- Volume 138, Issue 12, 1992
Volume 138, Issue 12, 1992
- Review Article
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- Biochemistry
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Secondary fatty alcohols of Mycobacterium xenopi
More LessSUMMARY: Secondary alcohols of Mycobacterium xenopi were studied by gas chromatography and gas chromatography-mass spectrometry. Mycobacterial cells were hydrolysed and the liberated alcohols separated by extraction and analysed both underivatized and as trimethylsilyl-, methyl ether- and pentafluorobenzoyl derivatives. Seven straight-chain secondary alcohols containing from 18 to 24 carbon atoms and two branched-chain secondary alcohols with 21 and 23 carbon atoms were present in all of the studied strains.
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Chemical modification studies of the active centre of Candida albicans chitinase and its inhibition by allosamidin
More LessSUMMARY: Allosamidin, a glycoside antibiotic, is shown to be a strong, competitive inhibitor of semi-purified chitinase from yeast cells of Candida albicans. The inhibitory potency of allosamidin was pH-dependent, with IC50 values of 280 nM at pH 5.0 and 21 nM at pH 7.5. At higher, micromolar, concentrations, allosamidin inactivated this chitinase in a time- and concentration-dependent manner. Kinetic studies of this inactivation provided evidence for the formation of a reversible complex between allosamidin and chitinase, characterized by K inact = 5 μM, followed by irreversible modification of the enzyme with velocity constant k 2 = 4.6 × 10-3.s-1. Chemical modification studies with the use of group-specific reagents suggested the presence of Glu/Asp carboxyl group(s) at or near the active site, that were important for enzyme activity. The carboxyl-specific reagent, 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide, inactivated the chitinase in a single step process, with apparent second-order rate constant of 0.014 M-1.s-1.
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Purification and characterization of a surface lectin from the nematode-trapping fungus Arthrobotrys oligospora
More LessSUMMARY: Several studies have indicated that the capture of nematodes by the nematophagous fungus Arthrobotrys oligospora is mediated by a lectin on the fungal surface. One of the major surface proteins of this fungus showed haemagglutinating activity and was isolated by affinity chromatography using a mucin Sepharose column. Biochemical analysis showed that the protein was a dimeric glycoprotein with a molecular mass of 36 kDa and an isoelectric point of pH 6.5, and contained no sulphur amino acids. The protein was N-terminally blocked; four internal peptides were sequenced, and showed no significant similarity to sequences in the Swiss-Prot or PIR databases. The haemagglutinating activity of the isolated protein was not inhibited by any of the mono- or disaccharides tested, but it was inhibited by the glycoproteins fetuin and mucin. The haemagglutinating activity changed after incubating the protein in buffers of different pH, with maximal activity at pH 11.0 and no activity at pH 2.8. The lectin was tested for different enzymic activities but none were detected. Analysis of the haemagglutinating activity in various cell fractions indicated that the protein was associated with extracellular polymer layers and with the cell wall of the fungus. About the same amount of the haemagglutinating protein was recovered from samples of vegetative mycelium and of mycelium containing nematode-trapping cells.
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- Development And Structure
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Succinylated lipid A is a potent and specific inhibitor of endotoxin mitogenicity
More LessSUMMARY: Chemically modified lipopolysaccharides of Salmonella abortus-equi were tested for mitogenicity on mouse spleen cells as well as antagonism of the mitogenicity of intact lipopolysaccharide (LPS). All the lipopolysaccharide preparations deacylated by different alkaline treatments suffered a drastic loss of mitogenicity. The mitogenic activity of lipid A was also lost when succinic residues were introduced on hydroxyl groups. Partially deacylated alkaline-treated preparations (but not completely deacylated preparations) inhibited the activation of splenic B-cells by LPS. They were found to be toxic to spleen cells, however, and to suppress not only the mitogenicity of LPS but that of concanavalin A as well. This inhibitory action was not exhibited when all of the fatty acid was eliminated. Succinylated lipid A, on the other hand, was not toxic to the cells and inhibited the B-cell mitogenicity of lipopolysaccharide (but not the T-cell mitogenicity of concanavalin A). Chemical analysis revealed that about 4.6 mol of succinic acid had been introduced into lipid A by succinylation, and that the fatty acid and phosphate composition was unchanged by this treatment. Macrophages do not seem to participate in this inhibition. Inhibition was observed when succinylated lipid A was added either at the same time or after lipid A mitogen, but optimal inhibition was expressed when it was added to the culture 3 h before LPS. Inhibition was not affected by washing the cells before adding LPS. Inhibition increased as the ratio of suppressor to mitogen increased, suggesting that the succinylated lipid A competes with intact LPS.
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Structural studies of an emulsion-stabilizing exopolysaccharide produced by an adhesive, hydrophobic Rhodococcus strain
T. R. Neu, T. Dengler, B. Jann and K. PorallaSUMMARY: The primary structure of an emulsion-stabilizing exopolysaccharide from the adhesive, hydrophobic Rhodococcus strain No. 33 was elucidated by NMR spectroscopy, methylation analyses, periodate oxidation and oligosaccharide analyses. The polysaccharide PS-33 consisted of rhamnose, galactose, glucose and glucuronic acid in molar ratios of 2:1:1:1. The main chain contained 3-substituted α-D-glucuronic acid linked to the 3-position at α-L-rhamnose, in addition to 3-substituted residues of β-D-galactose and α-D-glucose. The α-L-rhamnose of the side chain was linked to position 4 of the galactose. In addition, the polysaccharide was O-acetylated, corresponding to one acetyl group per repeating unit. From the results two structural possibilities could be suggested. As the polysaccharide carries hydrophobic groups (methyl of rhamnose/O-acetyl), it is very likely that these are of general significance for the emulsifying activity of polysaccharides. It also seems to be possible that this polysaccharide is at least partially responsible for the hydrophobic cell surface properties of the Rhodococcus strain No. 33 and it may be involved in hydrophobic interactions when adhering to hydrophobic interfaces.
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A developmental mutation (npfL1) resulting in cell death in Physarum polycephalum
More LessSUMMARY: In Physarum, microscopic uninucleate amoebae develop into macroscopic multinucleate plasmodia. In the mutant strain, RA614, plasmodium development is blocked. RA614 carries a recessive mutation (npfL1) in a gene that functions in sexual as well as apogamic development. In npfL + apogamic development, binucleate cells arise from uninucleate cells by mitosis without cytokinesis at the end of an extended cell cycle. In npfL1 cultures, apogamic development became abnormal at the end of the extended cell cycle. The cells developed a characteristic rounded, vacuolated appearance, nuclear fusion and vigorous cytoplasmic motion occurred, and the cells eventually died. Nuclei were not visible by phase-contrast microscopy in most of the abnormally developing cells, but fluorescence microscopy after DAPI staining revealed intensely staining, condensed nuclei without nucleoli. Studies of tubulin organization during npfL1 development indicated a high frequency of abnormal mitotic spindles and, in some interphase cells, abnormally thick microtubules. Some of these features were observed at low frequency in the parental npfL + strain and may represent a pathway of cell death, resembling apoptosis, that may be triggered in more than one way. Nuclear fusion occurred during interphase and mitosis in npfL1 cells, and multipolar spindles were also observed. None of these features were observed in npfL + cells, suggesting that a specific effect of the npfL1 mutation may be an incomplete alteration of nuclear structure from the amoebal to the plasmodial state.
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Bacterial differentiation within Moraxella bovis colonies growing at the interface of the agar medium with the Petri dish
More LessSUMMARY: Moraxella bovis was found to colonize the interface between agar and the polystyrene Petri dish, producing circular colonies when the inoculum was stabbed at a single point. The bacteria occurred in a thin layer of nearly uniform thickness, and colonial expansion occurred in at least two temporal phases. In the first phase, the radial colonial expansion was slow and non-linear. In the second phase, the radial expansion was linear. The interfacial colonies possessed three characteristic concentric growth zones. At the periphery was a narrow ring zone that enclosed another wider ring zone, which, in turn, surrounded a central circular zone. Different bacterial phase variants were recovered from these zones. The two outer ring zones yielded bacteria that formed agar surface colonies of spreading-corroding morphology, while cells from the innermost zone always yielded colonies with a different morphology. The uniform thickness of the colonies implied that replication was restricted to the outermost ring, and that the bacteria within the inner ring and inner circle had entered a quiescent state. The inner ring appeared to represent the lag in time needed for the replicative form to differentiate into the quiescent form. A different kind of variant was associated with wedge-shaped sectors within the colonies. The greatest number of these clonal variants appeared shortly after inoculation and their frequency decreased after the onset of linear growth. The period of slowest colonization coincided with highest frequency of clonal variant expression. It is proposed that the proliferative rate of the parental bacterial population exerted selective pressure on the expression of new clonal variants.
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The periplasmic flagella of Serpulina (Treponema) hyodysenteriae are composed of two sheath proteins and three core proteins.
SUMMARY: The major components of the periplasmic flagella of the spirochaete Serpulina (Treponema) hyodysenteriae strain C5 were purified and characterized. We demonstrate that the periplasmic flagella are composed of five major proteins (molecular masses 44, 37, 35, 34 and 32 kDa) and present their location, N-terminal amino acid sequence and immunological relationship. The 44 kDa and the 35 kDa protein are on the sheath of the periplasmic flagellum, whereas the 37, 34 and 32 kDa protein reside in the periplasmic flagellar core. The two sheath flagellar proteins are immunologically related but have different N-terminal amino acid sequences. The N-terminus of the 44 kDa protein shows homology with the sheath flagellins of other spirochaetes, but the 35 kDa protein does not. The three core proteins are immunologically cross-reactive and their N-terminal amino acid sequences are almost, but not completely, identical, indicating that the core proteins are encoded by three distinct genes. The core proteins show extensive N-terminal sequence similarities and an immunological relationship with periplasmic flagellar core proteins of other spirochaetes.
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Physico-chemical and structural properties of the surfaces of Peptostreptococcus micros and Streptococcus mitis as compared to those of mutans streptococci, Streptococcus sanguis and Streptococcus salivarius
More LessSUMMARY: The surface properties of nine Streptococcus mitis and four Peptostreptococcus micros strains from the oral cavity were examined and compared with a large group of oral streptococci. Zeta potential and contact angle measurements were employed to determine physico-chemical cell surface properties. In addition, elemental surface concentration ratios were obtained via X-ray photoelectron spectroscopy, and surface structures were examined with transmission electron microscopy. The S. mitis and P. micros strains were found to have higher isoelectric points, higher hydrophobicities and higher N/C surface concentration ratios than some other oral streptococci. The combined data suggest that both species possess large amounts of surface protein. All the S. mitis strains displayed abundant surface fibrils in negative staining, but the P. micros strains were devoid of surface appendages indicating that surface protein is present in different forms in the two species. The surfaces of S. mitis and P. micros type strains differed significantly from the other strains examined.
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- Genetics And Molecular Biology
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Virulence plasmid pJM1 prevents the conjugal entry of plasmid DNA into the marine fish pathogen Vibrio anguillarum 775
More LessSUMMARY: Studies involving the introduction of cloned homologous genes into Vibrio anguillarum revealed that several plasmids could not be conjugally introduced into V. anguillarum 775(pJM1), but were transmissible to the pJM1-cured derivative H775-3. Recombinant pBR322 plasmids containing V. anguillarum genomic DNA inserts were mobilized from Escherichia coli donors, using pRK2013, into V. anguillarum H775–3 recipients at frequencies of 10-6 to 10-5 per recipient. When identical matings were performed with V. anguillarum 775(pJM1) recipients, the infrequent exconjugants recovered carried the pBR322-based plasmid but had lost the large virulence plasmid pJM1. Similar studies were carried out with plasmid RP4 and with recombinant derivatives of the closely related broad-host-range plasmid pRK290. While RP4 was transmissible from E. coli to V. anguillarum H775–3 at frequencies of 6.7 × 10-2 per recipient, transmission to V. anguillarum 775(pJM1) recipients occurred at frequencies of only 2.5 × 10-7. When pRK290 contained V. anguillarum DNA inserts, the only exconjugants recovered had lost pJM1, or contained pJM1 and a deletion derivative of the recombinant pRK290 plasmid where all of the DNA insert had been deleted. The use of Dam-, Dcm-, or EcoK- methylation-deficient E. coli donor strains failed to result in appreciable numbers of V. anguillarum 775(pJM1) exconjugants that contained the desired transferred plasmids. Following UV mutagenesis, a derivative of V. anguillarum 775(pJM1) was isolated that would accept conjugally transferred plasmid DNAs at frequencies similar to those observed when using V. anguillarum H775–3 recipients. These data suggest that virulence plasmid pJM1 mediates a restriction system that prevents conjugal transmission of plasmid DNA from E. coli donors into V. anguillarum 775(pJM1). This putative restriction system appears not to be directed towards Dam-, Dem-, or EcoK-methylated DNA, and appears not to involve a Type II restriction endonuclease.
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A gene suppressing the allelic protoplasmic incompatibility specified by genes at five different loci in Podospora anserina
More LessSUMMARY: Protoplasmic incompatibility (PI) in fungi is a phenomenon of immediate cell destruction resulting from the fusion of cells of unlike genotypes. In Podospora anserina, five loci contain genes determining PI as the result of allelic gene interactions. The present work shows that mutations of a gene called modD inhibit allelic PI irrespective of the locus responsible for the phenomenon. Other modD mutations show differential actions on the allelic interactions and on the expression of the two allelic incompatibility genes of the same locus. These results thus suggest that the modD gene product is involved in the trigger mechanism of allelic PI. The modD gene acts in differentiation: it has been previously shown to control proteolytic activities required for exit from stationary phase. There is thus a connection between this function and allelic PI. This leads to the suggestion that allelic incompatibility genes are involved in the control of stationary phase exit to promote differentiation.
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Isolation and characterization of new fluoroacetate resistant/acetate non-utilizing mutants of Neurospora crassa
More LessSUMMARY: Sixty-two mutants of the filamentous fungus Neurospora crassa were isolated on the basis of resistance to the antimetabolite fluoroacetate. Of these, 14 were unable to use acetate as sole carbon source (acetate non-utilizers, acu) and were the subject of further genetic and biochemical analysis. These mutants fell into four complementation groups, three of which did not complement any known acu mutants. Mutants of complementation group 3 failed to complement acu-8, demonstrated similar phenotypic properties and proved to be closely linked (less than 2% recombination) but not allelic. Representatives of groups 2 and 4 were mapped to independent loci; the single representative of group 1 could not be mapped. The four complementation groups were therefore designated as genes acu-10 to acu-13 respectively. All the mutants demonstrated normal acetate-induced expression of acetyl-CoA synthetase and the unique enzymes of the glyoxylate cycle and gluconeogenesis. The nature of these mutations is therefore quite different to those reported for other fungal species. Mutant acu-11 was unable to fix labelled acetate, indicating the loss of an initial transport function; partial enzyme lesions were observed for acu-12 (acetyl-CoA hydrolase) and acu-13 (acetate-inducible NAD+-specific malate dehydrogenase).
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Structure and function of the spoIIIJ gene of Bacillus subtilis: a vegetatively expressed gene that is essential for σG activity at an intermediate stage of sporulation
More LessSUMMARY: The spo-87 mutation is one of two sporulation mutations originally used to define the spo0J locus of Bacillus subtilis. We now show that it blocks sporulation after completion of prespore engulfment (stage III). Surprisingly, the operon is expressed vegetatively, probably from a σA-dependent promoter, and its expression is shut down at the transcriptional level at about the onset of sporulation. DNA sequencing reveals that the locus defined by spo-87, which we now designate spoIIIJ, consists of a bicistronic operon. However, only the first gene is essential for sporulation; the function of the second cistron is cryptic. The predicted SpoIIIJ product has an M r of 29409. It probably forms a lipoprotein and is rich in basic and hydrophobic amino acids. Mutations in spoIIIJ abolish the transcription of prespore-specific genes transcribed by the σG form of RNA polymerase but not transcription of the spoIIIG gene encoding σG. The SpoIIIJ product could be involved in a signal transduction pathway coupling gene expression in the prespore to events in the mother cell, or it could be necessary for essential metabolic interactions between the two cells.
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The use of bacterial luciferase for monitoring the environmental regulation of expression of genes encoding virulence factors in Listeria monocytogenes
More LessSUMMARY: A promoter probe vector, which utilized the luxAB genes from Vibrio fischeri as reporters of gene expression, was constructed for use in Listeria monocytogenes. Using this system gene expression can be monitored non-destructively and in real-time, simply by measuring cellular bioluminescence. Derivatives of the promoter probe were constructed that contained the cloned promoters from the hlyA and plcA genes of L. monocytogenes. The activity of these promoters was dependent on the transcriptional activator PrfA. Accordingly, in a strain containing an intact copy of the prfA gene, expression from both the hlyA and plcA promoters was 25–45-fold higher than in prfA mutants. Heat shock was identified as an environmental signal which induced expression of hlyA and plcA. Conversely, oxidative stress had no effect upon the expression of the virulence factors. In addition, the composition of the growth media was found to have a dramatic effect upon the expression of hlyA and plcA, suggesting the presence of an unidentified signal which may regulate induction of expression of virulence genes in L. monocytogenes.
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Distinct genetic groups of Giardia intestinalis distinguished by restriction fragment length polymorphisms
More LessSUMMARY: The taxonomic status of the parasitic protozoal species Giardia intestinalis depends on the morphological similarity of all Giardia isolated from humans and the presumption that Giardia are host-specific. On the basis of electrophoretic data derived from examination of 26 enzyme loci in Australian isolates, it has been proposed that G. intestinalis is a species complex comprising three or four genetically distinct (but morphologically cryptic) species. These received the tentative designations of genetic groups I-IV (R. H. Andrews, M. Adams, P. F. L. Boreham, G. Mayrhofer & B. P. Meloni. International Journal for Parasitology 19, 183–190, 1989). In the present study, two unrelated DNA probes (one specific for a gene encoding a trophozoite surface protein, the other detecting a non-coding repetitive sequence within the G. intestinalis genome) were used in Southern hybridization analyses to examine 10 axenic isolates of G. intestinalis, established from diverse geographical regions in Australia, together with the Portland-1 isolate from the USA. Both probes identified every isolate unambiguously as belonging to one or other of two genetic clusters. Electrophoretic analysis of the same samples indicated that these clusters correspond to the previously defined genetic groups I and II. No heterogeneity was apparent within the seven group I isolates using either probe. However, when probed with the repetitive sequence, the four isolates belonging to group II exhibited small differences in banding patterns, suggesting that this group may be less homogeneous than group I. The consistency of the Southern blot data with the allozyme groupings confirms group I and group II as distinct genetic subgroups of G. intestinalis and establishes that selected restriction sites can be used as taxonomic markers for the purposes of a genetically based systematics for G. intestinalis.
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Purification and amino acid sequence of sakacin A, a bacteriocin from Lactobacillus sake Lb706
More LessSUMMARY: Sakacin A, a bacteriocin produced by Lactobacillus sake Lb706 and which inhibits the growth of Listeria monocytogenes, was purified to homogeneity by ammonium sulphate precipitation and ion-exchange, hydrophobic-interaction and reversed-phase chromatography. The complete amino acid sequence of sakacin A was determined by Edman degradation. The bacteriocin consisted of 41 amino acid residues and had a calculated M r of 4308.7, which is in good agreement with the value determined by mass spectrometry. The structural gene encoding sakacin A (sakA) was cloned and sequenced. The gene encoded a primary translation product of 59 amino acid residues which was cleaved between amino acids 18 and 19 to yield the active sakacin A. Sakacin A shared some sequence similarities with other bacteriocins.
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A new colicin that adsorbs to outer-membrane protein Tsx but is dependent on the tonB instead of the tolQ membrane transport system
More LessSUMMARY: A new colicin, Col5, was synthesized by an Escherichia coli isolate of human origin from the ECOR Collection. It was unique because it adsorbed to the outer-membrane protein Tsx, but used the tonB rather than the tolQ membrane transport system, which is employed by the only other Tsx-specific colicin, ColK. Col5 was encoded by a 5.2 kb plasmid, p5. It was inducible by mitomycin C, and strains harbouring p5 exhibited quasi-lysis. The bactericidal protein had an M r of 56000.
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Characterization and purification of mesentericin Y105, an anti-Listeria bacteriocin from Leuconostoc mesenteroides
More LessSUMMARY: A Leuconostoc mesenteroides ssp. mesenteroides was isolated from goat's milk on the basis of its ability to inhibit the growth of Listeria monocytogenes. The antimicrobial effect was due to the presence in the culture medium of a compound, named mesentericin Y105, excreted by the Leuconostoc mesenteroides Y105. The compound displayed known features of bacteriocins from lactic acid bacteria. It appeared as a proteinaceous molecule exhibiting a narrow inhibitory spectrum limited to genus Listeria. The apparent relative molecular mass, as indicated by activity detection after SDS-PAGE, was 2.5–3.0 kDa. The bacteriocin was purified to homogeneity by a simple three-step procedure: a crude supernatant obtained from an early-stationary-phase culture in a defined medium was subjected to affinity chromatography on a blue agarose column, followed by ultrafiltration through a 5 kDa cut-off membrane, and finally by reverse-phase HPLC on a C4 column. Microsequencing of the pure bacteriocin and of tryptic fragments showed that mesentericin Y105 is a 36 amino acid polypeptide whose primary structure is close to that of leucocin A-UAL 187, which contains an extra residue at the C-terminus and displays only two differences in the overlapping sequence. However, unlike leucocin A-UAL 187, mesentericin Y105 displayed a bactericidal mode of action.
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- Pathogenicity And Medical Microbiology
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Pathogenic activities of live cells and extracellular products of the fish pathogen Pasteurella piscicida
More LessSUMMARY: The pathobiological activities in vivo and in vitro of live cells and extracellular products (ECP) of eleven Pasteurella piscicida strains of different origin were examined. Infectivity trials showed that P. piscicida did not possess strict host specificity since the majority of the isolates were virulent for gilthead seabream, rainbow trout and turbot, with LD50 values ranging between 103 and 106 live cells. However, none of the strains tested were pathogenic for mice (LD50 > 108 cells). In addition, the ECP were strongly toxic for fish (LD50 ranging from 1.0 to 4.6 μg protein per g fish), which clearly demonstrates their important role in the pathogenesis of pasteurellosis. All the ECP samples were cytotoxic for fish and homoiothermic cell lines, possessed notable phospholipase activity and displayed haemolytic activity for sheep, salmon and turbot erythrocytes (but not for trout erythrocytes). However, the production of proteolytic enzymes differed among the P. piscicida strains. Although no strain displayed elastase activity, five isolates (the Japanese and Italian strains) hydrolysed casein and gelatin. All these biological activities in vivo and in vitro were lost after heat treatment (100 °C for 10 min). The general enzymic patterns of both live cells and ECP evaluated by the API-ZYM system also revealed some variation among the P. piscicida isolates. Generally, whole cells showed a wider range of enzymic activities than ECP. The results presented here are important for the selection of strains in the development of effective polyvalent pasteurellosis vaccines containing both whole cells and ECP.
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Molecular analysis of isolates of Streptoccocus suis capsular type 2 by restriction-endonuclease-digested DNA separated on SDS-PAGE and by hybridization with an rDNA probe
SUMMARY: This study was undertaken to assess the discriminatory value of restriction endonuclease (RE) digestion patterns of Streptococcus suis chromosomal DNA using polyacrylamide gel electrophoresis (SDS-PAGE) and DNA-rDNA hybridization. For the RE digestion patterns, DNAs were digested separately with the enzymes BamHI and BglII and the resultant fragments were separated by SDS-PAGE. An Escherichia coli rDNA probe derived from pKK3535 was used for the hybridization. Twenty-three S. suis capsular type 2 isolates recovered from diseased and clinically healthy pigs, from a human case, and from a cow were compared in this study. The majority of isolates associated with septicaemia belonged to one restriction endonuclease analysis (REA) profile group. Isolates associated with pneumonia belonged either to the REA profile group of isolates associated with septicaemia or to a second REA profile group. The REA profiles of isolates from clinically healthy animals were more heterogeneous. The REA profile of the type 2 reference strain, S735, which was originally isolated from a pig, was very different from those of the porcine and bovine isolates but similar to the profile of the human isolate. The profiles obtained after rDNA hybridization were more homogeneous. Although different patterns were detected in the 23 isolates, there was no correlation between the source of the isolate and the patterns observed with this technique.
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Identification of an outer-membrane haemoglobin-binding protein in Neisseria meningitidis
More LessSUMMARY: Although Neisseria meningitidis can use haemoglobin as an iron source in vitro, the mechanism of haemoglobin-iron uptake is unknown. Using a biotinylated human haemoglobin probe in a solid-phase dot-binding assay, haemoglobin-binding activity was detected in total membranes derived from meningococci grown under iron-limited but not iron-sufficient conditions. In competition binding experiments, bovine and human haemoglobin could abrogate binding. In contrast, no binding inhibition was seen with ferric nitrate, protoporphyrin IX, and iron-loaded human transferrin. The ability of both haemin and catalase, a nonhaemoglobin haem-containing compound, to inhibit binding competitively suggested that the ligand recognized by the binding protein is the haem moiety. Scatchard plot analysis revealed a heterogeneous receptor population. Limited proteolysis with proteinase K abolished binding activity, suggesting a haemoglobin-protein interaction. Detection of activity in a whole-cell binding assay demonstrated that this haemin-binding protein was surface exposed. In a limited survey of meningococcal strains, the presence of haemoglobin-binding activity in all isolates indicated that expression of this binding protein is not serogroup specific.
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- Physiology And Growth
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Glycine betaine transport by Staphylococcus aureus: evidence for two transport systems and for their possible roles in osmoregulation
More LessSUMMARY: The transport of glycine betaine by Staphylococcus aureus was investigated. Two transport systems were found that could be differentiated on the basis of their affinity for glycine betaine and their activation by osmotic pressure. The high-affinity system was relatively independent of osmotic pressure and exhibited a K m of approximately 3 μM. This system was not inhibited by proline, for which a separate high-affinity transport system has been recently discovered. The low-affinity system was activated approximately 35-fold by an increase in osmotic pressure and exhibited a K m of approximately 130 μM for glycine betaine. This system is partially inhibited by excess proline and may be identical to the low-affinity system recently described for proline. Both glycine betaine transport systems are Na+-dependent.
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Energy production from L-malic acid degradation and protection against acidic external pH in Lactobacillus plantarum CECT 220
More LessSUMMARY: Malate degradation by Lactobacillus plantarum CECT 220 provides energy which enables this organism to remain viable for longer at low environmental pH values. Energy production was not coupled to H+-ATPase activity. This protective mechanism against acidic external pH is complemented by another system of ΔpH maintenance. The H+-ATPase did not seem to be involved in this system of pH maintenance as the system was not sensitive to N, N'-dicyclohexylcarbodiimide (DCCD) and was functional at very low pH values where ATPase activity was severely inhibited.
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Respiratory dependence of the CCCP-resistant luminescence of Vibrio harveyi
More LessSUMMARY: The relation between respiration and luminescence of Vibrio harveyi was studied in the presence of the proton conductor CCCP (carbonyl cyanide m-chlorophenylhydrazone). CCCP affected both luminescence and oxygen uptake rate, but did not alter the cellular ATP level. In the absence of CCCP, luminescence and oxygen uptake rate were almost constant over the pH range 6.5–8.5. However, at 10 μM-CCCP, both were apparently pH-dependent, with maxima observed at pH 8.5 and minima at pH 6.5. In the presence of CCCP (0.1–10 μM), the changes in the luminescence were in close correlation with those in the oxygen uptake rate regardless of the pH. HQNO (2-heptyl-4-hydroxyquinoline-N-oxide) and NaCN inhibited the CCCP-resistant luminescence. From these results, it is concluded that the CCCP-resistant luminescence is respiratory-dependent.
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Regulation of the secretion of Rhizopus oligosporus extracellular carboxyl proteinase
More LessSUMMARY: Secretion of the extracellular Rhizopus carboxyl proteinase (EC 3.4.23.6) by Rhizopus oligosporus is repressed in the presence of low-molecular-mass sources of nitrogen, sulphur and carbon. Proteinase is secreted when the medium is deficient in any one of these three nutrients. In the case of nitrogen metabolite repression, control is at the level of transcription. Induction of proteinase secretion by exogenous protein does not occur in any of the media examined.
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TPK gene products mediate cAMP-independent thermotolerance in Saccharomyces cerevisiae
More LessSUMMARY: Incubation of Saccharomyces cerevisiae with the plant cytokinin N 6-(Δ2-isopentenyl)adenine (2iP) resulted in an induction of thermotolerance similar to that induced by sublethal temperatures. Intracellular cAMP levels did not change significantly either during incubation at a sublethal temperature or in the presence of 2iP or ethanol. This suggested that stress-induced thermotolerance is triggered by a mechanism independent of cAMP activation. However, measurement of stress-induced thermotolerance in two mutant strains (tpk1, tpk2, TPK3; tpk1, TPK2, tpk3) each deficient in two of the catalytic subunits of the cAMP-dependent protein kinase (cAPK), revealed that sublethal heat induces thermotolerance by a mechanism part-mediated by the catalytic subunits of cAPK. In contrast, 2iP and ethanol induced thermotolerance by a mechanism fully dependent on the catalytic subunits of cAPK for expression. Therefore, this implies there must be an alternative novel mechanism, other than cAMP, for activating cAPK during stress. Sublethal heating resulted in large increases in intracellular trehalose levels which correlated with the induction of thermotolerance. However, incubation in 2iP or ethanol had no significant effect. This suggests trehalose synthesis is either coincidental with heat stress or that different stress factors induce thermotolerance by alternative mechanisms. Incubation with protein synthesis inhibitors reduced the levels of trehalose synthesized during sublethal heating, suggesting that synthesis of trehalose-6-phosphate synthase during heat stress could be accounting for the increased trehalose levels.
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Analysis of transcription and translation of glycolytic enzymes in glucose-limited continuous cultures of Saccharomyces cerevisiae
More LessSUMMARY: mRNA steady-state levels and activities of enzymes of intermediary carbon metabolism (hexokinase, phosphoglucoisomerase, phosphofructokinase, glucose-6-phosphate dehydrogenase, phosphoglucomutase) and glucose-regulated enzymes (pyruvate decarboxylase, pyruvate dehydrogenase, invertase, alcohol dehydrogenase) were determined in glucose-limited continuous cultures of an industrial strain of Saccharomyces cerevisiae at different dilution rates (D) ranging from 0.05 to 0.315 h-1. The activity of most enzymes measured remained constant over this range except for alcohol dehydrogenase I/II which decreased proportionally with increasing dilution rate. A decrease in phosphoglucomutase activity occurred with increasing dilution rate but reached a minimum at D 0.2 h-1 and from thereon remained constant. A decrease in pyruvate decarboxylase activity and a slight decrease in phosphoglucoisomerase activity was observed. At D 0.29/0.315 h-1, at the onset of the Crabtree effect, most glycolytic enzymes remained constant except for pyruvate decarboxylase and glucose-6-phosphate dehydrogenase which increased at D 0.315 h-1 and alcohol dehydrogenase I/II which decreased. The ADHI/II and PDC1 mRNA levels obtained at the different dilution rates were in accordance with the activity measurements. The mRNA level of HXK1 decreased with increasing dilution rates, whereas the transcription of HXK2 increased. Pyruvate dehydrogenase (PDA1) and PG.11 mRNA fluctuated but no significant change could be detected. These results indicate that there is no transcriptional or translational regulation of glycolytic flux between D 0.05 h-1 and 0.315 h-1 except at the branch point between oxidative and fermentative metabolism (pyruvate decarboxylase/pyruvate dehydrogenase) at D 0.315 h-1. Surprisingly regulation of the Crabtree effect does not seem to involve transcriptional regulation of PDA1. The concentrations of ATP and cAMP decreased slightly during the increase in dilution rate, but increased again at D 0.315 h-1. The concentrations of glucose 6-phosphate and fructose 6-phosphate did not increase when the dilution rate increased as expected from the activities of hexokinase, phosphoglucoisomerase and phosphofructokinase. Instead, a decrease in the glucose 6-phosphate and fructose 6-phosphate concentrations was observed. The concentration of glucose 1-phosphate also decreased with increasing dilution rate but increased again at D 0.29 and 0.315 h-1, whereas the fructose 1,6-diphosphate concentration increased from 0.05 h-1 to 0.315 h-1. These data indicate that glycolytic flux in S. cerevisiae is regulated mainly by allosteric regulation of glycolysis when growth rate is increased. Invertase was present (mRNA and activity) at every dilution rate which indicates that glucose-specific repression of enzyme systems is not present in glucose-limited continuous cultures, not even when the yeast produces ethanol. This also indicates that the Crabtree effect is not related to glucose repression.
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Differential extracellular enzyme production in colonies of Coriolus versicolor, Phlebia radiata and Phlebia rufa: effect of gaseous regime
More LessSUMMARY: The effects of different gaseous regimes on the growth rate and extracellular enzyme location in vitro of colonies of Phlebia radiata, Phlebia rufa and Coriolus versicolor are reported. The two Phlebia species showed similar growth, extracellular enzyme and pH responses to gaseous composition (N2, O2 and CO2), but the responses of C. versicolor differed. Whilst maximum extension rates were obtained for all species under atmospheric gaseous composition, maximum biomass production occurred at 5% (v/v) O2 with 20% (v/v) CO2 for the Phlebia species and at 5% O2 with 60% CO2 for C. versicolor. The Phlebia species had a coenocytic margin (5–6 mm width) under atmospheric conditions, which increased in width with increasing percentage of CO2. Laccase and peroxidase activity were present throughout the septate region, but not in the coenocytic zone. With C. versicolor laccase and peroxidase activities appeared throughout the colony, but were more intense in the peripheral region, under all gaseous regimes. A laser densitometer, normally used to visualize proteins on electrophoresis strips, was used to estimate profiles of biomass and laccase-α-naphthol activity within colonies. Surface pH changed little in colonies of C. versicolor but dropped by over 1 pH unit from the margin inwards with the Phlebia species. The significance of these results is discussed in terms of ecological strategy and developmental versatility.
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Adhesion to porcine squamous epithelium of saccharide and protein moieties of Lactobacillus fermentum strain 104-S
More LessSUMMARY: The mechanism by which Lactobacillus fermentum strain 104-S adheres to porcine squamous epithelium was investigated by studying the adsorption to epithelial cells, and control surfaces, of radioactively labelled material released from the bacterial cells by water extraction. The released material was fractionated by gel filtration and the adsorption of pronase-sensitive and -resistant material in the various fractions to porcine gastric tissue and the control surfaces of polystyrene and immobilized bovine serum albumin (BSA) was determined. The fraction with affinity for the epithelium was characterized by enzymic degradation, periodate oxidation, lipid extraction, and protein and carbohydrate analyses. The adsorption pattern of radioactively labelled crude released material mimicked the adhesion of whole labelled cells to polystyrene and to gastric squamous tissue pieces. On fractionation, the pattern of adsorption to polystyrene and BSA was different from that obtained for the tissue pieces. Considerably less labelled pronase-stable material bound to surfaces of polystyrene and BSA, as compared with the tissue, suggesting that the pronase-resistant component has a tissue-specific affinity. After pronase treatment of the fraction of M r about 20000 (20 K) containing labelled components with affinity for the epithelium, only saccharides were detected. Radioactivity was lost after hydrolysis with HCl, and therefore this pronase-resistant labelled component must be a saccharide. It is concluded that protein moieties in the extract have an affinity for several surfaces, including polystyrene, and that saccharide moieties have a specific affinity for the gastric squamous epithelium.
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Heterocyst envelope thickness, heterocyst frequency and nitrogenase activity in Anabaena flos-aquae: influence of exogenous oxygen tension
More LessSUMMARY: The heterocyst envelope of the N2-fixing cyanobacterium Anabaena flos-aquae thickened as exogenous O2 partial pressure (pO2) was increased from 5 to 40 kPa. The majority of the thickening occurred in the glycolipid layer area of the envelope. Such thickening appears to be an O2-induced mechanism for providing a greater O2 diffusion barrier against O2 inhibition of nitrogenase. Nitrogenase activity decreased at pO2 levels above ambient (20 kPa), indicating that a thicker envelope is not completely effective as a barrier to O2 diffusion. However, when cultures grown at 10 kPa and 40 kPa pO2 were transferred to ambient pO2, the 40 kPa pO2 cells showed higher nitrogenase activity 24 h after transfer compared to those grown at 10 kPa or ambient pO2, indicating O2-protection of nitrogenase by thicker heterocyst envelopes. Heterocyst frequency was lowest at 20 kPa O2.
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Regulation of nitrogenase activity in relation to the light-dark regime in the filamentous non-heterocystous cyanobacterium Trichodesmium sp. NIBB 1067
More LessSUMMARY: A periodicity in nitrogen fixation potential with respect to the light-dark regime was studied in the filamentous non-heterocystous cyanobacterium Trichodesmium sp. NIBB 1067. During a 12 h light/12 h dark cycle, potential nitrogenase activity measured by acetylene reduction in the light was insignificant in the dark period, but developed after illumination for 1 to 3 h. Maximum nitrogenase activity was found at the middle of the light period, and activity decreased near the end of the light period. Manipulation of the length of the light and dark periods, and use of the glutamine synthetase inhibitor L-methionine sulphoximine, led to the conclusion that (1) the periodicity in activity was not attributable to an endogenous rhythm, (2) development and maintenance of nitrogenase activity in Trichodesmium was regulated by the light period, and (3) the decrease in activity at the end of the light period was due to the accumulation of an intermediate(s) in nitrogen metabolism. The nitrogenase Fe- and MoFe-proteins were always present despite the changes in nitrogenase activity associated with the light-dark cycle. However, a change in apparent molecular mass of the Fe-protein on SDS-PAGE correlated with the change in nitrogenase activity. The results indicate that changes of nitrogenase activity in Trichodesmium under a light-dark regime can be attributed to activation and deactivation of the Fe-protein, and that the activation of the protein depends on light.
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The role of VirA and VirG phosphorylation in chemotaxis towards acetosyringone by Agrobacterium tumefaciens
More LessSUMMARY: The Ti-plasmid-encoded two-component sensor-regulator system comprising VirA and VirG confers upon Agrobacterium tumefaciens the ability to respond chemotactically to nanomolar concentrations of vir-inducing phenolics such as acetosyringone. Non-phosphorylatable, mutant VirA and VirG proteins are incapable of replacing their wild-type counterparts in conferring this phenotype. This indicates that, like vir-gene induction in response to acetosyringone, chemotaxis to the same ligand involves phosphorylation of VirA and VirG. However, unlike vir-induction, deletion of the periplasmic domain of VirA severely curtails acetosyringone chemotaxis, suggesting that acetosyringone may mediate effects through more than one region of VirA. When introduced into strains expressing wild-type VirA and VirG, the non-phosphorylatable versions suppress chemotaxis towards acetosyringone, implying that mutant copies of VirA and VirG compete with their wild-type counterparts in interactions between VirA and VirG.
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