- Volume 138, Issue 7, 1992
Volume 138, Issue 7, 1992
- Review Article
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- Biochemistry
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Dissociation by NH4Cl treatment of the enzymic activities of glutamine synthetase II from Rhizobium leguminosarum biovar viceae
Glutamine synthetase II (GSII) was purified to homogeneity from Rhizobium leguminosarum biovar viceae and characterized. The sequence of 26 amino acid residues from the amino-terminal end of the protein showed high similarity with the sequence of GSII from Bradyrhizobium japonicum or from Rhizobium meliloti. Non-denaturing PAGE showed that GSII, either in crude extracts or in the pure state, was a mixture of an octamer and a tetramer and that under specific conditions the octamer/tetramer ratio could be modified in either direction. The pure enzyme was used to raise an antiserum which was highly specific. Addition of NH4Cl to a bacterial culture derepressed for GSII caused a specific decrease in transferase activity, faster than the one observed when the amount of immunoreactive material was measured by different methods. On the other hand, biosynthetic activity, measured as the rate of ADP or glutamine formation, paralleled the rate of decrease in immunoreactive material. A partially purified enzyme preparation retained this dissociation of kinetic parameters, strongly suggesting a post-translational modification. These findings are discussed with respect to the possible role of GSII in the Rhizobium-legume symbiosis.
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Purification and characterization of L-2,4-diaminobutyrate decarboxylase from Acinetobacter calcoaceticus
More LessAcinetobacter calcoaceticus ATCC 23055 produces a large amount of 1,3-diaminopropane under normal growth conditions. The enzyme responsible, L-2,4-diaminobutyrate (DABA) decarboxylase (EC 4.1.1.-), was purified to electrophoretic homogeneity from this bacterium. The native enzyme had an M r of approximately 108000, with a pl of 5.0, and was a dimer composed of identical or nearly identical subunits with apparent M r 53000. The enzyme showed hyperbolic kinetics with a K m of 1.59 mM for DABA and 14.6 μM for pyridoxal 5'-phosphate as a coenzyme. The pH optimum was in the range 8.5–8.75, and Ca2+gave a much higher enzyme activity than Mg2+as a cationic cofactor. N-γ-AcetyIDABA, 2,3-diaminopropionic acid, ornithine and lysine were inert as substrates. The enzyme was different in subunit structure, N-terminal amino acid sequence and immunoreactivity from the DABA decarboxylase of Vibrio alginolyticus previously described.
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- Development And Structure
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Production and localization of restrictocin in Aspergillus restrictus
More LessThe production and secretion of restrictocin (a cytotoxin that cleaves ribosomal RNA) by cultures of the fungus Aspergillus restrictus was investigated. Previous studies have indicated that restrictocin production in liquid culture coincides with the appearance of differentiated cell structures. A study of the correlation between the appearance of differentiated structures and restrictocin production was conducted with A. restrictus grown on agar medium. Restrictocin was found to be associated with the cell mass of the agar-grown culture (in contrast to liquid cultures), and was first observed when aerial hyphae emerged. Restrictocin levels increased until the time of conidiation, after which they fell off sharply. No restrictocin could be found in the agar medium. The presence of restrictocin upon and within various cell structures was determined by immunofluorescent laser microscopy. This study showed that restrictocin became localized to the conidiophores and phialides during the process of conidiation. Prior to this, restrictocin was found within the hyphae in localized concentrations that may correspond to secretory vesicles.
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- Genetics And Molecular Biology
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Functional expression in Escherichia coli of proteins B and C from soluble methane monooxygenase of Methylococcus capsulatus (Bath)
More LessMethylococcus capsulatus (Bath) uses a soluble methane monooxygenase (sMMO) to catalyse the oxidation of methane to methanol. sMMO is comprised of three components; A, B and C. Protein C (the reductase) transfers electrons from NADH to protein A (the hydroxylase) which contains the active site, and protein B regulates this electron flow. The five genes encoding the sMMO proteins and their subunits are clustered and have been cloned in Escherichia coli. A DNA fragment containing mmoB, the gene encoding protein B, was subcloned into pT7–5, a plasmid of the T7 RNA polymerase promoter expression system. Upon induction, E. coli expressed protein B which was fully functional after purification. The gene encoding protein C, mmoC, was amplified with unique restriction sites at each end using the polymerase chain reaction and then subcloned into pT7–7 (a plasmid similar to pT7–5 but containing its own ribosome-binding site and ATG start codon). Protein C expressed in E. coli was also found to be functional. This is the first report of the functional expression of methanotroph methane monooxygenase genes in a heterologous host and represents a significant step forward in our analysis of the assembly and catalysis of sMMO.
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A monofunctional prephenate dehydrogenase created by cleavage of the 5′ 109 bp of the tyrA gene from Erwinia herbicola
More LessA cohesive phylogenetic cluster that is limited to enteric bacteria and a few closely related genera possesses a bifunctional protein that is known as the T-protein and is encoded by tyrA. The T-protein carries catalytic domains for chorismate mutase and for cyclohexadienyl dehydrogenase. Cyclohexadienyl dehydrogenase can utilize prephenate or L-arogenate as alternative substrates. A portion of the tyrA gene cloned from Erwinia herbicola was deleted in vitro with exonuclease III and fused in-frame with a 5' portion of lacZ to yield a new gene, denoted tyrA*, in which 37 N-terminal amino acids of the T-protein are replaced by 18 amino acids encoded by the polycloning site/5' portion of the lacZ α-peptide of pUC19. The TyrA* protein retained dehydrogenase activity but lacked mutase activity, thus demonstrating the separability of the two catalytic domains. While the K m of the TyrA* dehydrogenase for NAD+remained unaltered, the K m for prephenate was fourfold greater and the V max was almost twofold greater than observed for the parental T-protein dehydrogenase. Activity with L-arogenate, normally a relatively poor substrate, was reduced to a negligible level. The prephenate dehydrogenase activity encoded by tyrA* was hypersensitive to feedback inhibition by L-tyrosine (a competitive inhibitor with respect to prephenate), partly because the affinity for prephenate was reduced and partly because the K i value for L-tyrosine was decreased from 66 μM to 14 μM. Thus, excision of a portion of the chorismate mutase domain is shown to result in multiple extra-domain effects upon the cyclohexadienyl dehydrogenase domain of the bifunctional protein. These include alterations in apparent substrate specificity, isoelectric point, stability, catalytic properties and regulatory properties.
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Lack of homology between two haloacetate dehalogenase genes encoded on a plasmid from Moraxella sp. strain B
More LessTwo genes encoding haloacetate dehalogenases, H-1 and H-2, are closely linked on a plasmid from Moraxella sp. strain B. H-1 predominantly acts on fluoroacetate, but H-2 does not. To elucidate the molecular relationship between the two enzymes, we compared their structural genes. Two restriction fragments of the plasmid DNA were subcloned on M13 phages and their nucleotide sequences were determined. The sequence of each fragment contained an open reading frame that was identified as the structural gene for each of the two dehalogenases on the basis of the following criteria; N-terminal amino acid sequence, amino acid composition, and molecular mass. The genes for H-1 and H-2, designated dehH1 and dehH2, respectively, had different sizes (885 bp and 675 bp) and G + C contents (58.3% and 53.4%). Sequence analysis revealed no homology between the two genes. We concluded that the dehalogenases H-1 and H-2 have no enzyme-evolutionary relationship. The deduced amino acid sequence of the dehH1 gene showed significant similarity to those of three hydrolases of Pseudomonas putida and a haloalkane dehalogenase of Xanthobacter autotrophicus. The dehH2 coding region was sandwiched between two repeated sequences about 1.8 kb long, which might play a part in the frequent spontaneous deletion of dehH2 from the plasmid.
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Molecular genetics of the extracellular lipase of Pseudomonas aeruginosa PAO1
More LessThe structural gene (lipA) coding for the extracellular lipase of Pseudomonas aeruginosa PAO1 has been cloned on plasmid pSW118. Nucleotide sequence analysis revealed a gene of 936 bp. lipA codes for a proenzyme of 311 amino acids including a leader sequence of 26 amino acids. The mature protein was predicted to have a M r of 30134, an isoelectric point of 5.6, and a consensus sequence (IGHSHGG) typical of lipases. Furthermore it is highly homologous (>60%) to other lipases from various pseudomonads. The lipA gene failed to hybridize detectably with genomic DNA from other Pseudomonas species except P. alcaligenes, even under relaxed stringency. Located 220 bp downstream of the lipA gene, is an open reading frame (ORF2, lipH) which encodes a hydrophilic protein (283 amino acids; M r 33587) that shows some homology to the limA gene product of P. cepacia. In complementation tests of lipase-defective mutants, lipH was shown to be necessary for expression of active extracellular lipase in P. aeruginosa PAO1.
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Stability, frequency and multiplicity of transposon insertions in the pyoverdine region in the chromosomes of different fluorescent pseudomonads
More LessTn5 mutagenesis of different fluorescent pseudomonads was achieved by conjugational transfer of the suicide vector pSUP 10141. Pyoverdine negative (Pvd−) mutants were detected by the absence of fluorescence on King's B medium and by their inability to grow in the presence of the iron chelator EDDHA [ethylenediamine di(ohydroxyphenylacetic acid)]. In P. fluorescens ATCC 17400 and three rhizosphere isolates (one P. putida and two P. fluorescens), the percentage of Pvd−mutants ranged between 0 and 0.54%. In a P. chlororaphis rhizosphere isolate, this percentage was higher (4%). In these mutants both of the Tn5 antibiotic resistances (Km and Tc) were stable and the transposon could be detected by hybridization. In Pvd−mutants of P. fluorescens ATCC 17400, the transposon was found to be inserted twice in the chromosome while single insertions were detected in the DNA of other, randomly tested mutants. In P. aeruginosa PAO1, where 13.1% of the mutants were Pvd−, both antibiotic resistances were rapidly lost and accordingly no transposon insertion could be detected by hybridization. However, the Pvd−phenotype was generally stable in these mutants. The plasmid pNK862 containing a mini-Tn10 transposon was introduced by electroporation into P. aeruginosa PAO1 and Kmrmutants were recovered, 89% of which were Pvd−and confirmed to be P. aeruginosa by PCR amplification of the P. aeruginosa lipoprotein gene. The mini-Tn10insertions were also found to be unstable in PAO1.
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A survey of Col plasmids in natural isolates of Escherichia coli and an investigation into the stability of Col-plasmid lineages
More LessA survey of colicins in the ECOR reference collection of Escherichia coli is presented. Twenty-five of the 72 ECOR strains exhibited a phenotype consistent with colicin production and E. coli isolated from human hosts were more likely to be colicinogenic than those from animal hosts. Multiple representatives of two Col plasmids, lowmolecular-mass ColE1 plasmids and high-molecular-mass, conjugative ColIa plasmids were isolated from the ECOR collection and were examined with a combination of restriction fragment and Southern analysis. These data suggested that ColE1 plasmids comprise a stable (cohesive) plasmid lineage, while ColIa plasmids represent a family of distinct plasmid lineages united by the presence of the colicin Ia operon.
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Cloning, sequencing and expression of the gene encoding the cell-envelope-associated proteinase from Lactobacillus paracasei subsp. paracasei NCDO 151
More LessThe gene encoding the cell-envelope-associated proteinase of Lactobacillus paracasei subsp. paracasei NCDO 151 (formerly Lactobacillus casei NCDO 151) was cloned and sequenced. The gene was located on the chromosome and encoded a polypeptide of 1902 amino acids. The proteinase is N-terminally cleaved upon maturation. It shows extensive homology to the Lactococcus lactis subsp. cremoris Wg2 proteinase. Similar to the situation in Lactococcus, a maturation gene was found upstream of the proteinase gene. The cloned proteinase gene was expressed in Lactobacillus plantarum. However, no expression was observed when the gene was cloned in Lactococcus lactis.
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Molecular cloning and characterization of an alkalophilic Bacillus sp. C125 gene homologous to Bacillus subtilis sec Y
More LessA 1.8 kb HindIII DNA fragment containing the secY gene of alkalophilic Bacillus sp. C125 has been cloned into plasmid pUC119 using the B. subtilis secY gene as a probe. The complete nucleotide sequence of the cloned DNA indicated that it contained one complete ORF and parts of two other ORFs. The similarity of these ORFs to the sequences of the B. subtilis proteins indicated that they were the genes for ribosomal protein L15–SecY–adenylate kinase, in that order. The gene product of the alkalophilic Bacillus sp. C125 secY homologue was composed of 431 amino acids and its M r value has been calculated to be 47100. The distribution of hydrophobic amino acids in the gene product suggested that the protein was a membrane integrated protein with ten transmembrane segments. The total amino acid sequence of alkalophilic Bacillus sp. C125 secY homologue showed 69.7% homology with that of B. subtilis sec Y. Regions of remarkably high homology (78% identity) were present in transmembrane regions, and cytoplasmic domains (73% identity) with less homologous regions present in extracellular domains (43% identity).
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Stable inheritance of shuttle vectors based on plasmid pIM13 in a mutant strain of Clostridium acetobutylicum
More LessNew shuttle vectors for Clostridium acetobutylicum were constructed, using as replicons the Gram-positive plasmid pIM13, and derivatives of the Gram-negative plasmid pBR322, including pUC19. These vectors transformed C. acetobutylicum at a high frequency (up to 106transformants per μg DNA) by PEG-mediated protoplast transformation. A mutant host strain, NI-4082, was isolated on the basis of its ability to maintain plasmid pIM13 stably in the absence of selection pressure. The shuttle vectors showed no segregational or structural instability in this mutant strain. Moreover, the results suggested a relationship between segregational instability and the multimerization of pIM13 in C. acetobutylicum. The host/vector system described possessed all the properties required for efficient gene cloning in this species.
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Isolation and characterization of a conjugative plasmid from Legionella pneumophila
More LessThe conjugative properties of an indigenous 85 MDa plasmid (designated pCH1) from Legionella pneumophila were studied. To determine if pCH1 was transmissible by conjugation, mating experiments were performed between legionellae that harboured pCH1 and several plasmid-less recipients. Plasmid transfer was monitored by colony hybridization, using a cloned 21.0 kb SalI restriction fragment from pCH1 as a probe. The results from these experiments showed that pCH1 could be conjugatively transferred into several strains of L. pneumophila serogroup 1 but not into strain Bloomington-2 (serogroup 3) or Escherichia coli. Southern hybridization experiments in which pCH1 DNA was used as a probe showed that pCH1 does not share homology with other indigenous L. pneumophila plasmids. There was no detectable DNA homology between pCH1 and L. pneumophila chromosomal DNA. Additional mating experiments revealed that pCH1 was unable to mobilize the L. pneumophila chromosome. The conjugative transfer of pCH1 into plasmid-less avirulent or virulent serogroup 1 strains did not alter the intracellular growth characteristics of these strains in U937 cells, a human-monocyte-like cell line, or in the amoeba Hartmannella vermiformis. These results suggest that pCH1 does not contribute to the ability of L. pneumophila to enter or grow within eukaryotic cells.
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Characterization of bacteriophage BFK20 from Brevibacterium flavum
More LessBacteriophage BFK20 was isolated from a Brevibacterium flavum strain that had become contaminated during industrial fermentation. BFK20 has a polyhedral head 50 nm wide and a non-contractile tail 200 nm long and 10 nm in diameter. The genome of this bacteriophage consists of a linear double stranded DNA molecule of 44–45 kb with cohesive ends. The capsid of phage BFK20 contains nine polypeptides with molecular masses from 22.0–108.0 kDa. BFK20 DNA was used as a donor for fragments carrying promoters and transcription-terminators.
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Molecular characterization of two bacteriophages isolated from Desulfovibrio vulgaris NCIMB 8303 (Hildenborough)
More LessA preliminary endonuclease restriction map of a bacteriophage isolated from Desulfovibrio vulgaris has been established. BamHI cleaved whole phage DNA into four fragments while HindIII cut the same DNA into seven fragments. Mapping studies succeeded in linking the four BamHI fragments into two DNA segments; however, no linkage between the two segments was detected. These data imply that two phages were induced from cultures of D. vulgaris and that the two segments represented the DNA from these phages. Support for this hypothesis came from size approximation of restriction enzyme fragments, electron micrographs, and density gradients.
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Molecular characterization of the genes encoding acetohydroxy acid synthase in the cyanobacterium Spirulina platensis
More LessThe enzyme acetohydroxy acid synthase (AHS), which catalyses the first common step in the biosynthesis of isoleucine, leucine and valine, has been demonstrated to be present in Spirulina platensis in two isoenzymic forms. The complete nucleotide sequences of the genes ilvX and ilvW encoding these two enzymes have been determined. Sequence analysis revealed the presence of two open reading frames, of 1836 and 1737 nucleotides for ilvX and ilvW, respectively. The predicted amino acid sequences of the two isoenzymes, compared with the Synechococcus PCC 7942 AHS enzyme and the large subunits of the Escherichia coli AHSI, II, III isoenzymes, revealed a notable degree of similarity. A small subunit has not been identified for either of the S. platensis AHS isoenzymes. Analysis by Northern blot hybridization demonstrated that the ilvX and ilvW genes are transcribed to give mRNA species of approximately 2.15 kb and 1.95 kb, respectively.
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Generation of genetic recombinants in Trichosporon cutaneum by spontaneous segregation of protoplast fusants
More LessAuxotrophic mutants were isolated in two strains of Trichosporon cutaneum. Complementing auxotrophs were hybridized by protoplast fusion. Some of the fusants were apparently transient diploids and segregated to give recombinant marker combinations.
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Cloning and expression of multiple cellulase cDNAs from the anaerobic rumen fungus Neocallimastix patriciarum in Escherichia coli
More LessA cDNA expression library of the rumen fungus Neocallimastix patriciarum was made in Escherichia coli. Cellulolytic clones were identified by screening on a medium containing carboxymethylcellulose. Restriction mapping and Southern hybridization analysis of selected clones revealed three distinct cellulase cDNAs, designated celA, celB and celC. Studies on the substrate specificity showed that the enzyme encoded by celA had high activity towards amorphous and microcrystalline cellulose, while the celB and celC enzymes had relatively high activity on carboxymethylcellulose, with little activity on microcrystalline cellulose. Analysis of hydrolysis products from defined cellodextrins showed that the celB and celC enzymes hydrolysed β-1,4-glucosidic linkages randomly, whereas the celA enzyme cleaved cellotetraose to cellobiose, and cellopentaose to cellobiose and cellotriose. Cellobiose was also the only product detectable from hydrolysis of microcrystalline cellulose by the celA enzyme. Based on substrate specificity and catalytic mode, celA appears to encode a cellobiohydrolase, while celB and celC encode endoglucanases. Northern blot hybridization analysis showed that expression of the three cellulase transcripts in N. patriciarum was induced by cellulose.
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Regulation of restrictocin production in Aspergillus restrictus
More LessThe production of restrictocin (a cytotoxin that specifically cleaves ribosomal RNA) by cultures of Aspergillus restrictus grown in liquid medium was investigated. The function of restrictocin, the method of its accumulation and the mode of resistance to restrictocin in A. restrictus are unknown. Previous studies have indicated that restrictocin accumulates in the medium with culture age. These observations have been extended in this study by cloning the cDNA of the res gene and using this cDNA clone to probe the onset of messenger RNA synthesis in the cells. The results of the Northern analysis were compared to the production and accumulation of restrictocin and morphological differentiation of the cells in culture. Restrictocin was found in the medium at the same time that mRNA was detected in the cells. This suggests that the leader sequence encoded by the cDNA provides an efficient secretion system for the protein. Both the protein and the mRNA were detected coincident with the formation of differentiated cell structures. These structures develop into conidiophores with one layer of sterigmata and conidia forming from the sterigmata. These results suggest that restrictocin is either involved in the process of conidiation or is coordinately regulated with differentiation leading to conidiation.
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- Pathogenicity And Medical Microbiology
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Molecular cloning of a determinant coding for fimbrial antigen F1651, a Prs-like fimbrial antigen from porcine septicaemic Escherichia coli
The genetic determinant coding for F1651 fimbriae was cloned from the chromosome of the porcine Escherichia coli wild-type strain 4787 (O115:K−:H51:F165). The fimbrial determinant was further subcloned into the BamHI site of pACYC184 and a restriction map was established. On Southern hybridization, identity between the chromosomally encoded prs-like determinant of strain 4787 and its cloned counterparts was demonstrated. The cloned F1651 fimbriae and those of the wild-type strain possessed a major protein subunit of molecular mass 18.5 kDa. Strains expressing F1651 fimbriae were detected using an F165-specific polyclonal antiserum and caused mannose-resistant haemagglutination and agglutination of Forssman latex beads. Antiserum against the cloned F1651 fimbriae recognized a 18.5 kDa band in the parent strain 4787.
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Identification of erythrocyte-binding antigens in Helicobacter pylori
More LessThe surface antigens of Helicobacter pylori conferring erythrocyte-binding activity were obtained by adsorption onto formaldehyde-treated dog and goat erythrocytes from supernatant fractions of sonicated bacteria and elution using a high concentration of NaCl. The desorbed material was analysed by SDS-PAGE and immunoblotting with anti-whole-cell serum to agar-grown bacteria which had been absorbed with broth-grown, non-haemagglutinating cells (haemagglutination-associated antiserum). Two polypeptides with molecular masses of 25 and 59 kDa were revealed as erythrocyte-binding antigens. Strains which agglutinated both dog and goat erythrocytes possessed both these erythrocyte-binding antigens, whereas an antigenically cross-reactive 24 kDa polypeptide was present in a strain which only agglutinated goat erythrocytes. Haemagglutinin material was extracted from H. pylori using n-octylglucopyranoside and purified by Sepharose chromatography and sucrose density gradient ultracentrifugation. The purified extract directly agglutinated erythrocytes in a neuraminyl-lactose-sensitive and neuraminidase-sensitive manner. The 59 kDa polypeptide was not present in the purified haemagglutinin preparation. The haemagglutination-associated antiserum reacted strongly with the 25 kDa polypeptide band which was the most prominent polypeptide band on analysis of the purified haemagglutinin preparation by SDS-PAGE and silver staining. Thus, H. pylori possesses at least two adhesins, one of which recognises a N-acetylneuraminic acid (α2–3) moiety of receptors, the other being of unknown receptor specificity. Differences in the antigenicity and molecular masses of these adhesins in individual strains may underlie differences in receptor-binding specificities and haemagglutination profiles.
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Binding of purified Bacillus sphaericus binary toxin and its deletion derivatives to Culex quinquefasciatus gut: elucidation of functional binding domains
More LessHighly larvicidal strains of Bacillus sphaericus produce a binary toxin composed of 51 and 42 kDa proteins which binds to sharply delineated regions of the gastric caecum and posterior midgut of susceptible larvae of the mosquito Culex quinquefasciatus. To investigate the role of the individual subunits and the organization of functional binding regions within the toxin, plasmids were constructed for the expression in Escherichia coli of the toxin proteins and their NH2- and COOH-terminal deletion derivatives as fusions with glutathione S-transferase (GST). Toxin proteins were purified by affinity chromatography followed by cleavage from the GST carrier with thrombin. The LC50 values for the purified toxin proteins and their deletion derivatives were determined. The binding patterns of fluorescently labelled toxin suggested that the 51 kDa protein is the primary binding component of the toxin and mediates the regional binding and internalization of the 42 kDa protein. Examination of the toxin deletion derivatives revealed that the NH2-terminal region of the 51 kDa protein was required for binding to the larval gut, whilst the COOH-terminal region was responsible for interacting with the 42 kDa protein. Toxicity was strongly correlated with the subsequent internalization of the toxin, probably by endocytosis.
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Lack of correlation between colony morphology and lipooligosaccharide content in the Mycobacterium tuberculosis complex
More LessRough and smooth colony variants of the Mycobacterium tuberculosis complex were compared with respect to their composition in trehalose-containing glycolipid antigens in view of the results of a recent investigation suggesting that the chemical basis of rough and smooth colony morphology in mycobacteria may reside in the occurrence of lipooligosaccharides. A careful chemical characterization of the individual glycolipids of the selected strains allowed the identification of the major glycolipids. The comparative study of the glycolipid content of the smooth Canetti strain, its spontaneous rough variant, and 16 additional strains of M. tuberculosis, M. bovis and M. africanum showed that the presence of lipooligosaccharides was not related to the morphology of the colonies.
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Demonstration of peptidoglycan-associated Brucella outer-membrane proteins by use of monoclonal antibodies
More LessA monoclonal antibody (3D6) was produced which reacted only with Brucella sonicated cell extracts that had been lysozyme-treated after sonication. The monoclonal antibody (mAb) reacted with the three major outer-membrane proteins (OMPs) of B. melitensis B115 in Western blots. A large number of reactive bands ranging from 12 to 43 kDa were present in lysozyme-treated Escherichia coli and Yersinia enterocolitica sonicated cell extracts. In a latex agglutination inhibition immunoassay, mAb 3D6 showed better reactivity with purified peptidoglycan (PG) of B. melitensis B115 than with that of Escherichia coli. This mAb was also used in immunogold electron microscopy with whole Brucella cells and sections. No binding was observed on whole cells and immunogold labelling in sections was observed close to the outer membrane, in the periplasmic space and in the cytoplasm. These findings indicate that mAb 3D6 is specific for PG subunits. Immunoblot analysis of B. melitensis B115 rough sonicated cell extracts after SDS-PAGE, with or without lysozyme treatment, was performed using mAbs specific for Brucella OMPs of molecular masses of 10, 16.5, 19, 25-27, 31-34, 36-38 and 89 kDa, for PG and for rough lipopolysaccharide (R-LPS) and smooth lipopolysaccharide (S-LPS). mAbs specific for the 25-27, 31-34 and 36-38 kDa OMPs reacted with three to six bands. All of them except the band of lowest molecular mass reacted with the PG-specific mAb and not with R-LPS- and S-LPS-specific mAbs. Therefore we propose that variation in the apparent molecular mass of the major OMPs (25-27, 31-34, and 36-38 kDa) is due to varying numbers of PG subunit residues rather than attached R-LPS or S-LPS molecules. Our results suggest a very strong, possibly covalent, interaction of the major OMPs (25-27, 31-34 and 36-38 kDa) with PG. There is no evidence of association of the minor OMPs (10, 16.5, 19 and 89 kDa) with PG, since mAbs specific for these proteins reacted with single bands that were not apparent when reacted with the anti-PG mAb. Furthermore, lysozyme treatment did not affect the electrophoretic mobility of these proteins.
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- Physiology And Growth
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Isolation and characterization of polyene-resistant mutants from the maize smut pathogen, Ustilago maydis, defective in ergosterol biosynthesis
More LessUstilago maydis mutants resistant to polyene antibiotics were screened for defects in ergosterol biosynthesis. Slow-growing mutants recovered after selecting for amphotericin B resistance were devoid of ergosterol and accumulated the methylated sterols, 14α-methylfecosterol, obtusifoliol and eburicol, indicating that these isolates were impaired in C-14 sterol demethylation and were similar to the Erg40 mutant of U. maydis. By contrast, nystatin- and pimaricin-resistant isolates which exhibited reduced growth rates showed a dysfunction in C-8 sterol isomerization. In these mutants (Erg2) ergosterol was replaced by the Δ8-sterols, ergosta-5,8,22-trienol, ergosta-8,22-dienol, fecosterol and ergost-8-enol. Analysis of a random sample of polyene-resistant isolates that grew normally revealed that although most retained a typical wild-type sterol profile, two of the isolates failed to accumulate ergosterol. The major sterols detected in these isolates were ergosta-7,22-dienol and ergosta-7-enol, suggesting a lesion in C-5 sterol desaturation in these mutants (Erg3). Of four Erg2 mutants recovered, one mutant, selected on nystatin, contained low but detectable amounts of ergosterol. Ergosterol was not detected immediately after selection in the three other pimaricin-resistant Erg2 mutants. Although the growth of three of the Erg2 mutants remained unchanged during non-selective culture, one mutant reverted and began to grow at a greater rate than the rest; analysis of the sterols produced by this strain revealed that ergosterol was now present, but at lower concentrations than those in the wild-type strain. No changes in the type of sterol formed were observed in the other slower-growing Erg2 mutants even after prolonged culture. All the Erg2 mutants exhibited morphological abnormalities; sporidia were swollen and distorted, and the inability of sporidia to separate after cell division led to the development of highly branched, multicellular groups of cells.
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Effect of nitrogen source on the levels of nitrate reductase in the yeast Hansenula anomala
More LessLevels of nitrate reductase (NR) protein in Hansenula anomala and Hansenula wingei were determined using specific antiserum raised against the enzyme from H. anomala. Extracts from nitrate-grown cells contained NR protein, while in those from cells grown on ammonium, glutamine or peptone, no cross-reacting material could be observed. Enzyme activity correlated with the levels of cross-reacting material. When nitrate was used as nitrogen source, NR was always present, even in cultures with ammonium, glutamine or peptone, although in these cases both the levels of activity and protein were lower. NR activity was consistently two to four times higher in cells grown in glucose than in cells grown in ethanol. Nitrate was required for NR ind uction, and deprivation of nitrate from nitrate-grown cells resulted in a rapid loss of NR activity.
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Inhibition of lipopolysaccharide synthesis in Agrobacterium tumefaciens and Aeromonas salmonicida
More LessLipopolysaccharide (LPS) synthesis was inhibited, new lipid A metabolites accumulated, and growth ceased, when the plant pathogen Agrobacterium tumefaciens and the fish pathogen Aeromonas salmonicida were treated with an antibacterial agent which specifically inhibits CTP:CMP-3-deoxy-manno-octulosonate cytidylyltransferase (CMP-KDO synthase). The new lipid A metabolites were purified by chromatography on DEAE-cellulose and chemically analysed. Metabolites isolated from both bacterial species contained glucosamine and phosphate in a 1:1 molar ratio, and 3-OH-C14:0 was the major fatty acid present (1 mol and 1.4 mol per mol glucosamine for A. tumefaciens and A. salmonicida, respectively). Inhibition of LPS synthesis by CMP-KDO synthase inhibitor had no effect on the initial kinetics of A. tumefaciens attachment to cultured carrot cells, but did inhibit cell aggregation normally induced by bacterial cellulose synthesis. Bacteria treated with inhibitor remained viable and able to synthesize protein at 15% the rate of control cells, indicating that the lack of cellulose-induced aggregation was not due to the inability of bacteria to make protein, but rather the inability to respond normally to the bacterial-plant cell interaction.
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- Systematics
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Taxonomy of xanthomonads from cereals and grasses based on SDS-PAGE of proteins, fatty acid analysis and DNA hybridization
L. Vauterin, P. Yang, B. Hoste, B. Pot, J. Swings and K. KerstersStrains of all Xanthomonas species, X. campestris pathovars pathogenic for cereals and grasses and strains of X. campestris pv. campestris were compared by SDS-PAGE of proteins, gas chromatography of fatty acid methyl esters and DNA-DNA hybridization. The groupings derived using all three methods correlated well with each other. Strains of X. axonopodis and X. campestris pv. vasculorum were heterogeneous, each comprising two groups (A and B); group A contained the type strain and pathovar reference strain, respectively. Besides the recognized Xanthomonas species X. albilineans, X. campestris, X. fragariae, X. maltophilia, X. oryzae and X. populi, the following five centres of variation could be delineated at DNA-binding levels of above 60%: (i) a group consisting of X. campestris pathovars translucens, hordei, cerealis, secalis, undulosa (the ‘translucens group’), graminis, poae, arrhenatheri, phlei (the ‘graminis group’) and phleipratensis; (ii) X. axonopodis type A together with X. campestris pv. vasculorum type A and X. campestris pv. coracanae; (iii) a group of X. campestris pv. vasculorum type B and X. campestris pv. holcicola; (iv) Xanthomonas strains isolated from Bromus; and (v) strains isolated from sugarcane in Guadeloupe. The type B strains of X. axonopodis appeared to be misclassified in Xanthomonas. An interim proposal for an improved classification of Xanthomonas is presented on the basis of the data.
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The use of rRNA sequences and fluorescent probes to investigate the phylogenetic positions of the anaerobic ciliate Metopus palaeformis and its archaeobacterial endosymbiont
More LessThe polymerase chain reaction (PCR) was used to amplify small-subunit ribosomal DNA from the anaerobic ciliated protozoon Metopus palaeformis, and from its uncultured endosymbiotic bacteria. This was accomplished directly from total DNA extracted from protozoa without prior isolation or enrichment for symbiont cells. The double-stranded amplification products were precipitated and directly sequenced using the linear PCR reaction. Fluorescent oligonucleotide probes were designed and used in whole-cell hybridizations to provide direct visual evidence that the sequences originated from the host ciliate and from the endosymbiont. Phylogenetic analysis of the Metopus palaeformis sequence consistently placed it as a deep-branching lineage near the root of the ciliate tree. However, the present data were insufficient to resolve the detailed relationship between Blepharisma and Metopus and thus to determine if the heterotrichs are mono- or paraphyletic. Phylogenetic analysis of the symbiont partial sequence clearly demonstrated that it is an archaeobacterium and that it is closely related to, but distinct from, Methanobacterium formicicum.
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Cellular fatty acid composition of symbiotic cyanobacteria isolated from the aquatic fern Azolla
More LessThe cellular fatty acid composition of 10 isolates of symbiotic cyanobacteria from 7 different species of the aquatic fern Azolla was investigated. Sixteen major components accounted for 88.31% of total fatty acids: the saturated 14:0, 16:0 and 18:0 carbon chains; the unsaturated straight-chained 12:1, 14:1 cis-7, 16:1 cis-7, 16:1 cis-9, 16:1 cis-11, 18:2 cis-9, 18:3 cis-9, 18:1 cis-and trans-9, and 20:4 cis-5; and the branch-chained iso-16:0. Also included was an unsaturated 16-carbon (equivalent carbon chain length of 15.5), with unsaturation sites undetermined. The most abundant component was the 16:0 (mean of 38.10% of the total). Thirty-six minor fatty acids, comprising 10.30% of the total, were detected and identified. These included hydroxy-substituted fatty acids (1.10%), branched chains in addition to the iso-16:0 (1.96% of a class total of 2.96%) and cyclopropane fatty acids (0.89%). A comparison of the fatty acid profile of Azolla cyanobionts with those previously published for free-living cyanobacteria of the genera Anabaena and Nostoc indicated that there were at least 19 individual fatty acids, class totals or ratios that were statistically different and could be used as differentiating factors. Nine of the 19 factors were characteristically unique to Azolla cyanobionts and different from both Anabaena and Nostoc. Five were different from only Anabaena, and five from only Nostoc. Based on one taxonomic interpretation of fatty acid analysis, the Azolla cyanobionts appeared to be equally distinct from Anabaena and from Nostoc.
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