Showing papers in "Current Microbiology in 1991"
TL;DR: Acidobacterium is proposed as a new genus for the acidophilic, chemoorganotrophic bacteria containing menaquinone isolated from acidic mineral environments.
Abstract: Acidobacterium is proposed as a new genus for the acidophilic, chemoorganotrophic bacteria containing menaquinone isolated from acidic mineral environments.Acidobacterium capsulatum is proposed for the singleAcidobacterium species which consists of eight strains (Biogroup 5). The members of this species are gram-negative, aerobic, mesophilic, non-spore-forming, capsulated, saccharolytic, and rod-shaped bacteria. They are motile by peritrichous flagella. They can grow between pH 3.0 and 6.0, but not at pH 6.5. They give positive results in tests for esculin hydrolysis, catalase, and β-galactosidase. Oxidase and urease are negative. They can use glucose, cellobiose, starch, maltose, or β-gentiobiose as a sole carbon source, but cannot use elemental sulfur and ferrous iron as an energy source. The DNA base composition is 59.7–60.8 guanine plus cytosine (G+C) mol%. The major isoprenoid quinone is menaquinone with eight isoprene units (MK-8). The major fatty acid is 13-methyltetradecanoic acid. DNA relatedness between this species and the species ofAcidiphilium, Acidomonas, andDeinobacter was 18 to 2%. From phenotypic and chemotaxonomic characters, these member do not belong to any known taxa of gram-negative bacteria. A culture of the type strain (strain 161) has been deposited in the Japan Collection of Microorganisms as JCM 7670.
226 citations
TL;DR: An extraction method that yields up to 25 µg of DNA from 1 gram of soil is described, which can be digested by restriction enzymes and extracted with Geneclean.
Abstract: We describe an extraction method that yields up to 25 µg of DNA from 1 gram of soil. Samples are processed within 48 h. The purified DNA is 20–25 kb in size and can be digested by restriction enzymes. The soil is vortex-mixed with 6 mL mixing buffer in an Oakridge tube. Polyvinyl polypyrrolidone is added to adsorb the humic compounds that interfere with restriction enzyme activity. The entire soil homogenate is treated with lysozyme and Novozym, followed by sodium dodecyl sulfate to lyse the cells. The crude DNA extract is separated from the soil debris by low-speed centrifugation. Finally, the DNA is purified by CsCl gradient centrifugation and extracted with Geneclean.
105 citations
TL;DR: Results indicate that ELISAs for SLT I and SLT II antibodies are comparable to HeLa cell cytotoxicity neutralization assays, and may be more useful than assays for antitoxic antibodies in detecting persons withE.
Abstract: Shiga-like toxin-producingEscherichia coli O157:H7 are important causes of bloody diarrhea and hemolytic uremic syndrome. To facilitate the epidemiologic study of these organisms, we developed enzyme-linked immunosorbent assays (ELISAs) for antibodies to Shiga-like toxin I (SLT I), Shiga-like toxin II (SLT II), andE. coli O157 lipopolysaccharide (LPS). We tested serum samples from 83 patients in two outbreaks ofE. coli O157:H7 diarrhea and from 66 well persons. Forty-three patients (52%) had at least one serum sample positive for anti-O157 LPS antibodies; among 26 culture-confirmed patients, 24 (92%) had at least one positive serum sample. Two (3%) of 66 control sera had positive anti-O157 LPS titers. ELISA results for SLT I and II were compared with those of HeLa cell cytotoxicity neutralization assays on both patient and control sera. Neutralization assays detected anti-SLT I antibodies in at least one serum sample from each of 17 (20%) patients and 7 (10.6%) controls, while 16 (19%) patients and 7 controls had positive titers by anti-SLT I ELISA. Although all serum samples, including control sera, showed nonspecific neutralization of SLT II, no antibody titers to SLT II were detected by either neutralization or ELISA. These results indicate that ELISAs for SLT I and SLT II antibodies are comparable to HeLa cell cytotoxicity neutralization assays. Both the ELISAs and neutralization assays are insensitive in detecting infected patients. However, the ELISA for antibodies toE. coli O157 LPS is both sensitive and specific, and may be more useful than assays for antitoxic antibodies in detecting persons withE. coli O157:H7 infection.
80 citations
TL;DR: Chitobiase (EC 3.2.1.29), from the culture filtrate of Trichoderma harzianum, was purified in sequential steps by ammonium sulfate precipitation, ion exchange chromatography, and gel filtration.
Abstract: Chitobiase (EC 3.2.1.29), from the culture filtrate ofTrichoderma harzianum, was purified in sequential steps by ammonium sulfate precipitation, ion exchange chromatography, and gel filtration. The physical and biochemical properties of the enzyme have been determined. The native enzyme has a molecular weight of 118 kDa when determined by gel filtration, and 64 kDa by SDS-PAGE. The enzyme catalyzed the hydrolysis of N,N-diacetylchitobiose andp-nitrophenyl-β-N-acetyl glucosamine with apparent Km of 575 µM and 235 µM, respectively. The pH optimum for the enzyme was pH 5.5, and maximum activity was obtained at 50°C. Glucosamine and N-acetylglyucosamine strongly inhibited the enzyme.
64 citations
TL;DR: In this article, the precipitation of calcium carbonate by 27 strains ofDeleya halophila using solid and liquid media containing different NaCl concentrations (2.5, 7.5 or 20%, wt/vol) as sole salt, and two incubation temperatures (22° and 32°C) have been studied.
Abstract: The precipitation of calcium carbonate by 27 strains ofDeleya halophila using solid and liquid media containing different NaCl concentrations (2.5, 7.5, or 20%, wt/vol) as sole salt, and two incubation temperatures (22° and 32°C) have been studied. All the strains tested were able to precipitate calcium carbonate under the different environmental conditions assayed. Crystals formed were calcite and vaterite; the ratio of calcite to vaterite was dependent on total salts and on the type of medium.
59 citations
TL;DR: A sulfate-reducing bacterium (SRB) was isolated from a continuous anaerobic digester, which converted the furfural-containing wastewater to methane and CO2 as discussed by the authors.
Abstract: A sulfate-reducing bacterium (SRB) was isolated from a continuous anaerobic digester, which converted the furfural-containing wastewater to methane and CO2. This SRB isolate could use furfural, furfuryl alcohol, and 2-furoic acid as sole source of carbon and energy in a defined mineral sulfate medium. Acetic acid was the major end product of furfural degradation. This organism also used wide varieties of other carbon sources, including ethanol, pyruvate, lactate, succinate, propanol, formate, and malate. The SRB isolate contained the electron carrier desulfoviridin. It used SO4, NO3, and thiosulfate as electron acceptors. This isolate used ammonium chloride, nitrate and glutamate as nitrogen source. The characteristics of the SRB isolate were closely similar toDesulfovibrio sp.
54 citations
TL;DR: A soil microcosm, containing a mixture of sand and a well-characterized phaeozem soil from loess, was developed for biodegradative applications and survival of Alcaligenes xylosoxidans ABIV in the presence and absence of selective pressure by the xenobiotic compound could be demonstrated as well as its capacity to maintain degradation of DCPA.
Abstract: A soil microcosm, containing a mixture of sand and a well-characterized phaeozem soil from loess, was developed for biodegradative applications. It was inoculated with soil-borneAlcaligenes xylosoxidans AB IV, degrading 2,2-dichloropropionate (DCPA), by a plasmid-encoded haloalkanoic acid halidohydrolase. In long-term microcosm experiments, survival ofAlcaligenes xylosoxidans ABIV in the presence and absence of selective pressure by the xenobiotic compound could be demonstrated as well as its capacity to maintain degradation of DCPA. At the same time its plasmid, pFL40, containing the degradative gene,dhlC, was horizontally transferred toPseudomonas fluorescens in sterilized soil and also to different indigenous soil bacteria in nonsterilized soil.
54 citations
TL;DR: NMR spectroscopy of cell extracts revealed glycine betaine to be the predominant intracellular organic solute accumulated within cells grown at high osmolarity, and highest growth rates were obtained when the defined medium was supplemented with glycine Betaine.
Abstract: The foodborne pathogenStaphylococcus aureus is distinguished by its ability to grow within environments of extremely high osmolarity (e.g., foods with low water activity values). In the present study, we examined the accumulation of intracellular organic solutes withinS. aureus strain ATCC 12600 when cells were grown in a complex medium containing high concentrations of NaCl. Consistent with previous reports [Measures JC (1975) Nature 257:398–400; Koujima I, et al. (1978) Appl Environ Microbiol 35:467–470; and Anderson CB, Witter LD (1982) Appl Environ Microbiol 43:1501–1503], intracellular proline was found to accumulate to high concentrations. However, NMR spectroscopy of cell extracts revealed glycine betaine to be the predominant intracellular organic solute accumulated within cells grown at high osmolarity. In additional experiments, we examined the growth rate ofS. aureus in a defined medium of high osmolarity and found it to be stimulated significantly by the presence of either exogenous proline or glycine betaine. Highest growth rates were obtained when the defined medium was supplemented with glycine betaine.
52 citations
TL;DR: Adhesion to roots is not necessary for the initial phase of enhanced root elongation that is induced byP.
Abstract: Inoculation of canola seeds withPseudomonas putida GR12-2 stimulates root elongation under gnotobiotic conditions. Transformation ofP. putida GR12-2 with the broad-host-range plasmid pGSS15 abolishes the enhancement of root elongation. With scanning electron microscopy it was found that both transformed and nontransformedP. putida GR12-2 are capable of binding to canola seed coats. In addition, it was observed that 4 days after the initial inoculation the roots of bothP. putida GR12-2- and GR12-2/pGSS15-treated seedlings were free of adhering bacteria despite the fact that it was subsequently shown that both bacterial strains are capable of binding to roots. Thus, adhesion to roots is not necessary for the initial phase of enhanced root elongation that is induced byP. putida GR12-2 under gnotobiotic conditions.
50 citations
TL;DR: M. methylutens, a methylotrophic methanogen isolated from submarine sediments, is suggested to be a new strain ofMethanococcoides methylutENS, an anaerobic methanogenic bacterium isolated from oxygenated coastal waters.
Abstract: Enrichment cultures containing marine plankton from oxygenated coastal waters (50–108% saturated) with supersaturated levels of methane (>700% saturated) yielded a strictly anaerobic methanogenic bacterium. Nonmotile, non-spore-forming, regular to slightly irregular cocci (0.5–0.8µm) were evident by phase contrast, epifluorescence, and scanning electron microscopies. The unpurified isolate required NaCl for growth, with maximal methanogenesis at 240 mM NaCl at 22°C. The optimal temperature range for growth was 22–31°C, and the optimal range for methanogenesis was 26–35°C. Mono-, di-, and trimethylated amines or methanol were substrates for methanogenesis; sodium acetate and H2:CO2 were not. The DNA base composition was 42 ±1% guanine plus cytosine. Serology suggested the isolate may be a new strain ofMethanococcoides methylutens. Morphology, growth physiology, DNA base content, and serology are all consistent with the type description ofM. methylutens, a methylotrophic methanogen isolated from submarine sediments.
47 citations
TL;DR: In the young rabbit, the celluloytic population appeared in the cecum and colon microflora between 12 and 16 days after birth and increased rapidly with age to stabilize at a level of about 107 bacteria a few days after weaning; in the adult animal the xylanolytic and pectinolytic populations established at levels between 109 and 1010 bacteria g−1.
Abstract: In the young rabbit, the celluloytic population appeared in the cecum and colon microflora between 12 and 16 days after birth. It increased rapidly with age to stabilize at a level of about 107 bacteria a few days after weaning. In the adult animal the xylanolytic and pectinolytic populations established at levels between 109 and 1010 bacteria g−1. The colic and cecal populations were comparable in size. When the feed cellulose content rose from 11 to 17% (P/V), the cellulolytic population increased about tenfold, and the range of individual variations decreased. All the fibrolytic strains isolated were strictly anaerobic;Eubacterium cellulosolvens was the most frequently isolated cellulolytic species. The eight xylanolytic strains and the twelve pectinolytic strains identified were assigned to the speciesBacteroides ruminicola.
TL;DR: The entire biosynthetic pathway for the synthesis of the antibiotic pigment violacein is encoded on a 14.5 kilobase fragment of thechromobacterium violaceum genome, which is in a wide range of Gram-negative bacteria.
Abstract: The entire biosynthetic pathway for the synthesis of the antibiotic pigment violacein is encoded on a 14.5 kilobase (kb) fragment of the Chromobacterium violaceum genome. When cloned into Escherichia coli, pigment synthesis is strongly expressed, as it is in a wide range of Gram-negative bacteria. Transposon mutagenesis resulted in a number of different phenotypes that correlated with transposon insertion sites in the gene cluster.
TL;DR: A simple, rapid, small-scale assay for infection of tobacco seedlings by Phytophthora parasitica var.nicotianae is developed, which may facilitate the rapid screening of potential biological and chemical control agents and may be useful for studying mechanisms of infection and control ofPhydophthora spp.
Abstract: We developed a simple, rapid, small-scale assay for infection of tobacco seedlings byPhytophthora parasitica var.nicotianae. One 7-day-old tobacco seedling was placed in each well of a 96-well microtiter plate and inoculated with 500 zoospores ofP. parasitica var.nicotianae. After 72 h all of the inoculated seedlings of the susceptible cultivar, KY14, were infected, and the pathogen had produced sporangia that were visible on the surfaces of the seedlings. Sporangia did not develop on seedlings that were inoculated simultaneously with zoospores and either 1 µg/mL of the chemical fungicide metalaxyl or 5 µL of filtrate of a sporulated culture of the biocontrol agent,Bacillus cereus UW85. Seedlings of tobacco cultivar KY17 were infected byP. parasitica var.nicotianae, although mature plants of this variety are resistant to the pathogen. This microassay may facilitate the rapid screening of potential biological and chemical control agents and may be useful for studying mechanisms of infection and control ofPhytophthora spp. under hydroponic conditions.
TL;DR: The ARhodobacter sphaeroides gene (pps) inducestrans suppression of bacteriochlorophyll and carotenoid levels in both R. sp Haeroides and R. capsulatus induces suppression of Crt levels in a Paracoccus denitrificans strain carrying the Crt genes ofR.
Abstract: ARhodobacter sphaeroides gene (pps) inducestrans suppression of bacteriochlorophyll (Bch) and carotenoid (Crt) levels in bothR. sphaeroides andR. capsulatus. It also induces suppression of Crt levels in aParacoccus denitrificans strain carrying the Crt genes ofR. sphaeroides. The gene is located approximately 11 kilobases fromcrtA in the photosynthetic gene cluster. Crt suppression bypps is quantitatively different from that caused by an absence of mature Bch.
TL;DR: The results indicate that besides the siderophore-mediated mechanism, V. anguillarum can also obtain iron from other sources presumably available from the host, although its importance for growth in vivo is so far unknown.
Abstract: Utilization of several iron sources available from the host was investigated in different strains ofVibrio anguillarum. We tested the ability to use transferrins, heme, hemoglobin, and haptoglobin-hemoglobin as iron sources in strains ofV. anguillarum possessing different iron uptake systems mediated by siderophores. Only the wild-type pathogenic strains with an intact siderophore-mediated iron transport system were able to obtain iron from transferrins. None of the low-virulence derivatives lacking siderophore production could grow in the presence of transferrins. However, all strains, wild-type and iron-deficient derivatives, could utilize heme, hemoglobin, and haptoglobin-hemoglobin as iron sources when added to iron-deficient media. The ability to grow in fish serum was also evaluated. Although only wild-type strains could grow in fresh serum, derivative strains lacking siderophore production also were able to grow when serum was heat inactivated or when a utilizable siderophore was present in serum. The results indicate that besides the siderophore-mediated mechanism,V. anguillarum can also obtain iron from other sources presumably available from the host, although its importance for growth in vivo is so far unknown.
TL;DR: In this paper, cellulosome-like complexes were identified in the broth and sonic extracts of cellobiose-and cellulose-grown cells of Bacteroides cellulosolvens.
Abstract: Cellulosome-like complexes were identified in the broth and sonic extracts of cellobiose-and cellulose-grown cells ofBacteroides cellulosolvens. The extracellular fractions contained three to four major polypeptides and several minor polypeptide bands that were localized in two major gel filtration peaks indicating average molecular weights of about 700 kDa and >10 MDa. A relatively large molecular weight component (Mr 230 kDa) was found to contain carbohydrate, but no apparent enzymatic activity of its own could be detected. The cell sonicate displayed a more complicated polypeptide profile, and glycosylated polypeptides were larger (ca. 310 and 290 kDa) than that of the extracellular fraction. The 230-kDa extracellular component interacted strongly with the GSI isolectin fromGriffonia simplicifolia, exhibited immunochemical cross-reactivity with the S1 subunit of the cellulosome fromClostridium thermocellum, and displayed anomalous pH- and salt-dependent migratory behavior in SDS-PAGE. Taken together, this evidence strongly suggests a structural similarity between the glycoconjugates of these two distinct cellulolytic bacteria. A major 84-kDa polypeptide was identified as a xylanase, and a 50-kDa polypeptide displayed endoglucanase activity. Additional biochemical and cytochemical evidence indicated that cellulosome-like cellulolytic complexes are associated with the cell surface in this bacterium.
TL;DR: The cells of Lactobacillus amylovorus (NRRL B-4540) secreted an amylase activity that rapidly solubilized cornstarch as discussed by the authors.
Abstract: The cells ofLactobacillus amylovorus (NRRL B-4540), grown in a medium containing 2% cornstarch as the sole carbon source, secreted an amylase activity that rapidly solubilized cornstarch. Fourier transform infrared (FTIR) spectroscopic analyses showed that 80–90% of starch was consumed by bacteria in a 10-day-old culture medium. The remnant of starch granules digested in the culture medium inoculated with the cells ofL. amylovorus have also lost their characteristic iodine-binding capacity as visualized by starch dye-binding microplate assay and light microscopy. Scanning electron miscroscopy (SEM) of granules from a 48-h culture medium showed hollow and fragmented granules with a pitting phenomenon characteristically produced byα-amylase activity. Analysis of an enzyme preparation from a culture medium ofL. amylovorus revealed that the putative enzyme appears to be a single protein band of unusually high Mr (150,000) on SDS gels stained for amylase activity. Analysis of starch digestion products by thin layer chromatography (TLC) showed enzyme activity typical ofα-amylase.
TL;DR: Four induced proteins were analyzed by microsequencing techniques and were identified as the homologues for GroEL, DnaK, enolase, and glyceraldehyde-3-phosphate dehydrogenase, which are heat shock proteins in other systems.
Abstract: Heat shock inBacillus subtilis may induce as many as 66 proteins after temperature upshift from 37° to 48°C. Four induced proteins were analyzed by microsequencing techniques. These were identified as the homologues for GroEL, DnaK, enolase, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which are heat shock proteins in other systems. The identities of GroEL and DnaK were confirmed additionally by Western blot analysis. As a control, a protein whose synthesis was repressed approximately threefold by heat shock was identified by microsequencing as flagellin.
TL;DR: I-labeled type I collagen (Cn-I) binding of 92 fresh isolates and 18 type culture collection strains of lactobacilli was tested and it was found that the binding was inhibited by excess of unlabeled Cn- I, gelatin, and was sensitive to proteinase K.
Abstract: 125I-labeled type I collagen (Cn-I) binding of 92 fresh isolates and 18 type culture collection strains of lactobacilli was tested. More than 75% of the strains bound Cn-I. The binding was inhibited by excess of unlabeled Cn-I, gelatin, and was sensitive to proteinase K. Other proteins such as fibronectin and albumin and various carbohydrates such asD-galactose,D-fucose, andD-mannose did not inhibit the binding. Therefore, we propose binding of Cn-I to lactobacilli involving specific surface protein(s).
TL;DR: An α-amylase secreted by Lactobacillus amylovorus was partially purified and characterized, and was renaturable following treatment with SDS and heat.
Abstract: An α-amylase (E.C. 3.2.1.1.) secreted byLactobacillus amylovorus was partially purified and characterized. This high-molecular-weight enzyme [Imam SH, Burgess-Cassler A, Cote GL, Gordon SH, Baker FL (1991) Curr Microbiol 22:365–370] was quantified with a clinical α-amylase assay adapted to a microplate format. It was isolated from concentrated cell-free culture medium by ammonium sulfate precipitation, ion exchange, and hydrophobic interaction chromatographies. The enzyme was not particularly thermostable, but like three other microbial α-amylases tested for comparison, was renaturable following treatment with SDS and heat. The pH optimum and pI were 5.5±0.5 and 5.0, respectively; its temperature optimum was 60–65°C, and the molecular weight on SDS gels was 140±10 kDa.
TL;DR: The characterization of the phages described herein provides the basic information required for their use in the construction of vectors for the transfer of genetic material, and indicates that they were circularly permuted and terminally redundant.
Abstract: Two bacteriophages (phgrBrb01 and phgrBrb02), lytic toBacteroides ruminicola ssbrevis AR20, were isolated from sewage water. Both phages possessed polyhedral heads and long noncontractile tails, and were classified as Siphoviridae of morphotype B1. Bacteria resistant to phages phgrBrb01 and phgrBrb02 arose following lysis of broth cultures. Survivors of phgrBrb01 infection were capsulated but remained susceptible to phgrBrb02 infection. Survivors of phgrBrb02 infection were noncapsulated and were resistant to attack by both phgrBrb01 and phgrBrb02. Neither phage lysogenized the host. Both phages contained double-stranded DNA, and their restriction endonuclease digestion patterns indicated that the phage genomes were circularly permuted and terminally redundant. Phage phgrBrb01 genome was examined in greater detail and confirmed to be circularly permuted, of size 33 kb, with a terminal redundancy of 2 kb, or 6% of the length of the genome. Circularly permuted genomes in phages of rumen bacteria do not appear to have been reported previously. At present, there is considerable interest in the genetic manipulation of rumen bacteria. The characterization of the phages described herein provides the basic information required for their use in the construction of vectors for the transfer of genetic material.
TL;DR: It seems that manganese and/or zinc may be more important for enzyme activity than calcium in the thermophilic streptomyceteStreptomyces thermoviolaceus, although calcium promoted increased thermotolerance such that around 65% of the activity remained after 100 min at 70°C.
Abstract: Production of secondary metabolites was investigated in the thermophilic streptomyceteStreptomyces thermoviolaceus grown at 45°C in a fermenter. Extracellular protein was secreted into the culture medium at the same time as an antibiotic granaticin; both were synthesized during the second slower phase of biphasic growth, which is most apparent at 45°C for this organism. Protease, assayed as azocaseinase, was identified as one component of, and marker for, the excreted protein. The effects of different growth temperatures revealed that the synthesis of extracellular protein, like that of the antibiotic, was maximal in cultures grown between 37° and 45°C, whereas protease activity was greatest in 50°C grown cultures. A method was devised, based on acetone precipitation, for concentrating the protease activity from culture supernatants. Characterization of the concentrated activity using inhibitors suggested the presence of a serine and a metallo-type protease. A peptide substrate specific for the metallo-protease showed that it had a pH optimum for activity of 6.5–7.0. Approximately 50% of the activity was lost after 80 min of incubation at 70°C. Although calcium (5 mM) promoted increased thermotolerance such that around 65% of the activity remained after 100 min at 70°C, it seems that manganese and/or zinc may be more important for enzyme activity.
TL;DR: In this paper, anaerobic glycerol degradation by a mixed microbial culture from a fermenter fed with industrial alcohol distillation waste water was investigated in the absence or presence of sulfate, at 37°C and at a constant pH of 7.2.
Abstract: Anaerobic glycerol degradation by a mixed microbial culture from a fermenter fed with industrial alcohol distillation waste water, was investigated in the absence or presence of sulfate, at 37°C and at a constant pH of 7.2. In the absence of sulfate, glycerol utilization was found to be characterized by the transient formation of 1,3-propanediol prior to propionate and acetate accumulation. In the presence of sulfate, 1,3-propanediol production was minor, and the carbon balance reflected a considerable accumulation of intermediate(s). A study of the role of sulfate reduction and methanogenesis on anaerobic 1,3-propanediol degradation showed that consumption of this substrate by the mixed microbial culture required a terminal electron acceptor. The number of fermentative and sulfate-reducing bacteria with glycerol or 1,3-propanediol as carbon and energy source revealed that sulfate-reducing bacteria outcompete fermentative bacteria for these substrates. The possible ecological role of sulfate-reducing bacteria in the metabolism of these reduced substrates is discussed.
TL;DR: An extracellular proteinase from aXanthomonas maltophilia strain isolated from soil was purified by ammonium sulfate precipitation, gel exclusion, and ion exchange gel chromatography and appeared homogeneous upon polyacrylamide-gel electrophoresis.
Abstract: An extracellular proteinase from aXanthomonas maltophilia strain isolated from soil was purified by ammonium sulfate precipitation, gel exclusion, and ion exchange gel chromatography. The enzyme appeared homogeneous upon polyacrylamide-gel electrophoresis. Its molecular weight was estimated to be 36,000 by the gel filtration assay and 40,000 by polyacrylamidegel electrophoresis after denaturation: it was a monomeric protein. Highly active at alkaline pH with casein as the substrate, it was inactivated by serine-site inhibitors, such as phenylmethylsulfonyl fluoride and diisopropylfluorophosphate. Like serine proteases, it hydrolyzed esters such as the β-naphthyl acetate. Moreover, like metalloproteases, the enzyme was rapidly inactivated by ethylene diamine tetraacetate, and the activity was partially restored by calcium. These original characteristics have not often been reported among other gram-negative bacteria.
TL;DR: Results indicate that the B. sphaericus larvicide acts as a binary toxin in mosquitos, whereas only the 39-kDa protein is required for full toxicity to tissue culture-grown cells.
Abstract: Bacillus sphaericus 2362 produces a parasporal crystal containing 42 and 51 kilodalton (kDa) proteins. Both of these proteins are required for toxicity to mosquito larvae; neither is toxic alone. When overexpressed inB. subtilis, these two proteins accumulate as amorphous inclusions (AIs). Bioassays involving larvae ofCulex pipiens and different ratios of these AIs indicated that maximal toxicity was observed at a ratio of approximately one 42-kDa protein to one 51-kDa protein. Purified preparations of these proteins, as well as derivatives similar to those which accumulate in the gut of mosquito larvae, were also toxic when combined, but not toxic singly. Different results were obtained when the toxicity of these preparations was tested for tissue culture-grown cells ofC. quinquefasciatus. Under these conditions, the 39-kDa derivative of the 42-kDa protein was alone sufficient for toxicity, which was not increased by the addition of the 51-kDa protein or its derivatives. These results indicate that theB. sphaericus larvicide acts as a binary toxin in mosquitos, whereas only the 39-kDa protein is required for full toxicity to tissue culture-grown cells.
TL;DR: Escherichia coli-Anacystis nidulans shuttle vectors pHIEX14, pSMG1, and pANHI, containing the leftward promoter of bacteriophage lambda and the gene for the temperaturesensitive repressor, cI857, were constructed and used to transform A. niduans.
Abstract: Escherichia coli-Anacystis nidulans shuttle vectors pHIEX14, pSMG1, and pANHI, containing the leftward promoter, PL, of bacteriophage lambda and the gene for the temperaturesensitive repressor, cI857, were constructed and used to transformA. nidulans. The transformation efficiencies and restriction endonuclease maps of these plasmids are reported. The use of these shuttle vectors should allow temperature regulation of foreign gene expression inA. nidulans.
TL;DR: Transfer of exponentially growing colonies of Schizophyllum commune to nitrogen-deficient media for 6–60 h resulted in increased general proteolytic activity and decreased extractable protein when compared with controls.
Abstract: Transfer of exponentially growing colonies ofSchizophyllum commune to nitrogen-deficient media for 6–60 h resulted in increased general proteolytic activity and decreased extractable protein when compared with controls. A concomitant increase in free radiolabeled leucine was detected in nitrogen-deprived colonies. Radiolabel was found in new surface hyphae following transfer of previously labeled colonies to media containing no label. The concentration of label present in the new growth indicates that this label is likely to have originated from proteolytic release of label from proteins in the older, central portions of the colony. The release of substantial amounts of label into the growth medium suggests that an extra-mycelial pathway may account, at least in part, for translocation of amino acids released by proteolysis.
TL;DR: Data indicate that substantial genetic diversity exists within the ruminal species S. ruminantium, but that at least one strain may represent up to 20% of isolates.
Abstract: Diversity in the ruminal bacterial speciesSelenomonas ruminantium has been investigated by DNA fingerprinting, DNA-DNA hybridization, plasmid analysis, bacteriophage sensitivity, and monoclonal antibody-based immunoassay. Twenty different isolates from the sheep rumen were initially classified morphologically and by carbon source utilization. DNA fingerprint analyses and quantitative genomic DNA hybridizations showed that limited grouping of these isolates was possible, with the largest group comprising four isolates, and two other groups comprising two isolates each. The remaining isolates were unique. Plasmids in four different size classes, 2.5, 3.7, 6.5 and 12.0 kbp, were identified, but these did not appear in all isolates. There was no apparent relationship between DNA fingerprint pattern and plasmid content. Only three isolates were sensitive to theS. ruminantium-specific temperate bacteriophage S-1. These data indicate that substantial genetic diversity exists within the ruminal speciesS. ruminantium, but that at least one strain may represent up to 20% of isolates.
TL;DR: Contact angles with polar liquids, water and formamide, were considerably higher on RAG-1 than on MR-481, in accordance with their relative hydrophobicities as measured by MATH, but no significant differences in contact angles were observed between the two strains with apolar liquids like diiodomethane,α-bromonaphthalene, and hexadecane.
Abstract: Acinetobacter calcoaceticus RAG-1 and MR-481, two standard strains used in microbial adhesion to hydrocarbons (MATH), were characterized by contact angles, pH-dependent zeta potentials, elemental surface composition by X-ray photoelectron spectroscopy (XPS), and molecular composition by infrared spectroscopy (IR). Negatively stained (methylamine tungstate) and ruthenium red-stained cells were studied by transmission electron microscopy to reveal the absence or presence of surface appendages. Despite the fact thatA. calcoaceticus RAG-1 is known to be extremely hydrophobic in MATH, whereas MR-481 is a completely non-hydrophobic mutant, neither XPS nor IR indicated a significant difference in chemical composition of the cell surfaces. Contact angles with polar liquids, water and formamide, were considerably higher on RAG-1 than on MR-481, in accordance with their relative hydrophobicities as measured by MATH. However, no significant differences in contact angles were observed between the two strains with apolar liquids like diiodomethane,α-bromonaphthalene, and hexadecane. Fibrous extensions on RAG-1, observed after ruthenium red staining, were absent on the non-hydrophobic mutant MR-481. Tentatively, these extensions could be held responsible for the hydrophobicity ofA. calcoaceticus RAG-1.
TL;DR: The ability ofEscherichia coli strains isolated from various human infections to bind fibronectin (Fn), collagen type I (Cn), laminin (Ln), and vitronECTin (Vn) was studied and cells seem to have specific binding sites for Fn, Cn type I, Vn, and possibly also Ln.
Abstract: The ability ofEscherichia coli strains isolated from various human infections to bind fibronectin (Fn), collagen type I (Cn), laminin (Ln), and vitronectin (Vn) was studied. Binding of the proteins was shown to be specific and to be inhibited after protease treatment and heating of bacterial cells at 80°C. Binding of Fn, Cn type I, and Ln was not expressed in CFA broth and Voccani's medium with sodium chloride concentration above 0.5%. Fn binding was specifically enhanced for cells grown in CFA broth and Voccani's medium containing CaCl2 (up to 10 mM final concentration), whereas binding of Cn type I was decreased. Growth in liquid medium yielded cells with maximal binding of Fn, Cn type I, Vn, and Ln after 20–22 h of growth. The binding of Fn, Cn type I, and Vn to cells of strain NG7C was inhibited for cells preincubated with antisera raised to the homologous strain;E. coli NG7C cells seem to have specific binding sites for Fn, Cn type I, Vn, and possibly also Ln.