- Volume 134, Issue 2, 1988
Volume 134, Issue 2, 1988
- Biochemistry
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The Interaction between Methanol Dehydrogenase and Cytochrome c in the Acidophilic Methylotroph Acetobacter methanolicus
More LessAcetobacter methanolicus contains only c- and b-type cytochromes. One of the b-type cytochromes is probably an o-type oxidase. During growth on methanol at pH 4 the soluble cytochromes c are induced fivefold compared with growth on glycerol. A. methanolicus contains a methanol dehydrogenase (MDH) that is more stable to low pH values than other quinoprotein MDHs but is similar in other respects. Its electron acceptor is an autoreducible cytochrome c L which differs from others in this class in being reduced at pH 4 by MDH in the presence of methanol and in being autoreduced at relatively low pH (pH 7·0). MDH-stimulated autoreduction occurs at the pH of the periplasm (pH 4·0) as predicted if the process of autoreduction is of physiological importance, and the rate is fast enough not to be rate-limiting in electron transport from methanol to the electron transport chain.
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Purification and Properties of Trimethylamine N-Oxide Reductase from Shewanella sp. NCMB 400
More LessTwo major trimethylamine-N-oxide reductases were detected in the periplasmic fraction of the marine bacterium Shewanella sp. NCMB 400 grown in the presence of trimethylamine N-oxide (TMAO). The high-M r enzyme was purified to homogeneity and consisted of a single polypeptide of M r 86000 as judged by SDS-PAGE. The second enzyme had an M r of 47000. On isoelectric focusing, multiple forms of the purified enzyme were revealed with isoelectric points of 5·1 and 5·2. The K m values for the N-oxides of trimethylamine, pyridine and γ-picoline were 0·02, 2·41 and 6·95 mm, respectively. The purified TMAO reductase is a molybdoenzyme containing 1·32 mol Mo (mol enzyme)−1.
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The Secreted Antigens of Mycobacterium tuberculosis and Their Relationship to Those Recognized by the Available Antibodies
Proteins secreted by strains of Mycobacterium tuberculosis during short-term, zinc-sufficient batch culture were identified in order to define antigens likely to be relevant to the pathogenesis of human disease. [35S]Methionine-labelled proteins in supernatants of 4-7 d cultures were separated by PAGE under both denaturing and non-denaturing conditions, and the position of labelled material was determined. Secreted protein patterns of M. tuberculosis were quite similar to those of Bacillus Calmette-Guérin (BCG) but differed by the absence of the 46 kDa dimeric protein specific to BCG and by the presence in large amounts of a 23 kDa protein which, when denatured, gave 13 kDa subunits. This 13 kDa subunit protein constituted up to 20% of secreted proteins in classical strains of M. tuberculosis of phage type B but was not detected in phage type I strains from South India. This may be relevant to the different pathogenicity of these strains. Western blot analysis showed that antigens defined in supernatants of short-term (3 d) cultures of M. tuberculosis constituted a small subset of those seen in supernatants of organisms cultured for longer periods. One of the secreted proteins has the interesting property of binding to fibronectin. The available monoclonal antibodies and antisera have been used to identify lines on immunoblots corresponding to the secreted/released antigens of M. tuberculosis. The present findings suggest that there are major secreted antigens to which antibodies do not yet appear to have been produced experimentally.
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- Genetics And Molecular Biology
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Genetic Regulation of the Quinic Acid Utilization (QUT) Gene Cluster in Aspergillus nidulans
More LessA large number of quinic acid non-utilizing qut mutants of Aspergillus nidulans deficient in the induction of all three quinic acid specific enzymes have been analysed. One class, the qutD mutants, are all recessive and are non-inducible at pH 6·5 due to inferred deficiency in a quinate ion permease. Two regulatory genes have been identified. The QUTA gene encodes an activator protein since most qutA mutants are recessive and non-inducible although a few fully dominant mutants have been found. The QUTR gene encodes a repressor protein since recessive mutations are constitutive for all three enzyme activities. Rare dominant non-inducible mutants which revert readily to yield a high proportion of constitutive strains are inferred to be qutR mutants defective in binding the inducer. The gene cluster has been mapped in the right arm of chromosome VIII in the order: centromere − >50 map units − ornB − 12 map units qutC (dehydratase) − 0·8 map units qutD (permease), qutB (dehydrogenase), qutE (dehydroquinase), qutA (activator) − 4·4 map units − qutR (repressor) − 20 map units galG. This organization differs from that of the qa gene cluster in Neurospora crassa, particularly in the displacement of qutC and qutR.
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Aspects of the Regulation of Adenylate Cyclase Synthesis in Escherichia coli K12
A. Roy, P. Glaser and A. DanchinIn Escherichia coli K12 expression of the adenylate cyclase gene is subject to multiple controls. In order to gain understanding of the regulation of adenylate cyclase synthesis, operon and protein fusions were constructed by in vitro recombination either into bacteriophage λ or low-copy-number plasmids, or directly on the chromosome at the cya locus. The fusions were used in physiological experiments as probes to study transcriptional and translational controls of cya expression. It was found that adenylate cyclase synthesis was insensitive to glucose effects. As already described by other workers, the CAP-cAMP complex had a moderate negative control on cya expression. In addition it was observed that concomitant with a severe slackening of growth rate, specific to the growth of cya strains in rich medium, cya expression was considerably enhanced. This increase of adenylate cyclase synthesis did not appear to be directly dependent on the presence of a functional cAMP receptor (CAP), and seemed to be controlled at the level of transcription. Finally, translation of the cya message was very weak when compared to cya transcription (the mRNA level was the same in protein and operon fusions).
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Genetic Analysis in Streptomyces ambofaciens
More LessA chromosomal linkage map of ten markers was established for Streptomyces ambofaciens by the four-factor cross method and allele-gradient analysis. Mutants were obtained by nitrous acid treatment as well as by UV mutagenesis. The fertility of crosses was enhanced over 100-fold by pSAM2, a plasmid present in some strains of S. ambofaciens, and over 1000-fold by the conjugative plasmid pIJ303.
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A Transposon Insertion in the Escherichia coli uvrC Gene; UvrC Protein is Absolutely Required for the Incision Step in Excision Repair
More LessThe formation of single-stranded breaks in DNA following UV irradiation is assessed in uvrC34 mutants. By altering the SOS DNA-repair system, either by additional mutations or by using drugs affecting transcription or translation, it is shown that such single-stranded breaks require one or more DNA-damage-inducible functions. A UV-sensitive strain is characterized as carrying a Tn10 insertion into the uvrC gene. The absence of post-irradiation incision in this strain demonstrates that uvrC function is absolutely required in vivo for the incision stage of excision repair, and suggests that other uvrC mutants are ‘leaky’.
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Transfer of the Ti Plasmid from Agrobacterium tumefaciens into Escherichia coli Cells
More LessWe have screened strains of Agrobacterium tumefaciens for spontaneous mutants showing constitutive transfer of the nopaline Ti plasmid pTiC58 during conjugation. The Ti plasmid derivatives obtained could be transferred not only to A. tumefaciens but also to E. coli cells. The Ti plasmid cannot survive as a freely replicating plasmid in E. coli, but it can occasionally integrate into the E. coli chromosome. However, insertion in tandem of plasmids carrying fd replication origins (pfd plasmids) into the T-DNA provides an indicator for all transfer events into E. coli cells, providing fd gene 2 protein is present in these cells. This viral protein causes the excision of one copy of the pfd plasmid and allows its propagation in the host cell. By using this specially designed Ti plasmid, which was also made constitutive in transfer functions, we found plasmid exchange among A. tumefaciens strains and between A. tumefaciens and E. coli cells to be equally efficient. A Ti plasmid with repressed transfer functions was transferred to E. coli with a rate similar to the low frequency at which it was transferred to A. tumefaciens. The expression of transfer functions of plasmid RP4 either in A. tumefaciens or in E. coli did not increase the transfer of the Ti plasmid into E. coli cells, nor did the addition of acetosyringone, an inducer of T-DNA transfer to plant cells. The results show that A. tumefaciens can transfer the Ti plasmid to E. coli with the same efficiency as within its own species. Conjugational transmission of extrachromosomal DNA like the narrow-host-range Ti plasmid may often not only occur among partners allowing propagation of the plasmid, but also on a ‘try-all’ basis including hosts which do not replicate the transferred DNA.
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Over-production and Characterization of the nifA Gene Product of Klebsiella pneumoniae — the Transcriptional Activator of nif Gene Expression
R. Tuli and M. J. MerrickThe nifA gene of Klebsiella pneumoniae, which encodes the transcriptional activator of nif gene expression, was cloned into a number of plasmid vectors to obtain high-level synthesis of nifA product (NifA). When over-produced, NifA was very insoluble and it precipitated with the cell debris after cell lysis. Localization of β-galactosidase activity from a nifA-lacZ translational fusion confirmed the insoluble nature of NifA. Analysis of two translational fusions in which the last six C-terminal amino acids of NifA were deleted suggests that these residues are required for activity.
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Chromosomal Mapping and Cloning of the Lipase Gene of Pseudomonas aeruginosa
More LessVarious mutants (lip) of Pseudomonas aeruginosa PAO 2302 that lacked extracellular lipase activity were isolated. They were selected on a calcium-triolein agar. The phenotypic characteristics of two of these mutants suggested that they were defective in the gene coding for lipase: both lip mutants produced no lipase in liquid- and on solid medium. They were non-pleiotropic with regard to various other exoproducts. None of the mutants released any putatively cell-bound lipase after treatment of cells with Triton X-100 or alginate. The electrophoretic protein- and LPS-profiles of outer membranes derived from lip mutants and the parental strain were identical. The lip locus was mapped on the chromosome of P. aeruginosa PAO 1 by FP5- and R68.45-mediated crossings and by transduction with phage G101. The lip locus was cotransduced with pyrF only (60%) indicating a map position at about 57 min. The lipase gene was cloned on a 3.1 kb Sal I fragment using vector pKT248. The newly constructed plasmid was able to complement the lipase deficiency of the two lip mutants of P. aeruginosa.
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Mutations Affecting the Synthesis of NADP-dependent Glutamate Dehydrogenase in Pseudomonas aeruginosa
More LessNADP-dependent glutamate dehydrogenase (NADP-GDH) was purified to homogeneity from Pseudomonas aeruginosa strain 8602 (PAC 1). The M r determined by Sephadex gel filtration was 280000; the subunit M r determined by SDS-PAGE was 45000. Mutant strains lacking NADP-GDH and glutamate synthase (Gdh−Glt−) required glutamate for growth. Transductants that lacked only NADP-GDH were indistinguishable from the wild-type strain in growth properties. It was concluded that NADP-GDH is not essential for growth of the wild-type organism and that glutamate formation via NAD-dependent glutamate dehydrogenase does not occur to a significant extent. A mutant strain, 39, producing high NADP-GDH activity, synthesized normal NADP-GDH and had the same intracellular glutamate concentrations as its parent. The mutation responsible for the synthesis of high levels of NADP-GDH was shown, by transduction, to be closely linked to the NADP-GDH structural gene (gdhA).
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Functional Analysis of the Adsorption Protein of Two Filamentous Phages with Different Host Specificities
More LessThe gene 3 coding for one minor coat protein (adsorption protein) of phage IKe was cloned into an expression plasmid and overproduced. The presence of a promoter for this gene could be demonstrated as well as the incorporation of the IKe gene 3 protein (g3p) into the cytoplasmic membrane of host cells. When 110 carboxy-terminal amino acids were deleted, the truncated protein was translocated across the cytoplasmic membrane into the periplasm. Thus the deleted amino acids bear a membrane anchor domain. In contrast to the partly homologous g3p of the Ff phages, IKe g3p did not alter the membrane properties of its host. IKe g3p was not incorporated into Ff phage particles in amounts detectable by our assays although the presence of IKe g3p may affect the efficiency of Ff phage production. The existence of a structural feature necessary for the specific recognition of the respective g3p during phage assembly is deduced.
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A Specific DNA Probe for the Identification of Campylobacter jejuni
More LessA 6·1 kb DNA probe for the human enteric pathogen Campylobacter jejuni has been isolated from a genomic library constructed in the plasmid vector pBR322 in Escherichia coli. The DNA sequence used as a probe was identified from recombinant plasmids following immunological screening of transformants using polyclonal antisera to whole cells and to membrane antigens of C. jejuni. Restriction endonuclease fragment mapping of C. jejuni DNA inserts from three of the recombinant plasmids showed an overlapping DNA fragment. One of these recombinant plasmids, when used as a DNA probe in Southern hybridization, specifically hybridized with chromosomal DNA from all of the C. jejuni strains tested. Hybridization was not detected at high stringency between the DNA probe and chromosomal DNA from any other Campylobacter species tested except weakly with the chromosomal DNA of strains of Campylobacter coli. Hybridization was also not detected with chromosomal DNA from a range of other enteric bacteria likely to be encountered in faecal material. The intensity of hybridization with C. coli could be increased by reducing the stringency of hybridization.
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- Immunology
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Immunological Reaction of Guinea-pigs Following Intranasal Mycoplasma pneumoniae Infection and Immunization with the 168 kDa Adherence Protein
More LessHumoral responses to Mycoplasma pneumoniae proteins, especially the 168 kDa protein, were demonstrated by Western blotting in sera and bronchial washings of all groups of infected or immunized guinea-pigs. However, infection was not prevented by these local and systemic antibodies. Hilar lymphocytes of infected and immunized guinea-pigs were stimulated in vitro by sonicated M. pneumoniae antigen and by the 168 kDa protein. Stimulation was significantly lower in animals which had been infected twice or had been preimmunized and challenged by infection. Histologically the most severe lesions were seen in the twice-infected group followed by the preimmunized group which was subsequently infected.
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- Pathogenicity And Medical Microbiology
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Inactivation of Human α-1-Antitrypsin by a Tissue-destructive Protease of Legionella pneumophila
More LessThree extracellular proteases produced by Legionella pneumophila during growth in liquid medium were examined for their effects on human α-1-antitrypsin (α-1-AT). One of these proteases, tissue-destructive protease (TDP) destroyed completely the trypsin-inhibitory capacity of α-1-AT at protease: inhibitor molar ratios down to 0.002:1. After inactivation by TDP, the M r of α-1-AT was reduced by 5000 in SDS-PAGE. This suggested that inactivation entailed only limited cleavage.
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A Phospholipase C from the Dallas 1E Strain of Legionella pneumophila Serogroup 5: Purification and Characterization of Conditions for Optimal Activity with an Artificial Substrate
More LessPhospholipase C from the Dallas 1E strain of Legionella pneumophila serogroup 5 was purified from buffered yeast extract culture supernate by ion-exchange chromatography followed by fractionation by manganous chloride and ammonium sulphate precipitation steps. Enzyme activity was assayed by hydrolysis of p-nitrophenylphosphorylcholine and confirmed by release of radioactivity from tritiated L-α-dipalmitoylphosphatidylcholine labelled in the methyl groups of choline. After SDS-PAGE, the purified preparation yielded a single band upon Coomassie-blue staining. This protein migrated with an apparent M r of 50000–54000. Phospholipase C activity was maximal at pH ≥ 8.4 and was enhanced in the presence of sorbitol and of several nonionic detergents but was eliminated by SDS. EDTA, Cu2+, Fe2+ and Zn2+ inhibited enzyme activity, whereas Ba2+, Ca2+, Co2+, Mg2+ and Mn2+ restored activity to EDTA-treated material. No haemolytic activity was demonstrated with the purified enzyme.
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Protein Changes Associated with Induced Resistance of Neisseria gonorrhoeae to Killing by Human Serum Are Relatively Minor
More LessSerum-susceptible (SS) Neisseria gonorrhoeae were induced to resistance (SR) to complement-mediated killing by fresh human serum (FHS) by a small-M r factor(s) from guinea-pig blood in 3 h at 37 °C, but not in the presence of bacteriostatic concentrations of chloramphenicol or neomycin, indicating that proteins mediated the acquisition of resistance. SDS-PAGE protein profiles of lysates of equal numbers of gonococci showed only two qualitative differences between SR and SS organisms, both in minor components (a protein A of about 205 kDa in the former and not the latter and vice versa for a protein B of about 16 kDa). Many proteins, however, including the three principal outer-membrane proteins, were present in larger amounts in SR gonococci. The lack of major changes in proteins when resistance is acquired was confirmed by immunoblotting the two protein profiles with the IgG of hyper-immune rabbit anti-SR and anti-SS sera, of rabbit anti-SR serum after absorption by SS organisms and of FHS used alone and after absorption with SS organisms. The IgM of FHS, which is responsible for most of the bactericidal activity, showed only faint reactions with a few proteins common to both SS and SR gonococci and no reactions when the FHS was absorbed with SS gonococci. This is in contrast to the strong and different reactions given with lipopolysaccharide (LPS) components of SS and SR organisms, which, prepared from the former organisms, neutralize the bactericidal activity of FHS. Hence, the relatively small protein changes accompanying induction are less likely to be directly responsible for serum resistance than the more profound LPS changes.
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Rapid Damage to Membranes of Neisseria gonorrhoeae Caused by Human Neutrophil Granule Extracts
More LessNeisseria gonorrhoeae were exposed to extracts of human neutrophil granules and effects on gonococcal growth and membranes were determined. Enumeration of gonococci by phase-contrast microscopy at 0 and 60 min revealed that they underwent very limited cell division after exposure to granule extract. At 60 min, treated gonococci tended to clump, and some lost their refractivity under phase-contrast optics, indicating membrane damage. Treated and untreated gonococci utilized oxygen at similar rates at time 0; treated gonococci utilized oxygen at a relatively constant rate for 60 min, even though colony-forming ability (i.e. viability) decreased by 90%, whereas untreated gonococci showed a steadily increasing rate of oxygen consumption over the same period, which essentially paralleled increase in colony-forming ability. Membrane ultrastructure of untreated and treated gonococci was compared in thin section by transmission electron microscopy. Extract treatment resulted in a time-related increase in disruption of the bacterial outer membrane, which became apparent almost immediately after treatment. This was accompanied by increasingly aberrant septum structure. Extract treatment also increased the resolution of peptidoglycan by electron microscopy, as early as 10 min after treatment. These data suggest that extract treatment of gonococci caused a rapid loss of the ability to form colonies on agar concomitant with alteration of gonococcal peptidoglycan and outer-membrane structure, but with little alteration of inner-membrane function.
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- Physiology And Growth
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Temperate Phages and Bacteriocins of the Gliding Bacterium Cytophaga johnsonae
More LessA collection of 30 independently isolated strains of Cytophaga johnsonae was screened for the presence of temperate bacteriophages. Two strains were found to harbour phages. The newly isolated phages differ in several respects from the 43 previously isolated phages for C. johnsonae. Both phages are polyhedral, approximately 60 nm in diameter, and have no apparent tail structure. They are chloroform sensitive, and plaque formation is inhibited by agar. Both are capable of establishing a stable association with host cells. Twenty-nine of the 30 strains produced diffusible substances that specifically inhibited the growth of other C. johnsonae strains or closely related species and that could not be propagated. These substances appear to be bacteriocins, some of which, like bacteriophages, are active only against motile cells, while others inhibit nonmotile as well as motile cells. One of each of these two types of bacteriocins was partially characterized and both were found to be proteinaceous in nature and bactericidal in effect.
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Monoclonal Antibodies against a Protein Missing from Nonmotile Mutants of the Gliding Bacterium Cytophaga johnsonae
More LessTo identify components of the gliding bacterium Cytophaga johnsonae that are involved in gliding motility, we generated hybridomas that produced monoclonal antibodies (MAbs) against membranes from wild-type cells and screened for MAbs that failed to bind to cells of nonmotile mutants. Of 22 hybridomas generated, three produced MAbs that were positive in ELISA with wild-type cells or their membranes and were negative in ELISAs with 57 of 63 mutants tested. Immunoblots of polyacrylamide gels of wild-type membrane proteins showed that all three of these motility-related MAbs recognized the same antigen: a 40 kDa major membrane protein of wild-type cells. Immunoblots of nonmotile mutant cells and membranes showed that those giving negative ELISA results with the three MAbs actually produced the protein, but in only trace amounts compared with the parental strain. These results show that synthesis of the 40 kDa protein is related somehow to the ability of the cells to move, but the nature of the relationship is still unknown. The protein may be required for motility, or regulation of its synthesis and assembly may be linked to motility although it has no direct role in motility. Results of other experiments on the distribution of an immunologically related protein among other gliding bacteria and on the effects of the three motility-related MAbs on gliding of C. johnsonae did not distinguish between these two possibilities.
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