- Volume 139, Issue 5, 1993
Volume 139, Issue 5, 1993
- Biochemistry
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Characterization of the trypsin-like enzymes of Porphyromonas gingivalis W83 using a radiolabeled active-site-directed inhibitor
More LessThe trypsin-like enzyme activity of Porphyromonas gingivalis is an important virulence determinant of this organism in destructive periodontitis. An active-site-directed inhibitor, tyrosyl-alanyl-lysyl-arginine chloromethyl ketone (YAKR-CK) was radio-iodinated and used with SDS-PAGE and autoradiography to determine the number and molecular masses of enzymes with trypsin-like specificity produced by P. gingivalis W83. Two forms (I & II) were detected in both crude culture supernatant and whole cell sonicates. Protease I was a sharp band (47 kDa) on reducing SDS-PAGE; Protease II electrophoresed as a diffuse band in the range 70–90 kDa. The specificity with which the inhibitor bound to Protease I was established in competition experiments using other active-site-directed agents. YAKR-CK inhibited P. gingivalis whole cell haemagglutination, supporting the possible role of trypsin-like proteases of this organism in adhesion mechanisms.
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Isolation and characterization of urease from Aspergillus niger
More LessSummary: Urease was purified (4126-fold) from Aspergillus niger (NRRL 003) to a homologous enzyme preparation with a specific activity of 1341 μmol min−1 (mg protein)−1. One species of urease was detected in A. niger, with K m = 3.0 mM, native molecular mass 250 000 Da, pH optimum of 8.0 and a high specificity for urea. Hydroxyurea was a strong competitive inhibitor of urease activity, while N-methylurea acted as a weak uncompetitive inhibitor, based on Lineweaver-Burk and Eadie-Hoftstee plots. The activity of urease was enhanced by, but not dependent on, the presence of Na2EDTA, DL-dithiothreitol (≤ 0.1 to 5.0 mM), Ca2+, Ba2+ and citrate (2 to 20 mM). Urease activity was not affected by Na+, K+, Cl−, Br−, acetate or nitrate (2 to 20 mM), but was significantly decreased in the presence of Li+, Ni2+, Mg2+, Zn2+ or I−. Urease activity decreased 26.0% after 30 min at 65 °C, and 86.5% and 100.0% after 5 and 1 min at 80 and 100 °C, respectively. Urease activity decreased 30.5% after 90 d at 4 °C and 21.0% after 28 d at −20 or −80 °C.
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Pyrification and properties of an extracellular α-glucosidase from the thermophilic fungus Malbranchea sulfurea
More LessSummary: An extracellular α-glucosidase from the thermophilic fungus Malbranchea sulfurea was purified 31.6-fold with a yield of 11.68%. The size of the enzyme was 110 kDa. The optimum pH and temperature for the enzyme activity were pH 4.8 and 60°C, respectively; the half-life of the activity was 45 min at 65°C, 125 min at 60°C, and 6 h at 55°C. The substrate preference of the α-glucosidase was p-nitrophenyl-α-D-glucoside > sucrose > maltose > maltotriose. No activity was recorded against isomaltose, methyl-α-D-glucoside and starch.
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- Environmental Microbiology
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Archaeal halophiles (halobacteria) from two British salt mines
More LessSamples were taken from the Winsford salt mine in Cheshire, England, which exploits bedded deposits from the Triassic Period (195–225 million years ago, MYA) and from Boulby potash mine in Cleveland, England, which is Permian (225–270 MYA) and is mined for the mineral sylvite (KCl). Halobacteria and obligately halophilic eubacteria were isolated from several different sample types. The halobacteria were characterized by chemotaxonomic methods and most but not all were shown to be very similar but not identical to those halobacterial types that dominate in highly concentrated surface brines. There was a high degree of similarity between the two mine populations, but some strains were particular to each mine.
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- Genetics And Molecular Microbiology
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Regulation of carotenoid and bacteriochlorophyll biosynthesis genes and identification of an evolutionarily conserved gene required for bacteriochlorophyll accumulation
More LessThe temporal expression of ten clustered genes required for carotenoid (crt) and bacteriochlorophyll (bch) biosynthesis was examined during the transition from aerobic respiration to anaerobiosis requisite for the development of the photosynthetic membrane in the bacterium Rhodobacter capsulatus. Accumulation of crt A, crtC, crtD, crtE, crtF, crtK, bchC and bchD mRNAs increased transiently and coordinately, up to 12-fold following removal of oxygen from the growth medium, paralleling increases in mRNAs encoding pigment-binding polypeptides of the photosynthetic apparatus. The crtB and crtI genes, in contrast, were expressed similarly in the presence or absence of oxygen. The regulation patterns of promoters for the crt A and crtI genes and the bchCXYZ operon were characterized using lacZ transcriptional fusion and qualitatively reflected the corresponding mRNA accumulation patterns. We also report that the bchI gene product, encoded by a DNA sequence previously considered to be a portion of crtA, shares 49% sequence identity with the nuclear-encoded Arabidopsis thaliana Cs chloroplast protein required for normal pigmentation in plants.
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The ftf gene encoding the cell-bound fructosyltransferase of Streptococcus salivarius ATCC 25975 is preceded by an insertion sequence and followed by FUR1 and clp P homologues
Analysis of the region downstream of the ftf gene of Streptococcus salivarius led to the detection of two open reading frames (ORFs). The deduced amino acid sequences of these ORFs were homologous to proteins encoded by genes not previously described and/or sequenced in Gram-positive bacteria. The deduced amino acid sequence of the first of these (orf2) showed strong homology to the product of the FUR1 gene of Saccharomyces cerevisiae, which codes for a uracil phosphoribosyltransferase. The over-expression of the product of this gene appeared to be the source of the detrimental effect observed with phagemids carrying orf2 in Escherichia coli hosts. The deduced amino acid sequence of the second ORF (orf3) was homologous to the ClpP family of proteases. Examination of the upstream region of the ftf gene led to the discovery of a new insertion sequence-like element which has been designated IS1161.
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Isolation, characterization and sequence analysis of the scrK gene encoding fructokinase of Streptococcus mutans
More LessA gene encoding an ATP-dependent fructokinase from Streptococcus mutans GS-5 was identified within a 2 kb DNA fragment immediately downstream from the scrA gene.The gene cloned in Escherichia coli also expressed mannokinase activity. Insertional inactivation of this gene in S. mutans markedly decreased both fructokinase and mannokinase activities. Nucleotide sequence analysis of the 2 kb fragment revealed an ORF starting 199 bp downstream from the scr A gene, preceded by potential ribosome-binding (Shine-Dalgarno) and promoter-like sequences. This ORF specified a putative protein of 293 amino acids with a calculated M r of 31681. The deduced amino acid sequence of the fructokinase gene, scrK, from S. mutans exhibited no significant similarity to fructokinase genes from Klebsiella pneumoniae, E. coli plasmid pUR400 or Vibrio alginolyticus, but was similar to a comparable gene from Zymomonas mobilis.
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Cloning, sequence analysis and expression in Escherichia coli of a gene encoding an alginate lyase from Pseudomonas sp. OS-ALG-9
More LessSummary: A gene (aly) encoding alginate lyase (ALY; EC 4.2.2.3) was isolated from a library constructed with the cosmid vector pHC79 and Sau3AI-digested genomic DNA of Pseudomonas sp. OS-ALG-9. Successive subcloning of the aly-containing cosmid enabled us to locate the gene on a 2.3 kb HpaI fragment. Nucleotide sequencing of this fragment revealed a single open reading frame (ORF) of I365 bp. The directly determined N-terminal amino acid sequence of the ALY protein purified from Pseudomonas sp. OS-ALG-9 was found in the amino acid sequence deduced from this ORF between nucleotides 282 and 366. Expression of aly was induced by IPTG in Escherichia coli and leakage of the enzyme into the extracellular milieu was significantly enhanced by addition of glycine to the growth medium. The ALY enzyme had a greater specificity for the homopolymer of mannuronate than for that of guluronate.
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Functional and physiological characterization of the Tn21 cassette for resistance genes in Tn2426
More LessSummary: The Tn21 subgroup of class II transposons plays an important role in the dissemination of resistance genes and especially in the epidemic spread of multi-resistance. This ability reflects the variety of resistance genes that associate with the streptomycin/spectinomycin-resistance gene (aadA) of Tn21. Deletion experiments with Tn2426, a typical member of the Tn21 subgroup, and sequencing of the region that accommodates additional resistance genes revealed significant structural characteristics. Each resistance gene was flanked by short, directly repeated recombinationally active sequences with unexpected variability in their sequence and length. The consensus for a recombinationally active sequence appeared to be 13 bp in length (TAAAACAANGNNA), compared to previous estimates of 54 bp. This sequence, in combination with the product of the integrase gene, is responsible for the genetic variability of members of the Tn21 family of transposable elements and the dissemination of multi-resistance.
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A protoplasting technique with general applicability for molecular karyotyping of hymenomycetes
More LessPulsed-field gel electrophoresis (PFGE) has been used to facilitate genetic fingerprinting within a natural hymenomycete population. A protoplasting technique has been developed which shows that the basidial cells of the commercially important mushrooms Agaricus bisporus, A. bitorquis and Lentinus edodes (Shiitake), as well as from many wild growing Agarics, are sensitive to commercially available fungal cell wall degrading enzymes. The technique produces a high yield of intact protoplasts within short time periods. Using the basidial cell protoplasting technique, chromosomal-sized DNA from a wild fruit body of Coprinus comatus has been prepared and resolved into distinct bands using PFGE. This protoplasting technique will allow the application of PFGE to provide electrophoretic karyotypes within natural and commercial populations of hymenomycetes.
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Wide diversity of genome size among different strains of Clostridium acetobutylicum
More LessThe sizes of the genomes of five strains of Clostridium acetobutylicum were estimated by summing the sizes of macro-restriction fragments produced after cleavage of their DNA with SmaI and ApaI. The five C. acetobutylicum strains fell into three discrete groups. The NCIMB 8052 and NI-4081 strains have 6·5 Mbp genomes and restriction profiles of their DNA were indistinguishable. The sizes of the genomes of the ATCC 824 and DSM 1731 strains were about 4·0 and 3·5 Mbp (minimum values), respectively, and the restriction profiles of their DNA were very similar. One notable difference was the presence of an additional 532 kbp SmaI fragment in the DNA of strain ATCC 824. The NCP 262 strain of C. acetobutylicum has a genome size of 2·85 Mbp. Restriction fragment profiles of DNA from strains belonging to different groups were not obviously similar. Probes made from several cloned ATCC 824 genes hybridized with DNA fragments of different sizes from strains representative of each group. These data add further weight to the considerable body of evidence indicating that the species C. acetobutylicum comprises a heterogeneous collection of strains.
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Expression of a plasmid gene of Chlamydia trachomatis encoding a novel 28 kDa antigen
Summary: Plasmid pCT is present in essentially all isolates of Chlamydia trachomatis and may encode factors important for survival in the natural environment. However, no pCT-associated phenotype has been described so far. With the purpose of investigating the possibility of a role of pCT in C. trachomatis pathogenicity, we examined the expression of an ORF (ORF3), potentially encoding a 28 kDa polypeptide (pgp3). Analysis of RNA extracted from chlamydia-infected Vero cells detected ORF3-specific transcripts, from 20 h post-infection onwards, mainly as discrete RNA species of 1390 nucleotides comprising the downstream ORF4 sequence. ORF3 DNA was cloned and expressed in Escherichia coli as a 39 kDa fusion protein (MS2/pgp3). Antibodies raised against purified MS2/pgp3, specifically recognized a 28 kDa protein on Western blots of protein from purified chlamydial elementary bodies (EBs). The same antibodies detected chlamydial inclusions in methanol-fixed infected cells by immunofluorescence. Western blot analysis of EBs extracted with 2% Sarkosyl, showed that a large proportion of the 28 kDa antigen is associated with the detergent-insoluble (‘membrane’) fraction. Antibodies recognizing pgp3 epitopes were detected in sera from patients with chlamydial infections, but not in sero-negative control sera. The findings support the hypothesis that pCT may provide a function related to chlamydial cell physiology.
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Cloning and analysis of the leuB gene of Leptospira interrogans serovar pomona
More LessThe leuB gene of Leptospira interrogans serovar pomona strain kenniwicki has been cloned on a 9·5 kb plasmid, pWVL1, by complementation of Escherichia coli leuB mutants. Subcloning and Tn5 mutagenesis showed that the region required for complementation was approximately 1·2 kb in length. Enzyme assays showed that the product of the cloned gene was a β-isopropylmalate dehydrogenase. Defects in the leuA, leuC and leuD genes of E. coli were not complemented by pWVL1. The nucleotide sequence of the leuB-complementing region and surrounding DNA has been determined. Three open reading frames were found which encode proteins of 40·9, 38·8 and 15 kDa. Analysis of subclones containing nucleotide deletions of varying sizes showed that only the 38·8 kDa protein was necessary to obtain complementation of E. coli leuB mutations. The PIR data base was searched and the enzyme 3-isopropylmalate dehydrogenase from six different micro-organisms was found to share significant amino acid sequence similarity (43–57%) with the 38·8 kDa L. interrogans leuB gene product. The organization of the leucine biosynthetic genes in L. interrogans differs from that found in E. coli, Salmonella typhimurium and Bacillus subtilis.
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The melA gene is essential for melanin biosynthesis in the marine bacterium Shewanella colwelliana
More LessSummary: The surface-adhering, Gram-negative marine bacterium Shewanella colwelliana synthesizes a red-brown melanin in the late stage of exponential growth in laboratory culture. Previous studies identified a single gene, melA, from S. colwelliana that could impart the ability to produce melanin to an E. coli host. However, these studies did not demonstrate a requirement for melA during melanization in S. colwelliana. In this paper, genetic analyses, using a broad host range conjugation system to generate specific lesions, reveal that melA null mutants fail to synthesize pigment. The wild-type melA gene provided in trans on a low copy number plasmid complemented these null mutations, as well as a spontaneous pigment variant, to wild-type melanin synthesis. Polyclonal antibodies, raised against a MelA-LacZ fusion protein, were used to confirm the presence of the melA gene product in wild-type S. colwelliana and verify its absence in the non-pigmented mutants. In addition, detection of the MelA protein over the course of growth in batch culture revealed a constant steady-state level of MelA protein, suggesting that the timing of melanizarion and the quantity of melanin synthesized is not controlled at the level of melA expression.
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- Pathogenicity And Medical Microbiology
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Biochemical properties of Streptococcus sobrinus reisolates from the gastrointestinal tract of a gnotobiotic rat
More LessStreptococcus sobrinus strain 6715–13–201 was inoculated into the oral cavity of a gnotobiotic rat and then reisolated from different portions of the gastrointestinal tract. Fourteen isolates, selected on the basis of their colonial morphology, were then screened for their ability to adhere to salivacoated hydroxyapatite (SHA) in vitro, and their ability to produce extracellular polysaccharide from sucrose, and low pH in glucose broth. Certain isolates were also tested for their cariogenic potential as monoinfectants in gnotobiotic rats. All isolates differed in their abilities to adhere to SHA, with most showing an increased level of adhesion in the presence of sucrose, but this did not correlate with their ability to be aggregated by dextran. Most isolates were capable of producing glucosyltransferases (with only one exception) and dextranases (also one exception). There was more variability in the production of dextranase inhibitor. No isolate was capable of producing dextranase inhibitor in the absence of dextranase production. There were no correlations between the ability of isolates to adhere in vitro or produce/utilize polysaccharides and their ability to produce caries in vivo. Due to the differences between strains in their abilities to adhere, produce polysaccharides, utilize polysaccharides or produce a low pH and the lack of correlation between any of these parameters and cariogenicity, the results suggest that the ability of strains to colonize and produce caries depends on a number of different characteristics, no one of which is essential.
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A critical appraisal of positive cooperativity in oral streptococcal adhesion: Scatchard analyses of adhesion data versus analyses of the spatial arrangement of adhering bacteria
Positive cooperativity is a mechanism proposed to account for the adhesion of bacteria to surfaces. In this paper, two methods that both claim to assess experimentally cooperative phenomena, viz. Scatchard analysis of adhesion data (using adhesion to vials) and analysis of the spatial arrangement of adhering cells (using a flow chamber), were compared and critically evaluated. Three oral strains were used and the substrata involved were glass (hydrophilic) and silicone-coated glass (hydrophobic) employed with or without a salivary coating. Scatchard analysis and near-neighbour analysis of adhering cells yield similar conclusions with regard to the mechanism of adhesion of the cells, provided that adhering cells are sufficiently immobilized under the experimental conditions. In the case of incomplete immobilization, near-neighbour collection results from sliding of adhering cells rather than from cooperative phenomena. Also, the agreement between the conclusions from both methods seems to be better, the more reversibly the cells adhere. Positive cooperativity can be absent or present on saliva-coated substrata with a distinct effect of the substratum hydrophobicity, despite the presence of an adsorbed film. This suggests that a different pellicle develops on a hydrophobic substratum than on a hydrophilic substratum. This is confirmed by our observation that the amino acid composition of salivary films in different on both types of substratum.
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- Physiology And Growth
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Antioxidant role of carotenoids in Phaffia rhodozyma
More LessThe role of carotenoids in protecting the yeast Phaffia rhodozyma against reactive oxygen species was studied. Addition of the O2 −-generator duroquinone (DQ) to yeast-malt broth increased total carotenoid content as well as the relative amounts of xanthophylls present, while the reactive oxygen scavenger mannitol reversed this effect. Resistance to DQ increased in the stationary phase, particularly in the carotenoid-hyperproducing strain studied. Assay of superoxide dismutase (SOD) in P. rhodozyma indicated the presence of Mn-SOD and the complete absence of Fe-SOD and Cu/Zn-SOD. Catalase activity in P. rhodozyma was significantly lower than in Saccharomyces cerevisiae, particularly in stationary cultures. In young cells, H2O2 resistance was directly related to carotenoid levels, and selection of cultures based on peroxide resistance resulted in slightly increased carotenoid production. These results indicate that carotenoids play an antioxidant role during ageing in P. rhodozyma.
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In situ localization of the secretion of lignin peroxidases in colonies of Phanerochaete chrysosporium using a sandwiched mode of culture
More LessProtein secretion and growth were investigated in Phanerochaete chrysosporium by using cultures sandwiched between perforated polycarbonate membranes. Labelling of colonies with radioactive N-acetylglucosamine and l-methionine indicated a close correlation between growth and general protein secretion, even in a central area of the colony secreting the idiophase enzymes lignin peroxidase (LiP) and manganese-dependent lignin peroxidase (MnP). Comparison of the sites of release into the medium of newly synthesized proteins and immuno-detected lignin peroxidases suggested that diffusion of the enzymes from the walls was a limiting step in the release of peroxidases into the medium. Microautoradiography of colonies exposed to N-acetyl[3H]glucosamine revealed the apical growth of thin hyphae and branches (4 to 5 μm diameter on average) in the central secreting area. These secondary hyphae showed peroxidase activity and reacted with lignin peroxidase antibodies. Although it was not possible to directly visualize secretion at hyphal tips, the results suggest that peroxidases (LiP and MnP) are initially secreted at the apex of secondary growing hyphae and later slowly released into the surrounding medium.
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Resistance to spiramycin in Streptomyces ambofaciens, the producer organism, involves at least two different mechanisms
More LessSummary: During its stationary phase, Streptomyces ambofaciens produces the macrolide antibiotic spiramycin, and has to protect itself against this antibiotic. Young mycelia, not yet producing spiramycin, are sensitive to it, but they become fully resistant when production begins. In a sensitive mycelium, resistance could be induced by exposure to sub-inhibitory concentrations of spiramycin, and these induced mycelia, like producing mycelia were resistant not only to spiramycin but also to several other macrolide antibiotics. Ribosomes extracted from these resistant mycelia were shown in vitro to be more resistant to spiramycin than ribosomes extracted from sensitive mycelium, indicating that S. ambofaciens possesses a spiramycin-inducible ribosomal resistance to spiramycin and to macrolide antibiotics. Studies with spiramycin non-producing mutants showed that, in these mutants, resistance to spiramycin also varies during cultivation, in that an old culture was much more resistant than a young one. But with these non-producing mutants, the spectrum of resistance was narrower, and in vitro data showed that resistance was not due to ribosomal modification. These results suggest that S. ambofaciens presents at least two distinct mechanisms for spiramycin resistance; a spiramycin-inducible ribosomal resistance, and a second resistance mechanism which might be temporally regulated and which could involve decreased permeability to, or export of, the antibiotic. The two mechanisms are probably at work simultaneously in the producing mycelium, the spiramycin-inducible resistance being induced by endogenous spiramycin. In non-producing mutants, in the absence of self-induction by spiramycin, only the second mechanism is observed.
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The induction of oxidative enzymes in Streptomyces coelicolor upon hydrogen peroxide treatment
More LessStreptomyces coelicolor (Müller) became resistant to killing by hydrogen peroxide (H2O2) when pretreated with non-lethal concentrations of H2O2. When rapidly growing cells were pretreated with 100 μm-H2O2, they became 7–10-fold more resistant to 20 mm-H2O2 than were naive cells. Activities of several oxidative defense enzymes were measured in cells treated with 100 μm-H2O2 in either exponential or stationary phase growth. The specific activity of catalase in crude extracts of cells pretreated in either phase increased about 40%, Peroxidase activity, in cell extracts and culture supernatants, respectively, of cells treated in the stationary growth phase increased two times and four times. Glucose-6-phosphate dehydrogenase increased by 60% at the exponential growth phase. Glutathione reductase increased 80% after treatment in the exponential phase and 4-fold in the stationary growth phase. However, superoxide dismutase activity decreased by 70%. Two mutants resistant to H2O2 were isolated after mutagenesis of spores with N-methyl-N′-nitro-N-nitrosoguanidine. In addition to a dramatic increase in the survival rate in 20 mm-H2O2, both mutants exhibited increased activities of all the above enzymes except superoxide dismutase. The pleiotropic phenotype of the mutants suggests that there exists a global regulation of oxidative response in S. coelicolor.
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