- Volume 135, Issue 5, 1989
Volume 135, Issue 5, 1989
- Biochemistry
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Use of a Fractionated, Coupled Transcription-Translation System in the Study of Ribosomal Resistance Mechanisms in Antibiotic-producing Streptomyces
More LessThe coupled transcription-translation system, formerly involving extracts of Streptomyces lividans, has been developed such that it functions with ribosomes (or their subunits) from at least 20 different Streptomyces species. This fractionated system has been used to investigate the antibiotic responses of ribosomes from various Streptomyces which synthesize inhibitors of protein synthesis. Of the 11 organisms included in this study, two strains possessed ribosomes that were specifically resistant to the autogenous antibiotic. These were Streptomyces pactum and Streptomyces karnatakensis, both of which produce pactamycin. Ribosomal subunit exchange analysis further demonstrated that resistance to pactamycin in those strains is due to some property of the 30S ribosomal subunits.
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The Hexokinase Isoenzyme PII of Saccharomyces cerevisiae is a Protein Kinase
More LessThe HXK2 gene product has an important role in controlling carbon catabolite repression in Saccharomyces cerevisiae. We have raised specific antibodies against the hexokinase PII protein and have demonstrated that it is a 58 kDa phosphoprotein with protein kinase activity. The predict amino acid sequence of the HXK2 gene product has significant homology to the catalytic domain of mammalian and yeast protein kinases. Protein kinase activity was located in a different domain of the protein from the hexose-phosphorylating activity. The hexokinase PII protein level remained unchanged in P2T22D mutant cells (hxk1 HXK2 glk1) growing in a complex medium with glucose. The protein kinase activity of hexokinase PII is regulated by the glucose concentration of the culture medium. Exit from the carbon catabolite repression phase and entry into the derepression phase may be controlled, in part, by modulation of the 58 kDa protein kinase activity by changes in cyclic AMP concentration.
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Overproduced β-Lactamase and the Outer-membrane Barrier as Resistance Factors in Serratia marcescens Highly Resistant to β-Lactamase-stable β-Lactam Antibiotics
More LessIn a clinical isolate of Serratia marcescens different states of low and high resistance to different β-lactam antibiotics considered to be β-lactamase-stable, viz. cefotaxime, ceftizoxime, ceftazidime, aztreonam, cefoxitin and imipenem, were found to be connected with the presence of constitutively overproduced, chromosomally encoded β-lactamase at concentrations in the bacterial periplasm of 0·4 and 0·9 mm, respectively. All the antibiotics were degraded by the β-lactamase. However, kinetic constants varied widely:K m from 92 to 0·012 µm, and k cat from 3·4 to 2 × 10−4 s−1. The relative contributions to resistance by the functioning of periplasmic β-lactamase, resynthesis of this enzyme, and limitation of antibiotic penetration by the bacterial outer membrane were analysed by computer simulations according to steady-state and non-steady-state models of interactions in the periplasm. Results for cefotaxime, ceftizoxime, ceftazidime, aztreonam and latamoxef revealed overproduced β-lactamase as the sole cause of the state of low resistance while antibiotic permeability was the same as in non-resistant S. marcescens strains. In contrast, high resistance was due to β-lactamase action and decreased permeability of antibiotics. For resistance to aztreonam, only, immobilization of the antibiotic as covalent acyl-enzyme by newly synthesized β-lactamase was essential. For cefoxitin, ampicillin and imipenem the analyses indicated that additional resistance factors may play a role, e.g. induction of β-lactamase.
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Purification and Characterization of Leucine Dehydrogenase from the Thermophile ‘Bacillus caldolyticus’
U. Kräst, H. Schütte, H. Baydoun and H. TsaiThe l-leucine dehydrogenase (LeuDH, EC 1.4.1.9) from the extremely thermophilic Bacillus caldolyticus (DSM 405) has been purified to homogeneity and crystallized. Its physical and biochemical properties, such as subunit structure, M r, amino acid composition, isoelectric point, heat stability, pH-optima, K m values for coenzymes and substrates, and pattern of inhibition have been determined. The native enzyme is an octamer (M r 320000) composed of identical subunits (M r 39000) which contain one intrachain disulphide bridge. Only aliphatic amino acids were accepted as substrates and a preference was exhibited for branched-chain residues. Inhibition occurred only upon incubation with thiol reagents such as HgCl2 and p-mercuribenzoate, and pyridoxal 5'-phosphate. The LeuDH was very thermostable, with 50% residual activity remaining after 30 min incubation at 80 C. Its properties, as well as amino acid composition and N-terminal sequence, were compared with those of the phylogenetically related LeuDH from the mesophile Bacillus cereus. Both enzymes displayed very similar properties: the quaternary structures were identical, amino acid composition alike, the N-terminal sequence showed 62% homology for the first 19 residues, and K m values for the different substrates differed by a factor of two or less, indicating that the active centres had a similar structure. Thus, the only major difference observed was the much higher stability of the thermophilic enzyme to denaturation by heat and organic solvents.
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- Development And Structure
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Multilayered Distribution of Peptidoglycan in the Periplasmic Space of Escherichia coli
More LessWhen a staining technique using phosphotungstic acid (PTA) in 10% (w/v) chromic acid was applied to cells of Escherichia coli, the periplasmic space was seen as a dark 15-nm-thick layer of uniform appearance and constant width. Our observations are consistent with peptidoglycan being the main material stained. Isolated sacculi as well as purified peptidoglycan (protein free) were also stained by the same procedure, the thickness of the peptidoglycan being 8·8 ± 1·8 and 6·6 ± 1·5 nm, respectively. The increased thickness of the PTA-stained layer in stationary phase cells correlated well with the increased thickness of isolated sacculi or purified peptidoglycan and with the increased amount of peptidoglycan in such cells. Thickness measurements on isolated peptidoglycan were compatible with a two to three layer structure for material from exponential phase cells and with a four to five layer structure for that from stationary phase cells. Furthermore, the results indicated an uneven distribution of peptidoglycan material in the periplasmic space, the peptidoglycan spanning the space from the inner to the outer membrane.
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- Genetics And Molecular Biology
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Identification of DNA Regions Homologous to Nitrogen Fixation Genes nifE, nifUS and fixABC in Azospirillum brasilense Sp7
A 30 kb DNA region from Azospirillum brasilense Sp7, containing the nitrogenase structural genes (nifHDK), has been cloned. The presence of nif genes, in the 20 kb located next to nifHDK, was explored by Tn5 mutagenesis after subcloning various restriction fragments in the broad-host-range suicide vehicle pSUP202. Over 25 mutations due to Tn5 random insertions were obtained in the 20 kb and each recombined into the genome of strain Sp7. Four new nif loci were identified, located at about 4, 9, 12 and 18 kb downstream from nifK respectively. Hybridization with heterologous nif probes from Klebsiella pneumoniae, Bradyrhizobium japonicum and Azorhizobium caulinodans was performed to characterize the new nif regions. The region proximal to nifK appears to contain nifE and the region distal to nifK contains genes homologous to nifUS and fixABC. nif gene(s) from the fourth locus were not identified. Mutants in this locus, which were devoid of nitrogenase activity when tested under nitrogen-free conditions, displayed a high nitrogenase activity when glutamate was added to the growth medium. This phenomenon was also observed with mutants of the fixABC homology region, but to a lesser extent. Homology between strain Sp7 total DNA and a nifB-containing probe from B. japonicum was detected, although the hybridizing region was not part of the nif cluster described above.
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Molecular Analysis of a Plasmid-encoded Phenol Hydroxylase from Pseudomonas CF600
More LessPseudomonas strain CF600 is able to utilize phenol and 3,4-dimethylphenol as sole carbon and energy source. We demonstrate that growth on these substrates is by virtue of plasmid-encoded phenol hydroxylase and a meta-cleavage pathway. Screening of a genomic bank, with DNA from the previously cloned catechol 2,3-dioxygenase gene of the TOL plasmid pWW0, was used in the identification of a clone which could complement a phenol-hydroxylase-deficient transposon insertion mutant. Deletion mapping and polypeptide production analysis identified a 1·2 kb region of DNA encoding a 39·5 kDa polypeptide which mediated this complementation. Enzyme activities and growth properties of Pseudomonas strains harbouring this fragment on a broad-host-range expression vector indicate that phenol hydroxylase is a multicomponent enzyme containing the 39·5 kDa polypeptide as one component.
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Expression and Flavinylation of Arthrobacter oxydans 6-Hydroxy-d-nicotine Oxidase in Bacillus subtilis
More Less6-Hydroxy-d-nicotine oxidase (6-HDNO) of Arthrobacter oxydans, an enzyme inducible by dl-nicotine, contains FAD covalently bound via an 8a-N(3)His linkage. Expression of the gene encoding 6-HDNO and flavinylation of the protein were studied in Bacillus subtilis. In this heterologous system the following findings were made. 1. An enzymically active covalently flavinylated 6-HDNO of normal size can be expressed in B. subtilis. 2. The natural promoter of the 6-HDNO gene appeared inefficient in B. subtilis. The B. subtilis sdh promoter, when inserted upstream of the A. oxydans promoter, increased 6-HDNO expression >50-fold. 3. Expression of the 6-HDNO gene from plasmids in B. subtilis was, independently of the promoter construct used, stimulated more than fivefold by dl-nicotine in the growth medium. It is concluded that flavinylation of 6-HDNO is possibly autocatalytic and mediated by factors generally found in bacterial cells.
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Genetic Basis of the Association of Sulphonamide Resistance with Methionine Auxotrophy in Neisseria gonorrhoeae
More LessSelection by sulphonamides was investigated in Neisseria gonorrhoeae because a sulphonamide-resistant (Sulr), methionine-requiring (Met−) phenotype that was common in the era of sulphonamide therapy became rare in the penicillin era. Cultures of wild-type (SulrMet+) gonococci on a conventional medium containing sulphadiazine (210 g ml-1) yielded numerous, nonidentical mutations of two met genes. The requirement of MetI- mutants was satisfied only by methionine, whereas MetI-1 mutants utilized either homocysteine or methionine. My theory that increased resistance to sulphonamides is a pleiotropic effect of methionine auxotrophy was confirmed by the return of sulphonamide susceptibility in all Met+ spontaneous mutants. Furthermore, the SulrMet- traits were introduced or eliminated together by DNA-mediated transformation. Sulphonamides are known to inhibit dihydropteroate synthase; consequently, they interrupt the entire sequence of reactions in the folate pathway including the methyl group transfer from N 5-methyltetrahydrofolate to homocysteine to form methionine. The increased sulphonamide resistance of these Met- mutants is discussed in terms of conservation of the pool of essential tetrahydrofolate derivatives. The ease with which spontaneous forward and reverse met mutations can be obtained is unique among gonococcal genes.
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Sequence of Pilin from Bacteroides nodosus 351 (Serogroup H) and Implications for Serogroup Classification
More LessThe nucleotide sequence of the pilin gene from Bacteroides nodosus strain 351, currently classified as serogroup H, subgroup 2 (H2) has been determined. The gene encodes a single polypeptide (prepilin) of 160 amino acids and M r 17 150. However, pilin isolated from B. nodosus 351 migrates as two distinct bands in sodium dodecyl sulphate-polyacrylamide gel electrophoresis, due to an internal peptide bond cleavage. Amino acid sequence studies of pilin from B. nodosus 351 have established that the cleavage occurs between 72Ala and 73Ser of the mature protein sequence. Comparisons of gene and amino acid sequences of pilin from B. nodosus 351 with the corresponding sequences from strains of serogroups D and H1 indicate that these sequences share a close relationship. However, the level of sequence identity between B. nodosus 351 pilin and pilin from strain 265 of serogroup H1 is lower than anticipated for strains within a serogroup and suggests that B. nodosus 265 and B. nodosus 351 should not be classified within the same serogroup.
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Plasmid Diversity within the Genus Chlamydia
More LessExamination of 12 Chlamydia psittaci strains recovered from nine different host species (three avian and six mammalian) revealed the presence of a 7·5 kb plasmid in all isolates except two ovine abortion strains, the human strain IOL207 and the Cal 10 strain. Restriction mapping analysis distinguished four different plasmids that were associated with avian, feline, equine and guinea-pig C. psittaci isolates, respectively. The restriction maps of these four C. psittaci plasmid types all differed from that of the plasmid recovered from C. trachomatis L2/434. Despite this plasmid diversity, which is likely to be of taxonomic importance, all four plasmids identified within the species C. psittaci were found to share some sequence homology, which was mapped to two separate regions in the plasmid molecules. One region, which showed a high degree of homology between C. psittaci plasmids and also detectable homology with the C. trachomatis plasmid, may represent a common replication control region for plasmids of this genus.
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Bidirectional Stimulation of the White-Opaque Transition of Candida albicans by Ultraviolet Irradiation
More LessMost strains of Candida albicans are capable of switching spontaneously and at high frequency between a number of phenotypes distinguishable by colony morphology. The switching frequency of Candida albicans strain WO-1 between two predominant phenotypes, ‘white’ and ‘opaque’, and a minor phenotype, ‘fuzzy’, increased dramatically with low doses of ultraviolet irradiation that killed less than 20% of the population. The ultraviolet irradiation effect continued to be expressed over many generations as evidenced by stimulated sectoring. Ultraviolet irradiation stimulated switching in both the white-to-opaque and opaque-to-white direction, suggesting that a common mechanism functions in both directions.
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Parallel Changes in Catabolite Repression of Haem Biosynthesis and Cytochromes in Repression-resistant Mutants of Saccharomyces cerevisiae
More LessEffects of three mutant genes, CAT1-2d, cat2-1 and hex2-3, on catabolite repression of mitochondrial cytochromes and the first two enzymes of haem biosynthesis were compared. The CAT1-2d mutation gave no resistance to glucose, whereas cat2-1 endowed both cytochromes and 5-aminolaevulinate dehydratase with resistance, but did not alter the effect of glucose on 5-aminolaevulinate synthase. The hex2-3 mutation caused repression resistance of cytochromes and of the two haem biosynthetic enzymes. hex2-3 strains also accumulated intracellular 5-aminolaevulinate. Co-inheritance of the latter traits, sensitivity to maltose inhibition and ability to grow on raffinose in the presence of 2-deoxyglucose, demonstrated that the pleiotropic phenotype is a function of the single gene hex2-3. Revertants which grew on maltose regained sensitivity to deoxyglucose and exhibited normal sensitivity of cytochromes and haem biosynthesis enzymes to repression. Addition of the hex1-18 mutation, which renders cytochromes resistant to repression, to a cat2-1 strain did not produce the same effect on 5-aminolaevulinate synthase as hex2-3. It is concluded that repression patterns of haem and cytochrome biosynthesis are substantially affected by hex2-3 and cat2-1 but not by CAT1-2d .
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Anaerobic Growth of Escherichia coli on Glycerol by Importing Genes of the dha Regulon from Klebsiella pneumoniae
More LessThe dha regulon of Klebsiella pneumoniae specifying fermentative dissimilation of glycerol was mobilized by the broad-host-range plasmid RP4:mini Mu and introduced conjugatively into Escherichia coli. The recipient E. coli was enabled to grow anaerobically on glycerol without added hydrogen acceptors, although its cell yield was less than that of K. pneumoniae. The reduced cell yield was probably due to the lack of the coenzyme-B12-dependent glycerol dehydratase of the dha system. This enzyme initiates the first step in an auxiliary pathway for disposal of the extra reducing equivalents from glycerol. The lack of this enzyme would also account for the absence of 1,3-propanediol (a hallmark fermentation product of glycerol) in the spent culture medium. In a control experiment, a large quantity of this compound was detected in a similar culture medium following the growth of K. pneumoniae. The other three known enzymes of the dha system, glycerol dehydrogenase, dihydroxyacetone kinase and 1,3-propanediol oxidoreductase, however, were synthesized at levels comparable to those found in K. pneumoniae. Regulation of the dha system in E. coli appeared to follow the same pattern as in K. pneumoniae: the three acquired enzymes were induced by glycerol, catabolite repressed by glucose, and glycerol dehydrogenase was post-translationally inactivated during the shift from anaerobic to aerobic growth. The means by which the E. coli recipient can achieve redox balance without formation of 1,3-propanediol during anaerobic growth on glycerol remains to be discovered.
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Protoplast Transformation of Bacillus stearothermophilus NUB36 by Plasmid DNA
Lijun Wu and N. E. WelkerAn efficient protoplast transformation system was established for Bacillus stearothermophilus NUB3621 using thermophilic plasmid pTHT15 Tcr (4·5 kb) and mesophilic plasmid pLW05 Cmr (3 kb), a spontaneous deletion derivative of pPL401 Cmr Kmr. The efficiency of transformation of NUB3621 with pLW05 and pTHT15 was 2 x 107 to 4 x 108 transformants per µg DNA. The transformation frequency (transformants per regenerant) was 0·5 to 1·0. Chloramphenicol-resistant and tetracycline-resistant transformants were obtained when competent cells of Bacillus subtilis were transformed with pLW05 [2·5 x 105 transformants (µg DNA)-1] and pTHT15 [1·8 x 105 transformants (µg DNA)-1], respectively. Thus, these plasmids are shuttle vectors for mesophilic and thermophilic bacilli. Plasmid pLW05 Cmr was not stably maintained in cultures growing at temperatures between 50 and 65 °C but the thermostable chloramphenicol acetyltransferase was active in vivo at temperatures up to 70 °C. In contrast, thermophilic plasmid pTHT15 Tcr was stable in cultures growing at temperatures up to 60 °C but the tetracycline resistance protein was relatively thermolabile at higher temperatures. The estimated copy number of pLW05 in cells of NUB3621 growing at 50,60, and 65 °C was 69,18, and 1 per chromosome equivalent, respectively. The estimated copy number of pTHT15 in cells of NUB3621 growing at 50 or 60 °C was about 41 to 45 per chromosome equivalent and 12 in cells growing at 65 °C.
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Mutants of Bacillus subtilis 168 Thermosensitive for Growth and Wall Teichoic Acid Synthesis
More LessA protocol designed to isolate mutants with thermosensitive (Ts) synthesis of the bacteriophage 𝜙29 receptor, which includes the major wall teichoic acid in Bacillus subtilis 168, yielded a significant enrichment for Ts growth mutants among colonies surviving 𝜙29 treatment. Nine mutants, Ts for both 𝜙29 susceptibility and cell growth, harboured mutations which were located in the tag locus by PBS1 transduction and recombination index with the tag-1 marker. Physical mapping revealed that they were distributed on a segment of more than 4 kb. Chemical analysis of cell walls showed a marked reduction in phosphate relative to diaminopimelic acid content of all mutants at the non-permissive temperature. Differences between mutants were correlated with the distribution of tag mutations on the genetic map. We conclude (i) that the newly identified markers affect several genes involved in poly(glycerol phosphate) synthesis, and (ii) that on phosphate-rich media, cell growth cannot occur without the synthesis of the latter polymer.
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Dissection of the Expression Signals of the spo0A Gene of Bacillus subtilis: Glucose Represses Sporulation-specific Expression
The expression of the spo0A-lacZ fusion gene was partially repressed in the presence of an excess of glucose. Expression was restored either by the mutation sigA47 (crsA47) or by addition of decoyinine, an inhibitor of GMP synthetase, to the medium. By constructing a lacZ fusion with a smaller fragment of the spo0A gene, we observed a β-galactosidase profile in which expression was completely repressed by an excess of glucose. This expression was restored by the addition of decoyinine. These results indicate that the expression of the spo0A gene is regulated by at least two different mechanisms, one sensitive to glucose, the other not. Furthermore, the glucose-sensitive regulation was shown to reside at the transcriptional level. It is likely that the reduced expression of the spo0A gene in the presence of glucose at an early stage of sporulation causes the repression of sporulation.
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Regulation of Development by the matA Complex Locus in Physarum polycephalum
More LessThe transformation of uninucleate amoebae of Physarum polycephalum into multinucleate plasmodia is under the control of a multiallelic mating-type locus, matA. Plasmodium formation usually occurs only when amoebal fusion brings together two different matA alleles within a single cell; such plasmodium formation is termed crossing. Mutations (gadA) in the matA locus permit haploid amoebae to self, that is to form plasmodia in clonal cultures without amoebal fusion. By constructing diploid amoebae heterozygous for gadA alleles, it was shown that the mutant alleles gadA5 and gadA111 were each dominant to gadA +. Non-selfing revertants of gadA mutants may be generated as a result of further mutations (npf) within the matA locus. Sixty-one revertants of this type were studied, derived from three matA3 gadA mutants. Some revertants were able to cross with a matA3 gadA + tester strain. These ‘class I’ revertants failed to cross with one another or with similar revertants of two matA2 gadA mutants; they appear all to lack a single gene function (npfC +) that is necessary in plasmodium development. Other revertants failed to cross with the matA3 gadA + tester. These ‘class II’ revertants also failed to cross with one another, but showed several different patterns of crossing when mixed with other strains. The behaviour of some of the class II revertants suggests that they lack a further gene function (npfB +) necessary in plasmodium development. The gadA mutations may affect a cis-acting regulator of npfB +.
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- Immunology
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Variation in Epitopes of the B Subunit of El Tor and Classical Biotype Vibrio cholerae O1 Cholera Toxin
More LessVariation in epitopes of the B subunit of cholera toxin (CT-B) produced by strains of El Tor and classical biotype Vibrio cholerae O1 was examined using monoclonal antibodies prepared to V. cholerae 569B CT. CT-B epitopes were markedly conserved for V. cholerae classical biotypes. In contrast, epitope variation was observed for El Tor biotypes, which produced both a classical-like CT-B and a unique CT-B lacking at least one epitope common to 569B CT-B. The missing epitope was located outside the GM1 ganglioside-binding site. From results of the study reported here, genetic divergence is exhibited in the El Tor biotype CT-B versus classical CT-B. Furthermore, at least five unique epitopes of V. cholerae 569B CT-B can be defined.
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- Pathogenicity And Medical Microbiology
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Properties of Adherence Factor Plasmids of Enteropathogenic Escherichia coli and the Effect of Host Strain on Expression of Adherence to HEp-2 Cells
EPEC adherence factor (EAF) plasmids from three strains of enteropathogenic Escherichia coli (EPEC) - E2347/69 (O127:H6), E20517 (O111:H2) and E24582 (O142:H6) - were examined. The EAF plasmids were all marked with ampicillin resistance by transposition of Tn801 to give pDEP1, pDEP2 and pDEP11, respectively. All three plasmids showed incompatibility with an FIme and an FIV plasmid and had some similarity in restriction enzyme digest patterns. Plasmid pDEP1 differed from pDEP2 and pDEP11 in being autotransferring and fertility-inhibition positive. An EAF probe consisting of a 1 kb BamHI-SalI restriction endonuclease fragment of the prototype EAF-associated plasmid pMAR2 hybridized to similar-sized SalI-BamHI fragments of pDEP1 and pDEP11 but to a different-sized fragment of plasmid pDEP2. Loss of the EAF plasmids from EPEC strains resulted in a marked reduction in the ability of these strains to adhere to HEp-2 cells. The EAF-plasmid-negative variants did not express a 94 kDa outer-membrane protein (OMP). When these EAF plasmids were reintroduced into EAF-plasmid-negative EPEC strains a high level of adherence equivalent to that of the parent EPEC strains was restored and a 94 kDa OMP was usually expressed. However, when EAF plasmids were transferred into E. coli K12 or non-EPEC E. coli the host strains either did not adhere or adhered poorly to the HEp-2 cells. These transconjugants did not express a 94 kDa OMP.
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Characterization of a Putative Colonization Factor (PCFO166) of Enterotoxigenic Escherichia coli of Serogroup O166
More LessEnterotoxigenic Escherichia coli (ETEC) of serogroup O166 gave mannose-resistant haemagglutination (MRHA) with bovine and human erythrocytes. The strains did not react with antisera prepared against the known colonization factors CFA/I, CFA/II, CFA/III, CFA/IV and PCFO159:H4. Strain E7476 of serotype O166:H27, which produced heat-stable enterotoxin (ST), was examined initially. It produced fimbriae about 7 nm in diameter. On SDS-PAGE two possible fimbrial polypeptides of molecular mass 15·5 and 17·0 kDa were seen. When variants of strain E7476 were isolated, loss of ST and MRHA together was associated with loss of a 98 MDa plasmid, while loss of ST alone correlated with plasmid deletion. An absorbed anti-strain E7476 antiserum reacted specifically with the 15·5 and 17·0 kDa polypeptides in Western immunoblotting and bound to the intact fimbriae by immuno-electron microscopy. When this antiserum was used in an ELISA to examine other strains of serogroup O166, a positive reaction was obtained with all the ST- and MRHA-positive strains. One strain of serotype O71:H27 and two strains of serotype O98:H− also reacted with the absorbed anti-strain E7476 antiserum. The antiserum did not react with ETEC carrying known colonization factors. E. coli K12 and a number of E. coli of different serotypes carrying a plasmid coding for ST transferred from strain E7476, all gave MRHA and reacted with the absorbed anti-strain E7476 antiserum. The term putative colonization factor O166 (PCFO166) is proposed to describe the adhesive factor(s) on ETEC of serogroup O166 because of the similarity of properties with those of known colonization factors.
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The Development of Chlamydia trachomatis Inclusions within the Host Eukaryotic Cell during Interphase and Mitosis
More LessThe dynamic nature of Chlamydia trachomatis inclusions was studied by video and 35 mm timelapse photomicrography of live cells, and by immunolocalization of inclusions in fixed cells. A serotype E isolate was used to infect the McCoy cell line and endometrial epithelia. Then resulting inclusions were observed over 4 d. They appeared as slowly expanding fluid-filled membrane vesicles whose growth varied considerably, and which were subject to great physical distortion by the host cell during interphase and mitosis. When this distortion became extreme the inclusion was observed to divide. However, as inclusions were mobile within the cytoplasm and thus able to come into contact with each other, there was a net tendency for the opposite process of inclusion fusion to occur when cells contained more than one inclusion. The proportion of infected cells decreased with time as a result of host cell proliferation, despite transmission of inclusions to progeny at the time of mitosis. Inclusion growth physically disrupted karyokinesis and cytokinesis so that host cell division became distorted or blocked on the second or third day of infection. Cell death eventually occurred by a very rapid lysis event.
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Protein-mediated Adhesion of Lactobacillus fermentum Strain 737 to Mouse Stomach Squamous Epithelium
More LessThe mechanism of adhesion of Lactobacillus fermentum strain 737 to mouse stomach squamous epithelium was investigated. Adhesion inhibition tests involving chelators, monosaccharides, periodate and concanavalin A and the use of bacteria grown in the presence of tunicamycin failed to clarify the adhesive mechanism. Washed bacterial cells had reduced adhesive capacity, except in the presence of spent broth culture supernatant fraction or cell washings. Spent culture supernatant fractions of erythrosine-supplemented broth did not enhance adhesion of washed cells. The adhesion-promoting factor(s) in the spent broth culture supernatant fractions and cell washings bound to both bacterial and epithelial cell surfaces, but did not promote adhesion of two other Lactobacillus strains which were not of mouse origin, thereby indicating host specificity for the adhesion-promoting activity. Chemical characteristics of the adhesion-promoting factor were determined by pretreatment of the dialysis retentate of spent broth culture supernatant fractions with proteolytic enzymes, concanavalin A-Sepharose or periodate before the adhesion assay. The adhesin was non-dialysable, pronase-sensitive, heat sensitive at 100 C, had no affinity for concanavalin A-Sepharose and contained no carbohydrate groups active in the adhesion process. The protein profiles of dialysis retentates of spent broth culture supernatant fractions after bacterial growth in the absence and presence of erythrosine were determined by 2-dimensional SDS-PAGE. Gel filtration by HPLC was used for purification of an adhesion-promoting fraction. The host-specific adhesion of L. fermentum strain 737 was mediated by a protein, with an M r of 12–13000, that was not detectable in cells grown in the presence of erythrosine. A model for the mode of binding of the adhesin to host epithelia and bacterial surfaces is proposed.
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Role of Myeloperoxidase in the Killing of Staphylococcus aureus by Human Neutrophils: Studies with the Myeloperoxidase Inhibitor Salicylhydroxamic Acid
More LessWe have used salicylhydroxamic acid (SHAM) to inhibit intraphagosomal myeloperoxidase activity in order to evaluate the role of this enzyme in the killing of Staphylococcus aureus by human neutrophils. 50 m-SHAM reduced the luminol-dependent chemiluminescence response stimulated during phagocytosis of unopsonized latex beads and opsonized S. aureus by over 80% and 60%, respectively. When opsonized S. aureus were incubated with neutrophils, 45% were killed within 15 min incubation and 60% by 1 h. However, in neutrophil suspensions incubated with 50 m-SHAM, only 13% were killed by 15 min whilst 71% still remained viable after 1 h. This inhibitor had no effect upon the number of bacteria phagocytosed or upon degranulation. In a cell-free system, 25 m-H2O2 alone killed 55% of the bacteria, whereas in the presence of myeloperoxidase (i.e. 10 mU myeloperoxidase and 25 m-H2O2) virtually all of the bacteria were killed: the addition of 50 m-SHAM abolished this myeloperoxidase-enhanced killing but did not affect the H2O2-dependent killing. We therefore conclude that in normal neutrophils whilst H2O2 is required for killing of this pathogen, both myeloperoxidase-dependent and -independent pathways exist.
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- Physiology And Growth
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Role of Erythrosine in the Inhibition of Adhesion of Lactobacillus fermentum Strain 737 to Mouse Stomach Tissue
More LessThe mechanism by which the food colour erythrosine inhibits the adhesion of Lactobacillus sp. to squamous epithelium in the mouse stomach was investigated using an in vitro adhesion assay. Inhibition of adhesion occurred only after growth of L. fermentum in erythrosine which bound to the bacterial cell surface. Erythrosine did not interfere with the receptor on the epithelial cell surface. Growth, but not the ATP content per cell, was affected by the presence of erythrosine in the growth medium. No consistent correlation between hydrophobicity and growth in two different broths was noted when erythrosine was present. Analyses of phenol/water extracts and transmission electron micrographs revealed no reduction in extracellular polysaccharide after growth in the presence of erythrosine. It was concluded that erythrosine affects bacterial metabolism thereby preventing production of the bacterial adhesin which is not the extracellular polysaccharide.
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Production of Outer-membrane Proteins and an Extracellular Fluorescent Compound by Iron-limited Azomonas macrocytogenes
More LessOuter membranes were isolated from iron-limited and iron-sufficient Azomonas macrocytogenes ATCC 12334 by sucrose density centrifugation or treatment with Sarkosyl. Analysis of the outer membranes by sodium dodecyl sulphate polyacrylamide gel electrophoresis revealed that iron-limited cells produced greater amounts of two proteins of apparent molecular mass 74 kDa and 70 kDa than iron-sufficient cells. The enhanced production of these two proteins was evident when media contained less than 9·7 μm added iron with maximum production occurring when media contained less than 1·1 μm added iron. Iron-limited growth conditions also caused A. macrocytogenes ATCC 12334 to excrete a visible yellow, blue-white fluorescent compound into culture supernatant fluids with maximum levels being produced in media that contained less than 1·1 μm added iron. This fluorescent compound had a distinctive pH-dependent absorption maximum characteristic of pyoverdin-type siderophores and appeared to bind iron. Furthermore, culture supernatant fluids containing the fluorescent compound promoted the uptake of 55Fe in A. macrocytogenes ATCC 12334 cells. Three other A. macrocytogenes strains, NCIB 10958, NCIB 8700 and NCIB 9129, produced iron-repressible outer-membrane proteins but only the former two strains produced the iron-repressible fluorescent compound. It is proposed that the iron-regulated proteins and fluorescent compound produced by A. macrocytogenes may function in high-affinity iron uptake.
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Role of Guanosine Kinase in the Utilization of Guanosine for Nucleotide Synthesis in Escherichia coli
More LessUsing purine auxotrophic strains of Escherichia coli with additional genetic lesions in the pathways of interconversion and salvage of purine compounds, we demonstrated the in vivo function of guanosine kinase and inosine kinase. Mutants with increased ability to utilize guanosine were isolated by plating cells on medium with guanosine as the sole purine source. These mutants had altered guanosine kinase activity and the mutations were mapped in the gene encoding guanosine kinase, gsk. Some of the mutants had acquired an additional genetic lesion in the purine de novo biosynthetic pathway, namely a purF, a purL or a purM mutation. A rev-recd map location of the gsk gene is presented and the gene order established as proC-acrA-apt-adk-gsk-purE.
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The Penetration of Antibiotics into Aggregates of Mucoid and Non-mucoid Pseudomonas aeruginosa
More LessCells of mucoid and non-mucoid Pseudomonas aeruginosa in colonies were at least one-thousandfold less sensitive to the antibiotics tobramycin or cefsulodin than were cells of the same bacteria in dispersed suspension. We did not detect any difference between the mucoid form and the non-mucoid form in the antibiotic sensitivity of colonies, from which we infer that the exopolysaccharide of the mucoid form does not contribute to colony-resistance by forming a barrier to antibiotic diffusion. Mathematical models were constructed in order to estimate time-courses of penetration of tobramycin and cefsolodin into biofilms and microcolonies of mucoid and non-mucoid P. aeruginosa. For tobramyen penetration, adsorption of antibiotic to the exopolysaccharide of the glycocalyx and antibiotic uptake by cells were taken into account in the calculations. The longest time-period for the concentration of tobramycin at the base of a biofilm 100 m deep to rise to 90% of the concentration outside the biofilm was predicted to be 24 h. For cefsulodin penetration, irreversible hydrolysis catalysed by lactamase was taken into account, using lactamase levels taken from the literature. The calculations predicted that the cefsulodin concentration at the base of antifilm 100 m deep would rise to 90% of the external concentration in 29 s when the -lactamase was synthesized at the basal level. For a similar biofilm of bacteria synthesizing enhanced levels of lactamase (derepressed), the concentration of cefsulodin at the base was calculated to rise to 41% of the external concentration in about 50s and then remain at that level. This was despite the fact that cefsulodin is a poor substrate for this -lactamase.
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Polyamines and Ornithine Decarboxylase Activity during Growth and Differentiation in Sclerotium rolfsii
More LessChanges in polyamine content and in the activity of ornithine decarboxylase (ODC) were followed during growth and differentiation of the plant pathogenic fungus Sclerotium rolfsii, in submerged mycelium liquid cultures and on solid agar cultures. Mycelial growth in submerged cultures was characterized by high putrescine content and ODC activity. Growth cessation, resulting from glucose exhaustion in the medium, was accompanied by a sharp decrease in putrescine content and ODC activity. Spermine, the level of which was initially low, was detected in high amounts after all of the glucose was consumed and when the fungus developed the potential for sclerotium formation. A decrease in spermidine, and especially putrescine content, and an increase in spermine content, were observed during the transition from mycelium to mature sclerotia on solid agar medium. Addition of spermine to solid agar medium increased the number of sclerotia by 40%. The changes in the content of the three polyamines were reversed when sclerotia were allowed to germinate. Moreover, α-difluoromethylornithine, the enzyme-activated inhibitor of ODC, greatly inhibited mycelium growth, sclerotium germination and ODC activity, and this inhibition was completely reversed by the addition of putrescine. Cycloheximide delayed sclerotium germination and initially inhibited ODC activity, but ODC inhibition was relieved as soon as sclerotia began to germinate. The data indicate that specific changes in polyamines are linked with two distinct developmental events in S. rolfsii. Mycelial growth and sclerotium germination are positively correlated, and possibly causally linked, with a marked increase in putrescine content and biosynthesis (while spermine cannot be detected). Differentiation (sclerotium formation), however, is accompanied by a major increase in spermine content.
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Production and Localization of Proteinases in Colonies of Timber-decaying Basidiomycete Fungi
More LessProteinase activity was detected in culture filtrates of eight wood-decaying basidiomycete fungi and compared in terms of ability to clear skimmed milk agar. All the fungi were proteolytic, but to differing extents. Five were compared using azocasein hydrolysis as a measure of proteolytic activity at the centres and margins of agar-grown colonies and it was found that in Coriolus versicolor the marginal mycelium was the more strongly proteolytic, while in all the other fungi proteolysis by central mycelium was greater. The time course of changes in proteolytic activity in culture filtrates of C. versicolor and Serpula lacrimans grown on the surface of liquid media was compared over 4 weeks, and differences were found which suggested that C. versicolor mycelium inactivates its proteinases after secreting them, but that S. lacrimans does not. The results are interpreted in terms of the likely role of proteinases in the nitrogen economy of these fungi when growing on their wood substrates.
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Pleiotropic Deficiency in the Control of Carbon-regulated Catabolic Enzymes in the ‘Slime’ Variant of Neurospora crassa
More LessA wall-less ‘slime’ strain of Neurospora crassa was shown to be pleiotropically altered in the production and secretion of several enzymes related to carbon catabolism, including invertase, alkaline protease, aryl-β-glucosidase and cellobiase. Invertase and protease were produced constitutively. The two β-glucosidases required induction by cellobiose but were poorly affected by glucose repression. Exoenzymes were released in larger amounts by the ‘slime’ strain than by the wild-type. Two other carbon-regulated enzymes, isocitrate lyase and phosphoenolpyruvate carboxykinase, were normally regulated in the ‘slime’ strain. A study of recombinants from a cross of a ‘slime’-containing heterokaryon and wild-type suggested that the anomalies observed were inherent in the ‘slime’ phenotype.
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Transport of Benzoic and Propanoic Acids by Zygosaccharomyces bailii
More LessUptake rates of benzoic and propanoic acid into Zygosaccharomyces bailii were proportional to the concentration of undissociated acid and showed no indication of limitation at high concentrations. Benzoic acid permeated 27 times faster than propanoic acid. Other low-M r fatty acids were taken up at rates approximately related to their lipophilicity. Glucose stimulated uptake rate and inhibitors of glucose transport or metabolism removed the stimulation. It was concluded that the principal mechanism of uptake was diffusion of undissociated acid. The Z. bailii membrane had lower permeabilities than reported for other cell types and lipid bilayers. Growth in the presence of benzoic acid reduced permeability to benzoic acid further. Preservative-resistant yeast species had lower uptake rates of propanoic acid than sensitive ones. Degradation of the cell wall did not change the permeability to propanoic acid.
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- Plant-Microbe Interactions
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Rhizobium Population Genetics: Genetic Variation Within and Between Populations from Diverse Locations
More LessPolyacrylamide gel electrophoresis (PAGE) of isoenzymes of chromosomal origin, and plasmid profile analyses, were used to investigate genetic variation within and between natural populations of Rhizobium leguminosarum biovar trifolii from diverse sources. PAGE showed that, within the biovar in general, relatively few alleles existed for the enzyme loci studied. There was a strong genetic disequilibrium between loci, suggesting that chromosomal recombination is rare. Both PAGE and plasmid profile analysis revealed differences in the degree of strain polymorphism at the sites examined. At sites which were acidic, improved (by liming) and neutral, strain variation was found to be low, intermediate and high, respectively.
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- Systematics
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Nucleic Acid Relatedness Studies on the Genus Carnobacterium and Related Taxa
More LessNone of the species Carnobacterium carnis, C. divergens, C. mobile or C. gallinarum showed significant DNA-DNA homology between themselves and other lactic acid bacteria. Nevertheless, the Carnobacterium species were found to belong to the same ribosomal RNA homology cluster. The species in this cluster were distant from the other bacteria tested.
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- Corrigendum
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