetamide-limited continuous culture at very low dilution rate (D 0·025 h−1) led to the selection of a hyperactive strain (MM6), the subsequent growth of which under acrylamide limitation led to the selection of another strain (MM8) which showed even higher activity. The amidase activities of strains MM6 and MM8 were respectively approximately four and twelve times higher than that of the wild-type organism following growth under similar conditions, whereas the concentrations of the enzyme as determined by SDS-PAGE and scanning densitometry were approximately four times higher than the wild-type organism in both strains. The amidase in strain MM8 exhibited a K m for acrylamide that was approximately one-third lower than that of the wild-type organism or of strain MM6. It is concluded that the hyperactivity of strain MM6 was due predominantly to the production of more wild-type enzyme, whilst the hyperactivity of strain MM8 was due to the production of approximately the same amount of enzyme as strain MM6 (up to 25% of the total cell protein depending on the nature of the limiting amide) but with a substantially enhanced catalytic activity (K cat). These changes were apparently the result of spontaneous mutations that occurred in response to growth at extremely low amide concentrations, giving the novel strains a strong selective advantage under these conditions (possibly by enhancing the rate of diffusion of amide into the cell).
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Uptake of Metabolites by Bacteroid-containing Vesicles and by Free Bacteroids from French Bean Nodules
G. Herrada, A. Puppo and J. RigaudThe use of a continuous Percoll gradient in a procedure for the rapid isolation of bacteroid- containing vesicles from French bean nodules is described. The purified vesicles appeared to be free from contamination by naked bacteroids and plant mitochondria and suitable for physiological studies. The metabolite uptake activities of vesicles isolated by this method were compared with those of free bacteroids. Succinate was transported through both peribacteroid and bacteroid membranes: the K m, values were 320μm. and 57μm, respectively. For glucose K m, values were 142μm. for the peribacteroid membrane and 102μm. for the bacteroid membrane, indicating that glucose could act as an energy-yielding substrate in functioning nodules. Experiments with inhibitors showed the involvement of ATPases in these transport processes and suggested that a proton-motive force was probably associated with them. The regulatory role of the peribacteroid membrane in the movement of the metabolites from the plant cytosol to bacteroids was demonstrated by its impermeability to glutamate, aspartate, citrate and benzoate.
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- Plant-Microbe Interactions
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Wheat Root Tips as a Vector for Passive Vertical Transfer of Azospirillum brasilense Cd
More LessThe ability of wheat root tips to serve as efficient vertical vectors for passive transfer of Azospirillum brasilense Cd was evaluated in sterilized growth chambers containing agar, sand or soil. Most root tips, whether from main or lateral roots, were colonized by A. brasilense Cd and were capable of transferring this bacterium to a depth of 290 mm from the inoculation site. The location of A. brasilense Cd was directly dependent on root tip location; whenever root tips passed through a bacterial layer, regardless of depth, they became inoculated. However, when root tips failed to reach the inoculation site they were not colonized by A. brasilense Cd. Seed inoculation resulted in an even distribution of A. brasilense Cd along the entire root system. Inoculation at various depths in the growth medium resulted in an uneven bacterial distribution and bacteria were concentrated mainly in the elongation and small root-hair zones as well as in the inoculation site. A. brasilense Cd multiplied on the root tip during its vertical movement. However, bacterial movement in the root vicinity was minimal. A. brasilense Cd did not spread from the root surface to the rhizosphere when exogenous nutrients were supplied in this area, but did so in the presence of chemoattractants. It is suggested that the prevalence of A. brasilense Cd deeper than the initial inoculation site is a result of their passive transfer by the growing root tip.
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Phage Induction of Lysogenic Rhizobium leguminosarum biovar trifolii in both the Free-living and the Symbiotic Form
More LessA lysogenic strain, Rhizobium leguminosarum biovar trifolii UK-1: φU, was isolated from a wild white clover nodule. It was symbiotically effective on white clover. A lysogenic phage (U-mole) was induced from this strain by treatment with either UV irradiation or mitomycin C. Phage U-mole had an icosahedral head (40 nm wide), a short tail (9 nm long) and tail fibres (15 nm long). Phage U-mole was induced with mitomycin C both from bacterial cells in infection threads and from bacteroids in nodules. Southern hybridization using EcoRI fragments of phage U-mole as probe indicated that phage U-mole DNA was integrated into the chromosome of strain UK-1: øU at a site involving the 60 kb EcoRI fragment of phage U-mole. Phage U-mole also lysogenized the wild-type strain R. leguminosarum biovar trifolii 4S. The resulting lysogenized strain, 4S:øU, had lost its 315 kb Sym plasmid and its nodulation ability.
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- Systematics
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A Reappraisal of the Terverticillate Penicillia Using Biochemical, Physiological and Morphological Features I. Numerical Taxonomy
Three-hundred-and-forty-eight strains representing the major species of terverticillate penicillia, and including representatives of other closely and distantly related species, were included in a numerical taxonomic study. One-hundred characters were derived from morphological features, physiological and biochemical activities and SEM micrographs. Strains were compared by both Gower’s coefficient and Pattern difference, and clustered using the average linkage algorithm. Thirty-seven species or species-complex clusters were recovered at approximately 70% similarity; they generally corresponded to existing taxonomic concepts. Several species were shown to contain variants or chemotypes which were often supported by differences in conidial shape and ornamentation. The use of different types of characters enabled a number of new and previously accepted species to be shown to be either variants or deteriorated examples of other species. Variation in properties both between and within species was considered, particularly in relation to strain stability.
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A Reappraisal of the Terverticillate Penicillia Using Biochemical, Physiological and Morphological Features II. Identification
More LessThe data from an integrated numerical classification was used to construct identification schemes for some fasciculate penicillia. The identification schemes were presented as a synoptic key and a frequency matrix for computer-assisted identification. Statistical testing of the frequency matrix showed that although character separation values were generally low, only four pairs of taxa showed overlap greater than that expected for a rectangular distribution. The identification schemes were tested practically with 52 previously studied strains and 51 further cultures. A synoptic key based on 10 and 90% cutoff limits was used to correctly identify 44 of the 51 additional strains, although this proved very sensitive to single test discrepancies. The frequency matrix was used to correctly identify 45 of the additional strains with a Willcox probability score and this was compared to identifications based on the modal likelihood fraction.
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A Reappraisal of the Terverticillate Penicillia Using Biochemical, Physiological and Morphological Features III. An Evaluation of Pectinase and Amylase Isoenzymes for Species Characterization
More LessPolyacrylamide gel electrophoresis of extracellular pectinase and amylase isozymes of 170 mainly terverticillate Penicillium strains was undertaken. The data were coded and subjected to numerical analysis. Variation in intensity of isozymes was observed in repeat analyses of some strains, although most were consistent. Variation was also observed between some representative strains of species. P. viridicatum was more variable than P. brevicompactum and P. hordei for intensity of pectinase activity. There was a correlation between the grouping of the strains on the basis of the isozymes and the species concepts only in some cases. The method proved useful for the identification of strains producing intense activity which provided clear patterns, for example, P. brevicompactum and P. chrysogenum and to a lesser extent P. solitum var. crustosum and P. hordei. The method was also exclusionary in that some species were restricted to a particular cluster or subcluster. Amylase patterns confirmed that strains referred to as single species are not all homogeneous genetically, and that some strains are not simply haploid homokaryons. The genetic heterogeneity of the strains explains some of the problems in the systematics of the terverticillate penicillia.
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Relationship between Mycoplasma mycoides subsp. mycoides (‘Large-colony’ Strains) and M. mycoides subsp. capri, as Indicated by Numerical Analysis of One-dimensional SDS-PAGE Protein Patterns
More LessTwenty-five strains classified as Mycoplasma mycoides subsp. mycoides LC or subsp. capri have been compared by one-dimensional SDS-PAGE of their cellular proteins. A computerized numerical analysis revealed that the protein patterns of all but two aberrant strains formed one large phenon that separated clearly from representatives of the four other members of the ‘M. mycoides cluster’ at a similarity level (S) of 66% and which remained undivided at up to 78% S. At higher similarity levels, these strains fell heterogeneously into mixed sub-phenons containing strains of both subspecies. Serological comparisons by immunofluorescence largely confirmed the subspecies designations of the test strains, but also showed that some were serologically intermediate between subsp. mycoides and subsp. capri, being cross-reactive with both. These results confirm and enlarge upon those of our earlier studies indicating the protein-pattern inseparability of subsp. capri and subsp. mycoides LC strains and their distinctiveness from the classical M. mycoides subsp. mycoides SC strains and other members of the ‘M. mycoides cluster’. As also recognized by other workers, subsp. mycoides LC and subsp. capri strains appear to comprise one large group, wherein those most readily identifiable as either type lie at either end of a serological spectrum that also contains serologically cross-reactive strains. Our observations therefore suggest the lines along which the three groups classified at present within the species M. mycoides (SC and LC strains of subsp. mycoides; subsp. capri) might eventually be reclassified, subject to direct genomic comparisons.
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