- Volume 132, Issue 8, 1986
Volume 132, Issue 8, 1986
- Biochemistry
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Photoactive Retinal Pigments in Haloalkaliphilic Bacteria
More LessSummary: Light-induced fast transient absorbance changes were detected by time-resolved spectroscopy in 38 of 51 haloalkaliphilic isolates from alkaline salt lakes in Kenya and the Wadi Natrun in Egypt. They indicate the presence of two retinal pigments, Pf and Ps, which undergo cyclic photoreactions with half-times of 2ms and 500ms respectively. Pf absorbs maximally near 580 nm and Ps near 500 nm. The pigments differ in their sensitivity to hydroxylamine and detergent bleaching and the photo reactions of Pf are strongly dependent on chloride concentration. Of the 38 pigment-containing strains, 29 possess both Pf and Ps, 9 possess only Ps. Inhibition of retinal synthesis with nicotine blocks pigment formation and addition of retinal restores it. Hydroxylamine-bleached pigments can be reconstituted with retinal or retinal analogues. Their similarity to the retinal pigments of Halobacterium halobium strongly suggests that they are also rhodopsin-like retinyledene proteins. Pf in all properties tested is almost identical to halorhodopsin, the light-driven chloride pump of H. halobium and may serve the same function in the haloalkaliphiles. Ps has photocycle kinetics similar to sensory rhodopsin and a far-blue-shifted long-lived photocycle intermediate, but its ground state absorption maximum is near 500nm instead of 587nm. We have not found a bacteriorhodopsin-like pigment in the haloalkaliphiles.
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Effect of Mitochondrial Cytochromes and Haem Content on Cytochrome P450 in Saccharomyces cerevisiae
More LessSummary: It is well established that the mitochondrial and the microsomal cytochromes in Saccharomyces cerevisiae are regulated differently. Mutations affecting the mitochondrial cytochromes aa 3 or c had no effect on the concentration of the microsomal cytochrome P450 even during haem limitation. Moreover, a defect in the cytochrome P450 gene did not affect mitochondrial cytochromes. However, a regulatory mutation present in strain SG1 decreased both mitochondrial and microsomal cytochrome contents. This mutation also affected the intracellular haem concentration. The haem precursor 5-aminolaevulinate increased both mitochondrial and microsomal cytochrome contents. Our results indicate that carbon source and haem concentration are involved in the regulation of cytochrome P450.
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Acyltransferase Activity of the Wide Spectrum Amidase of Brevibacterium sp. R312
More LessSummary: The wide spectrum amidase from Brevibacterium sp. R312, which can hydrolyse many amides tothe corresponding acids, was shown to transfer the acyl groups of amides, acids and esters to hydroxylamine. The transfer rates ofthese reactions in cytoplasmic fractions were measured and compared. The K m and V max were determined for different substrates in the presence of hydroxylamine. The enzyme was also shown to transfer the acyl group of the amide analogue N–methylacetamide to hydroxylamide and that of acetamide to the hydroxylamine analogue methylhydroxylamine.
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Purification and Characterization of the Secreted Alkaline Phosphatase of Bacillus licheniformis MC14: Identification of a Possible Precursor
More Lesssummary: The most abundantly secreted protein from Bacillus licheniformis MC14 is alkaline phosphatase (APase). A twofold purification yields a homogeneous enzyme. No discernible chemical-physical differences in the secreted APase distinguish this enzyme from the cell-bound APase(s) except a 10-fold higher specific activity. During the growth phase in which the bacterium was secreting APase into the medium an inactive cytosol protein antigenically similar but 3000 Da heavier than the subunit of the mature secreted APase was immunoprecipitated from the cytosol. A pulse-chase experiment showed the kinetics of disappearance of this protein from the cytosol to be correlated with the appearance of the secreted APase in the medium, suggesting that it may be a precursor to the secreted APase.
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- Development And Structure
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Relaxation Motions Induced in Bacillus subtilis Macrofibres by Cleavage of Peptidoglycan
More LessSummary: Bacillus subtilis macrofibres exposed to lysozyme underwent characteristic rotations, termed relaxation motions, in which their twist changed. Intact macrofibres and macrofibre fragments devoid of loop ends responded in the same way. Macrofibre strains for which the helix hand is temperature-dependent and also those of fixed-hand (both left and right) underwent initial relaxation motions towards the right-hand end of the twist spectrum, the only exception being those in which the initial twist state was at or near the right-hand maximum. Often when the initial relaxation motions were completed immediately before structure breakdown the macrofibres underwent one or a few rotations in the opposite direction (towards the left-hand end of the twist spectrum). Crude autolysin extract obtained from wild-type B. subtilis also caused macrofibre relaxation motions at pH 5·6 but at pH 8·0 macrofibre breakdown occurred as a result of septa) cleavage. This resulted in the release of helically shaped individual cellular filaments. These findings suggest that strain in the cell wall associated with helical shape was dependent on the integrity of the glycan backbone rather than peptide cross-bridges. In contrast, cleavage of peptide cross-bridges apparently was instrumental in the cell separation process. Left-and right-hand macrofibres, when exposed to lysozyme, exhibited different rates of relaxation, breakdown of fibre structure and protoplast formation. Similarly, the rate of macrofibre breakdown during the lag between temperature shift and inversion reflected the replacement of septal wall material by that of a new conformation corresponding to the new helix hand. The difference in the rates of protoplast formation indicates asymmetry in the overall rate of cleavage by lysozyme which may reflect the activity of left-twist protein(s).
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- Genetics And Molecular Biology
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Host Plant Effects on Hybrids of Rhizobium leguminosarum Biovars viceae and trifolii
More LessSummary: Rhizobium leguminosarum biovar viceae host-range plasmid pJB5JI was transferred to six R. leguminosarum biovar trifolii strains. Inheritance of pJB5JI enabled the R. leguminosarum biovar trifolii strains to nodulate both peas and clover species. Bacteria were isolated from nodules formed on peas (the ‘correct’ host for R. leguminosarum biovar viceae) and the hosts for R. leguminosarum biovar trifolii (white, red and subterranean clover). Isolates from peas and white clover appeared to have lost or changed the host-range plasmid conferring the ability to nodulate white clover or peas respectively. Isolates from subterranean and red clover could often nodulate all host plants. These results show that the host exerts some form of functional incompatibility when interacting with hybrid Rhizobium strains and that some hosts are more stringent than others.
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Properties of in vitro Recombinant Derivatives of pJV1, a Multi-copy Plasmid from Streptomyces phaeochromogenes
More LessSummary: The 10·8 kb plasmid pJVl, isolated from Streptomyces phaeochromogenes, has a high copy number (about 150) and a broad host range among Streptomyces spp. Several pJVl derivatives carrying the thiostrepton resistance gene (tsr) of S. azureus were made. One derivative, pWOR191, was shown to promote its own transfer and to mobilize chromosomal markers in S. lividans. Another derivative, pWOR109, was non-transmissible. Deletion in vitro of a segment of pWOR109 gave pWOR120 (5·6 kb), which has single BamHI and BglII sites shown to be capable of accepting ‘foreign’ DNA such as a previously cloned S. antibioticus DNA fragment encoding tyrosinase, giving vectors (pWOR125, pWOR126) with properties resembling the well-established multicopy vector pIJ702. Shuttle vectors capable of functioning in both S. lividans and Escherichia coli were also constructed. The region of pJVl essential for replication and maintenance was localized to a 2·5 kb segment. Stable maintenance of pWOR109 and pWOR12Q was observed in the presence of derivatives ofpIJl0l, the progenitor of pIJ702.
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The TraT Protein is able to Normalize the Phenotype of a Plasmid-carried Permeability Mutation of Salmonella typhimurium
More LessSummary: The isolation of different classes of antibiotic-supersensitive outer membrane permeability mutants of Salmonella typhimurium has been described previously ( Sukupolvi et al., 1984 , Journal of Bacteriology 159,704-712). One of these, the SS-A mutation, sensitizes the bacteria to gentian violet and to hydrophobic antibiotics. The phenotype of the SS-A mutant was restored to normal when a cloned fragment of the F plasmid, or the R plasmid R6-5, carrying the genes traS, T and D was introduced on a multicopy plasmid. The introduction of a plasmid carrying only the tra T gene showed thatthis gene was sufficient to restore the phenotype. Only clones with functioning traT (irrespective of copy number) restored the normal antibiotic-resistant phenotype in the SS-A mutant. An incompatibility test using a donor strain which carried transposon Tn10 in the 60 MDa plasmid of S. typhimurium and a recipient in which Tn5 was placed close to the SS-A mutation indicated that the SS-A mutation was located in the 60 MDa virulence plasmid (previously called the cryptic plasmid) of S. typhimurium. The introduction of the large virulence plasmid carrying the SS-A mutant allele into wild-type S. typhimurium or Escherichia coli resulted in strains with a phenotype identical tothat of the original SS-A mutant.
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Nuclear Suppressors of the Mitochondrial Mutation oxi1-V25 in Saccharomyces cerevisiae. Genetic Analysis of the Suppressors: Absence of Complementation between Non-allelic Mutants
More LessSummary: Ten informational nuclear suppressors of the oxil − mitochondrial mutation of Saccharomyces cerevisiae are recessive. They are linked to each other, but their allelism is uncertain. Some of them unfavourably affect functions of standard (mit +) mitochondrial genomes. One suppressor severely impairs or entirely prevents mitochondrial functions of the spore clones carrying it. The spectrum of mit − mutations on which these suppressors act is similar to that exhibited by nam3-l. In double heterozygotes nam x/NAM3+, nam3-l the oxil − (and box3-) mutation is suppressed, yet one of our suppressors (R705) and nam3-l show independent segregation in tetrads. This indicates that there may be absence of complementation between non-allelic suppressors.
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Regulatory Mutations Affecting the Synthesis of Pectate Lyase in Erwinia chrysanthemi
More LessSummary: Erwinia chrysanthemi secretes five isoenzymes of pectate lyase (PLa to PLe), which are involved in the phytopathogenicity of this bacterial species. We describe the isolation and characterization of mutants in the regulatory systems controlling PL synthesis. The regulation of PL synthesis appears to be complex, involving several controls. In one class of mutants, designated cri, the synthesis of various proteins became resistant to catabolite repression. This mutation mainly affected the synthesis of isoenzymes PLb and PLc. In a second class of mutants, designated gpiR, PL synthesis was no longer inducible in the presence of pectin derivatives; the regulatory gene responsible for the induction was evidently inactivated. Moreover gpiR mutations led to synthesis of PL independent of the growth phase, whereas in the wild-type strain PL synthesis increased only at the end of exponential growth. These mutations mainly affected the synthesis of PLa, PLd and PLe. Mutations in kdgR, the regulatory gene controlling 2-keto-3-deoxygluconate catabolism, had the same effects on PL synthesis as gpiR mutations.
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Genetic Transformation of Rifampicin Resistance in Lactobacillus acidophilus
More LessSummary: Lactobacillus acidophilus strain 100-33, originally isolated from swine faeces, was transformed to rifampicin resistance with DNA from spontaneous rifampicin-resistant mutants derived from it. Cells of the recipient strain were treated with lysozyme and mutanolysin, mixed with donor DNA and polyethylene glycol and grown on a regeneration medium overnight. After 48 h incubation, the numbers of rifampicin-resistant cells in the populations of regenerated cells were estimated from numbers of colonies. Efficiency of the lysozyme/mutanolysin treatment (the ratio of the number of osmotically fragile cells after the enzyme treatment to the initial cell number) was about 99%. The regeneration frequency of the enzyme-treated cells varied from 5 to 67%. The transformation frequency varied from about 0·2 × 10−8 to 8·0 × 10−8 transformants per regenerated cell per μg DNA. To our knowledge, this method for genetic transformation is the first to be reported for a Lactobacillus strain.
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Molecular Cloning of a Gene Coding for a Vibrio cholerae Haemagglutinin
More LessSummary: Recombinant plasmids encoding a Vibrio cholerae haemagglutinin were isolated from the highly virulent V. cholerae strain C5 by cosmid cloning. Both Escherichia coli HB101 containing the recombinant plasmids and V. cholerae C5 were able to agglutinate a variety of erythrocytes from human and animal origin ; this haemagglutination was not inhibited by D-mannose or L-fucose. Subcloning of the recombinant cosmid DNA revealed that a 1·3 kb DNA fragment was sufficient for haemagglutinin production in E. coli HB101. Under direction of this 1·3 kb Vibrio DNA fragment, two proteins were made inE. coli minicells, of 27 and 10 kDa. Haemagglutinin-encoding sequences were not detected in every V. cholerae strain.
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Suppression by the ColV, I-K94 Plasmid of a Growth Lesion in ompA Mutants of Escherichia coli
More LessSummary: Organisms of three independently isolated ompA mutants of Escherichia coli failed to form colonies on glucose minimal agar (glucose MA) at 44 °C after growth in glucose minimal salts medium at 37 °C, although all three strains formed colonies on nutrient agar at 44 °C. Supplementation of the glucose MA with individual amino acids including L-methionine and/or L-cysteine did not allow colony formation at 44 °C, although addition of 0·1% Casamino acids was effective; replacement of glucose with other energy sources or ammonium ions with glutamate also did not allow growth at 44 °C. The failure to form colonies at 44 °C was not due to killing of the organisms, because colonies were formed if plates of the ompA mutant initially incubated at 44 °C were shifted to 30 °C after 16 h. Introduction of the ColV, I-K94 plasmid into P678-54 ompA, 1131 ompA or an ompC ompA mutant suppressed the 44 °C growth lesion, but other plasmids (F lac, R483ColIa, RI, ColB-K98, R124) tested in P678-54 ompA did not. Growth of the ColV, I-K94+ derivative at 44 °C was due to a suppressing effect of the plasmid rather than to introduction of the plasmid into a variant with normal or altered OmpA protein. An attempt was made to ascertain which component(s) encoded by ColV, I-K94 was (were) responsible for allowing growth at 44 °C. Transfer components appeared unlikely to be involved and plasmids which conferred individual colicins (plus the corresponding immunity component) did not suppress. The findings that the ColV, I-K94-encoded VmpA protein resembles the OmpA protein (a) immunologically and (b) in being a transmembrane component of the outer membrane suggested that it might be the presence of the VmpA protein which allowed growth at 44 °C. Several experiments were in accord with this possibility.
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β-Galactosidase and A1ka1ine Phosphatase Do Not Become Extracellular When Fused to the Amino-terminal Part of Colicin N
More LessSummary: A series of plasmids encoding hybrid proteins comprising various lengths of the NH2-terminal region of colicin N coupled to almost complete β-galactosidase or alkaline phosphatase polypeptides was constructed by transposon mutagenesis of ColN plasmid derivatives. Synthesis of the hybrid proteins, like that of colicin N itself, was regulated by the SOS response. Large quantities of the hybrid proteins accumulated in the cytoplasm (β-galactosidase) or particulate fractions (alkaline phosphatase). When the gene fusions were expressed in cells that were producing colicin E2 and expressing the ColE2 lysis gene, only very low levels of the hybrid proteins were found in the medium. The results suggest that the amino-terminal part ofcolicin N does not contain sufficient biochemical information to promote the release of the hybrid proteins into the medium.
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A Detailed Study of gerJ Mutants of Bacillus subtilis
More LessSummary: A total of nine gerJ mutants have now been isolated in Bacillus subtilis. All are defective in their spore germination properties, being blocked at an intermediate (phase grey) stage. The dormant spores are sensitive to heating at 90°C and two of the mutants (generated by transposon insertion) produce spores sensitive at 80°C. The spores of these two more extreme mutants had a visibly defective cortex when studied by electron microscopy, as did some of the other mutants. During sporulation, the acquisition of spore resistance properties and the appearance of the sporulation-specific penicillin-binding protein PBP5* were delayed. A strain probably carrying a lacZ fusion to the gerJ promoter demonstrated increased expression between t 2 and t 4. We propose that the gerJ locus is involved in the control of one or more sporulation-specific genes.
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Enhanced Nitrogen Fixation and Competitiveness for Nodulation of Lotus pedunculatus by a Plasmid-cured Derivative of Rhizobium loti
More LessSummary: A plasmid-cured derivative of Rhizobium loti strain NZP2037, strain PN4010, was significantly more effective in N2 fixation and showed an improved capacity to compete with other root nodule bacteria for nodulation of Lotus pedunculatus, relative to that of strain NZP2037. Reintroduction of the plasmid pRlo2037 into PN4010 resulted in a return to the NZP2037 level of symbiotic effectiveness and competitiveness. The enhanced effectiveness and competitiveness of PN4010 for L. pedunculatus was associated with its increased capacity to form nodules. Strain PN4010 did not differ from NZP2037 in its growth characteristics in pure culture, in its resistance to L. pedunculatus root flavolan or in its ability to multiply in the L. pedunculatus rhizosphere in the presence of a competing strain of R. loti. An examination of 35 R. loti strains (including 25 field isolates) revealed two strains (NZP2014 and NZP2042) that did not contain a single large indigenous plasmid; both were significantly more effective in N2 fixation with L. pedunculatus than was NZP2037 or two other Nod+ Fix+ plasmid-containing strains of R. loti. Transfer of pRlo2037 into NZP2014 and NZP2042 reduced their effectiveness for L. pedunculatus.
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Molecular Cloning and Nucleotide Sequence of the Alkaline Cellulase Gene from the Alkalophilic Bacillus sp. Strain 1139
More LessSummary: The cellulase gene from the alkalophilic Bacillus sp. strain 1139 was cloned in Escherichia coli using pBR322. Plasmid pFK1 was isolated from transformants producing cellulase, and the cloned cellulase gene was found to be in a 4.6 kb HindIII fragment. The cellulase gene was subcloned in a functional state on a 2.9 kb DNA fragment and its nucleotide sequence was determined. The coding sequence showed an open reading frame encoding 800 amino acids. The pFK1-encoded cellulase had the same enzymic properties as the extracellular cellulase produced by the alkalophilic Bacillus sp. strain 1139, but its M r was slightly higher.
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Cloning and Expression of the pepD Gene of Escherichia coli
More LessPeptidase D of Escherichia coli, cleaving the unusual dipeptide carnosine, was found to be encoded by the ColE1 hybrid plasmid pLC44-11. From this plasmid the pepD gene was subcloned into small vectors. As shown by successive reduction of the flanking sequences of genomic DNA, the order of genes in the region at 6 min of the E. coli K12 map is phoE, pepD, in the clockwise orientation. Insertional inactivation of the pepD gene and expression of recombinant plasmids in maxicells allowed the identification of the pepD product as a 52 kDa protein. Comparison with the 100 kDa protein molecular mass determined by gel filtration suggests that active peptidase D is probably a dimer.
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- Pathogenicity And Medical Microbiology
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Cord Factor is Associated with the Maintenance of the Chronic Inflammatory Reaction Caused by Mycobacteria
More LessSummary: The distribution of an aqueous suspension of cord factor (CF) from Mycobacterium bovis BCG in several mouse organs was examined after intravenous injection, and the correlation between evolution of the inflammatory granulomatous reaction and the presence of CF in these organs was determined. CF was preferentially deposited in the lungs and liver, and the kinetics of the pulmonary and hepatic inflammatory reaction, evaluated by determining the indices for these organs, showed a gradual increase on day 2 after injection, reached a peak around the fifth day, and declined thereafter. Histological analysis showed that on day 5 both the lungs and the liver were diffusely damaged by a mononuclear inflammatory infiltrate arranged in a granulomatous manner and consisting predominantly of histiocytes. CF elimination was more marked in the liver than in the lungs: 2 d after injection 76% of the material deposited in the liver had been eliminated. Little or no CF was detected in the liver and lungs by day 16, when the inflammatory reaction was also substantially decreased. A second CF dose administered 8 d after the first exacerbated the inflammatory process in both the lungs and the liver, indicating that the intensity of this process depends on CF concentration in the lesion site.
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Composition and Immunochemical Properties of the Cell Surface Proteins of Vibrio cholerae
More LessSummary: The composition and immunochemical properties of cell surface proteins of Vibrio cholerae belonging to both the biotypes (classical and El Tor) and the serotypes (Ogawa and Inaba) were investigated. Proteins were isolated by extraction with EDTA/NaCl. When the extract was further treated with sodium deoxycholate, a product significantly enriched with the major protein was obtained. The surface localization of these proteins was confirmed by immunoelectron microscopy using protein A-colloidal gold particles as probes. Antisera to these proteins (a) possessed complement-mediated bactericidal activities towards V. cholerae strains belonging to both the biotypes and the serotypes, and (b) upon crossed immunoelectro-phoresis produced several immunoprecipitation reactions towards whole-cell sonicates belonging to all types of V. cholerae. These proteins were immunogenic in the rabbit intestine, as antibodies of two classes (IgG and IgA) were detected in the intestinal fluids. The intestinal immune response was greatly enhanced when cell surface proteins were administered with liposomes. These results suggest that cell surface proteins represent common antigens of V. cholerae and can be explored as vaccine candidates against cholera.
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