bluuebunny/crossref_metadata_2025 huggingface dataset:
Investigations of subterranean bacteria in deep crystalline bedrock and their importance for the disposal of nuclear waste
Authors: Pedersen Karsten
Published: February 2010

The diversity and distribution of bacteria in subterranean environments have been found to be extensive and to depend on the prevailing environmental conditions. In 1987, microbiology became a part of the Swedish scientific program for the safe disposal of high level nuclear waste (HLW). The goal of the microbiology program is to understand how subterranean bacteria will interact with the performance of a future HLW repository. It concerns several major processes that directly or indirectly may exert influence on waste canister corrosion and the mobility of radionuclides. Uptake and transport of radionuclides by bacteria seem to be negligible components for radionuclide migration, but the effect from bacterial production of complexing agent remains to be evaluated. Also, bacterial production and consumption of gases will influence radionuclide transport due to gas bubbles. Many important radionuclides are immobile at reduced conditions and mobile at oxidized conditions. Bacterial activity can, therefore, indirectly decrease the mobility of radionuclides due to consumption of oxygen and the reduction of electron acceptors to species such as ferrous iron and sulfide.Key words: 16S rRNA, diversity, microbial activity, nuclear waste, sulfate reduction.
Transport of bacteria through geologic media
Authors: Lawrence J. R. , Hendry M. J.
Published: February 2010

A review of the current literature on transport of bacteria through geologic media is presented. The review addresses the major controls on bacterial transport. These controls include the nature of the substratum, the solute, and the bacterial cell. Most knowledge on the transport of bacteria through geologic media has been gained from column studies. There is need for some standardization of approaches, particularly with regard to data collection and controls on factors such as ionic strength and flow velocity. Other systems including glass micromodels have been used in conjunction with microscopy and scanning confocal laser microscopy to examine the controls on transport at the pore scale rather than porous medium scale of column studies. Many inconsistencies exist regarding the effect of the numerous variables that impact bacterial sorption in porous media. These variables include the nature of the substratum (i.e., the presence or absence of coatings), chemical composition of the solute (particularly ionic strength), system hydrodynamics, and bacterial variables such as size, shape, hydrophobicity, and electrostatic charge. Mathematical models based on the advective–dispersion equation have been developed to simulate bacterial transport. Within specific limits, these models can approximate most aspects of bacterial transport; however, they neglect parameters such as growth and behavior of bacteria. There is a need for theoretical development, extensive laboratory investigation, and model development before the goal of prediction of bacterial transport at field scale may be realized.Key words: sorption, advection, dispersion, models, facilitated transport.
Production of β-glucosidase and diauxic usage of sugar mixtures byCandida molischiana
Authors: Freer Shelby N. , Skory Christopher D.
Published: February 2010

The fermentation of cellobiose is a rare trait among yeasts. Of the 308 yeast species that utilize cellobiose aerobically, only 12 species ferment it, and only 2 species, Candida molischiana and Candida wickerhamii, also ferment cellodextrins. Candida molischiana produced β-glucosidase activity on all carbon sources tested, except glucose, mannose, and fructose. When these sugars were added to cultures growing on cellobiose, the synthesis of β-glucosidase ceased. However, the total amount of enzyme activity remained constant, indicating that the C. molischiana β-glucosidase is catabolite repressed and not catabolite inactivated. When grown in medium initially containing glucose plus xylose, cellobiose, maltose, mannitol, or glucitol, C. molischiana preferentially utilized glucose and produced little β-glucosidase activity until glucose was nearly depleted from the medium. When grown in medium containing cellobiose plus either fructose or mannose, the yeast preferentially utilized the monosaccharides and produced little β-glucosidase activity. Candida molischiana produced β-glucosidase and co-utilized cellobiose and xylose, maltose, or trehalose. Glucose and fructose, mannose, or trehalose were co-utilized; however, no β-glucosidase activity was detected. Thus, the order of substrate preference groups appeared to be (glucose, trehalose, fructose, mannose) > (cellobiose, maltose, xylose) > (mannitol, glucitol).Key words: glucose repression, trehalase, diauxic utilization, yeast.
TheAzospirillum brasilense rpoNgene is involved in nitrogen fixation, nitrate assimilation, ammonium uptake, and flagellar biosynthesis
Authors: Milcamps Anne , Dommelen Anne Van , Stigter John , Vanderleyden Jos , Bruijn Frans J. de
Published: February 2010

The rpoN (ntrA) gene (encoding sigma 54) of Azospirillum brasilense Sp7 was isolated by using conserved rpoN primers and the polymerase chain reaction, and its nucleotide sequence was determined. The deduced amino acid sequence of the RpoN protein was found to share a high degree of homology with other members of the sigma 54 family. Two additional open reading frames were found in the Azospirillum brasilense rpoN region, with significant similarity to equivalent regions surrounding the rpoN locus in other bacteria. An rpoN mutant of Azospirillum brasilense Sp7 was constructed by gene replacement and found to be defective in nitrogen fixation, nitrate assimilation, and ammonium uptake. Lack of ammonium uptake was also found in previously isolated Azospirillum brasilense ntrB and ntrC mutants, further supporting the role of the ntr system in this process. In addition, the rpoN mutant was found to be nonmotile, suggesting a role of RpoN in Azospirillum brasilense flagellar biosynthesis.Key words: Azospirillum brasilense, sigma factor, nitrogen fixation, ammonium assimilation, motility.
Selection and characterization of strains of Streptomyces suppressive to the potato scab pathogen
Authors: Liu Daqun , Anderson Neil A. , Kinkel Linda L.
Published: February 2010

Ninety-three strains of Streptomyces were isolated from lenticels of potato tubers grown in naturally disease-suppressive and disease-conducive soils. Twenty-two strains showed more antibiotic activity against virulent Streptomyces scabies RB3II than the standard pathogen-suppressive strains PonR and PonSSII. These 22 suppressive strains were non-pathogenic on leaf-bud tubers in the greenhouse. These suppressive strains plus standard strains PonR and PonSSII, 4 Minnesota (MN) virulent S. scabies strains, 11 virulent S. scabies strains, and 2 virulent Streptomyces acidiscabies strains from the eastern United States were evaluated in all possible paired combinations for antibiotic activity and competitive interactions in antibiotic and co-plating assays. A significant positive correlation between the ability of a strain to inhibit others and the ability of that strain to resist inhibition was observed in antibiotic assays (r = 0.6, P < 0.01). On average, suppressive strains inhibited other strains more and were less inhibited by other strains than virulent strains. Suppressive strains significantly reduced scab and did not affect tuber yield in a field-pot test. Taxonomic tests were used to characterize the Streptomyces strains used in this study. All the virulent strains and 54% of suppressive strains were classified as S. scabies.Key words: Streptomyces, suppressive soil, potato scab.
Denitrification by a novel halophilic fermentative bacterium
Authors: Shieh Wung Yang , Liu Chia Ming
Published: February 2010

A novel halophilic heterotrophic bacterium, designated strain DN34, was isolated from seawater in Nanwan Bay of Renting National Park, Taiwan. It was Gram negative and facultatively anaerobic. Cells in late exponential to early stationary phase of growth were predominantly straight or curved rods, but Y- or V-shaped forms were also observed; straight and curved rods achieved motility by one to several lateral or subpolar flagella. The G+C content of the DNA was 51.7 mol%. Strain DN34 grew optimally at about 30 °C and pH 8.0. Growth depended on the presence of NaCl with optimal concentration at about 3%. Aerobically, strain DN34 grew much better and tolerated NaCl at a greater range of concentration with sufficient Mg2+and Ca2+than under deficient conditions; Mg2+or Ca2+was indispensable for growth under anaerobic conditions. The strain was capable of anaerobic growth by carrying out denitrifying metabolism using nitrate, nitrite, or nitrous oxide as terminal electron acceptors or, alternatively, by fermenting glucose or mannose as substrates. Halophilic heterotrophic bacteria capable of both denitrification and fermentation have not been reported previously.Key words: denitrification, denitrifying bacteria, halophilic bacteria, fermentative bacteria.
Cyanide degradation by anEscherichia colistrain
Authors: Figueira Marianne M. , Ciminelli Virgínia S. T. , Andrade Mabel C. de , Linardi Valter R.
Published: February 2010

Chemical formation of a glucose–cyanide complex was necessary for metabolic degradation of cyanide at concentrations up to 50.0 mg/L by a strain of Escherichia coli isolated from gold extraction circuit liquids. Ammonia accumulating during the culture log phase as the sole nitrogen by-product was further utilized for bacterial growth. Washed (intact) cells, harvested at different periods of bacterial growth on cyanide, consumed oxygen in presence of cyanide. These findings suggest that metabolism of cyanide involved a dioxygenase enzyme that converted cyanide directly to ammonia, without the formation of cyanate.Key words: cyanide, cyanide metabolism, cyanide oxygenase, Escherichia coli.
Evaluation of tributyltin resistance in marine sediment bacteria
Authors: Jude F. , Capdepuy M. , Quentin C. , Lascourrèges J. F. , Caumette P.
Published: February 2010

Tributyltin (TBT) resistance of aerobic and anaerobic bacteria isolated from TBT-contaminated coastal sediments in Arcachon Bay, France, was determined and compared with taxonomically similar bacteria originating from diverse non-TBT-contaminated environments. The minimum inhibitory concentrations (MICs) showed that TBT was toxic to most of the bacteria studied at concentrations above 2.1 μM (0.7 mg/L) for clostridia, 76 μM (25 mg/L) for pseudomonads, and 3 mM (1000 mg/L) for enterobacteria. The aerobic or facultative anaerobic Gram-negative bacteria (pseudomonads and enterobacteria) presented the highest TBT tolerance. No difference in TBT resistance was observed between strains originating from the polluted site and strains originating from non-TBT-polluted environments (thermal muds, Hospital of Bordeaux, etc.), suggesting that a TBT-polluted environment did not select for more specific resistant bacteria. It should be noted that the bacteria tested were found to be tolerant to TBT concentrations 1000 times higher than those found in the polluted site. Further, all strains presented the same pattern of metal and pharmaceutical antibiotic sensitivity despite the origin of the environment (TBT-polluted or non-TBT-polluted sediments). Six strains of Pseudomonas stutzeri isolated from Arcachon Harbor and one strain of Pseudomonas aeruginosa originating from the bacterial collection of Bordeaux Hospital have been selected for their TBT resistance. As antibiotic resistance, metal or organometal resistance can be plasmid mediated. However, among these TBT-resistant strains no plasmid was detected.Key words: TBT resistance, coastal environment, clostridia, pseudomonads, enterobacteria.
Isolation and characterization of a mutator strain of Streptomyces ambofaciens ATCC23877 exhibiting an increased level of genetic instability
Authors: Vandewiele Dominique , Volff Jean-Nicolas , Aigle Bertrand , Simonet Jean-Marc , Decaris Bernard
Published: February 2010

In Streptomyces ambofaciens ATCC23877, 0.7% of pigment-defective mutants (Pig−) can be observed in the progeny of wild-type colonies. A mutator (Mut−) strain was isolated from the offspring of the wild-type strain. The Mut− strain produced colonies that sported nonpigmented papillae. Furthermore, the frequency of Pig− colonies obtained in the progeny of this strain was fivefold higher than in the wild-type strain. This strain showed the same level of sensitivity to ultraviolet light and mitomycin C as the wild-type strain. This Mut− phenotype was found to be reversible at high frequency (3 × 10−3). Genomic analysis using pulsed-field gel electrophoresis (PFGE) showed that the Pig− mutants arisen from the Mut− strain were less frequently rearranged (32% were deleted) compared with the mutants arising from the wild type (59% were deleted). Moreover, the Pig− papillae mutants possessed no visible rearrangement as revealed by PFGE analyses.Key words: Streptomyces, genetic instability, mutator strain, papillae.
Characterization and transformation of plasmid pAA-1 found in an antarctic cryptoendolithic bacterium
Authors: Gliesche Christian G. , Jendrach Marina , Peissl Klaus , Siebert Jörg , Hirsch Peter
Published: February 2010

Sixty-three bacterial isolates from antarctic sandstone samples (Linnaeus Terrace, Asgard Range, McMurdo Dry Valleys) were screened for the presence of plasmid DNA. Twenty-seven percent of all the isolates (mainly Gram-positives) harbored one or more plasmids of low molecular mass (1.1–16.3 kb). Strain AA-341 contained plasmid pAA-1 (2.9 kb), as demonstrated by agarose gel electrophoresis and restriction endonuclease digests. This plasmid conferred resistance to chromium and ampicillin. It was not conjugative, but it could be transferred by electroporation to chromium- and ampicillin-sensitive strains AA-330, AA-338, and E. coli HB101. A restriction map of pAA-1 was constructed with HindIII, SalI, ScaI, AvaII, EcoRI, PvuII, BamHI, and DraI. Electrotransfer of this plasmid from E. coli HB101(pAA-1) to strain AA-330 was demonstrated. By natural genetic transformation, plasmid pAA-1 could be transferred into the sensitive strain AA-334, which thereby became resistant to chromium and ampicillin. The importance of such processes for the colonization of stressed environments is discussed.Key words: Antarctica, cryptoendolithic bacteria, plasmids, resistance to chromium, natural transformation.
Characterization of the indole-3-acetic acid (IAA) biosynthetic pathway in an epiphytic strain ofErwinia herbicolaand IAA production in vitro
Authors: Brandi M. , Clark E. M. , Lindow S. E.
Published: February 2010

An epiphytic strain of Erwinia herbicola (strain 299R) synthesized indole-3-acetic acid (IAA) from indole-3-pyruvic acid and indole-3-acetaldehyde, but not from indole-3-acetamide and other intermediates of various IAA biosynthetic pathways in enzyme assays. TLC, HPLC, and GC–MS analyses revealed the presence of indole-3-pyruvic acid, indole-3-ethanol, and IAA in culture supernatants of strain 299R. Indole-3-acetaldehyde was detected in enzyme assays. Furthermore, strain 299R genomic DNA shared no homology with the iaaM and iaaH genes from Pseudomonas syringae pv. savastanoi, even in Southern hybridizations performed under low-stringency conditions. These observations strongly suggest that unlike gall-forming bacteria which can synthesize IAA by indole-3-acetamide, the indole-3-pyruvic acid pathway is the primary route for IAA biosynthesis in this plant-associated strain. IAA synthesis in tryptophan-supplemented cultures of strain 299R was over 10-fold higher under nitrogen-limiting conditions, indicating a possible role for IAA production by bacterial epiphytes in the acquisition of nutrients during growth in their natural habitat.Key words: indole-3-acetic acid, Erwinia, tryptophan, indole-3-pyruvic acid, nitrogen.
Characterization of bacterial communities in heavy metal contaminated soils
Authors: Roane T. M. , Kellogg S. T.
Published: February 2010

Heavy metal pollution is a principle source of environmental contamination. We analyzed heavy metal impacted soil microbial communities and found that, in general, although lead adversely affected biomass, metabolic activity, and diversity, autochthonous lead- and cadmium-resistant isolates were found. In several metal-stressed soils, the microbial community consisted of two populations, either resistant or sensitive to lead. Additionally, a lead-resistant isolate was isolated from a control soil with no known previous exposure to lead, suggesting widespread lead resistance. Lead-resistant genera isolated included Pseudomonas, Bacillus, Corynebacterium, and Enterobacter species. Plasmids, ranging from 5 to 260 kb, were not detected through standard purifications from lead-resistant isolates. Positive correlations existed between antibiotic resistance and isolation habitat for lead-resistant strains, microbial metabolic activity and soil type, soluble lead concentration and microbial diversity, and arsenic concentration and total or viable cell concentrations.Key words: heavy metal, lead, cadmium, soil bacteria, stress, remediation.
Purification and characterization of a lectin fromXanthomonas campestrisNCIM 5028
Authors: Joshi Bhagyashree , Khire Jayant M. , SivaRaman Hephzibah , Khan M. Islam
Published: February 2010

A lectin was isolated from culture filtrates of Xanthomonas campestris NCIM 5028, by a simple procedure of hydrophobic chromatography on phenyl-Sepharose after ammonium sulphate precipitation. The lectin was a heterodimer, with subunit molecular masses of 30 000 and 28 000. Gel filtration on S-300 column, calibrated with markers, showed its molecular mass to be approximately 70 000. Its isoelectric point was 7.2. The agglutination of the rabbit erythrocytes by the lectin was inhibited by fetuin glycopeptides and host plant (Brassica oleracea) extracts.Key words: Xanthomonas campestris, lectin, purification.
Selection of ann-heptanol-resistant bacterium from an organic solvent-sensitive bacterium and characterization of its fatty acids
Authors: Tsubata Takuichi , Kurane Ryuichirou
Published: February 2010

We obtained an organic solvent-sensitive bacterium capable of degrading dibenzothiophene (DBT) under microaerobic (nitrogen exchange) conditions. The bacterium with the highest DBT-degradative activity was mutated and an n-heptanol-resistant mutant was selected. Although the parent strain did not grow in 0.1% v/v n-heptanol at all, the mutant did grow even in 50% heptanol and could still retain the ability to degrade and assimilate DBT in 5–10% heptanol, nonanol, or decanol under microaerobic conditions. The percentages of odd-chain fatty acids (C15and C17), unsaturated fatty acids (especially the trans form), and isooctadecanoic acid were significantly greater in the mutant strain than the parent strain.Key words: dibenzothiophene, n-heptanol tolerance, Pseudomonas putida, fatty acid.
The extreme N-terminus of the Caulobacter crescentus surface-layer protein directs export of passenger proteins from the cytoplasm but is not required for secretion of the native protein
Authors: Bingle Wade H. , Le Khai D. , Smit John
Published: February 2010

The paracrystalline surface layer (S-layer) of Caulobacter crescentus is composed of a single protein (RsaA, 1026 amino acids) that associates noncovalently with the lipopolysaccharide of the outer membrane. Like many other extracellular proteins of Gram-negative bacteria, the S-layer protein is not processed during transport to the cell surface. To study the secretion of RsaA, several N-terminal deletions of the protein were made by modifying the 5′-region of the rsaA gene. This analysis showed that portions of the N-terminus totalling the first 775 N-terminal amino acids (75% of the protein) could be removed from RsaA without abolishing secretion of the remainder of the protein. Although the RsaA N-terminus was not required for secretion, an N-terminal domain consisting of either 34 or 52 RsaA-derived amino acids promoted export of the alkaline phosphatase reporter (PhoA) and a cellulase reporter (ΔCenA) from the cytoplasm; using the cellulase reporter, the efficiency of hybrid protein export was estimated at 9%. No enzyme activity was detected in the cell-free culture fluids as the result of expressing any gene fusion, indicating that no hybrid protein was completely secreted from the cell. RsaA:PhoA hybrid proteins were also exported from the E. coli cytoplasm, a bacterium not expected to contain the necessary machinery for the secretion of RsaA. Taken together, these data indicate that the secretion pathway of RsaA relies on a C-terminal secretion signal and that once separated from the context of the native protein, the extreme N-terminus of RsaA can act as an inefficient cryptic export signal that is not used during native RsaA secretion.Key words: Caulobacter, S-layer, protein secretion.
Douglas-fir root-associated microorganisms with inhibitory activity towards fungal plant pathogens and human bacterial pathogens
Authors: Axelrood Paige E. , Clarke Alison M. , Radley Reed , Zemcov S. Janet V.
Published: February 2010

A microbial culture collection composed of 1820 bacterial strains, including 298 actinomycete strains, was established from the roots of Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) seedlings harvested from conifer nurseries and forest sites. Two hundred and thirty-four strains inhibited the growth of Fusarium, Cylindrocarpon, and (or) Pythium spp. in in vitro assays. A significantly greater proportion of bacterial strains from actinomycete genera exhibited antifungal properties compared with bacterial strains from nonactinomycete genera. Eighty-nine percent of identified inhibitory strains were Streptomyces, Streptoverticillium, Bacillus, Pseudomonas, or Burkholderia species. The actinomycete species were isolated almost exclusively from forest seedlings. Recovery of inhibitory strains representing 29 microbial species was enhanced using a variety of methods to isolate microorganisms from the roots of seedlings from nursery and forest sites. Bacterial strains (including actinomycete strains) with antifungal activity were tested for in vitro growth inhibition of six clinical human bacterial pathogens (Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Escherichia coli, Proteus mirabilis, and Pseudomonas aeruginosa). Forty-eight percent of the tested strains inhibited one or more human pathogens. Inhibitory activity towards fungal and bacterial pathogens was strain specific, not species specific, and many inhibitory strains exhibited broad-spectrum activity. Strains with antifungal activity against several conifer root pathogens were also more likely to inhibit multiple species of clinical bacterial pathogens.Key words: in vitro, antimicrobial, conifer rhizosphere.
Improved method for colony counts of the microaerophile Spirillum volutans
Authors: Alban Patrick S. , Krieg Noel R.
Published: February 2010

Studies of adaptive responses of Spirillum volutans to various stresses such as heat shock have been hampered by an inability to obtain reliable colony counts of the organism by the spread plate method, due in part to differences among various lots of the casein hydrolysate component of the medium. Colony counts approaching direct microscopic counts (DMCs) were obtained by inoculating culture dilutions into a semisolid version of the medium and using this to overlay a thicker layer of sterile medium, and by supplementing the medium with pyruvate, which destroys hydrogen peroxide. Both the pyruvate and the overlay were necessary for optimal results. Use of the new overlay–pyruvate method for colony counts revealed that exposure of S. volutans to 40 °C for 100 min results in a greater survival at 45 °C compared with cells having no prior exposure to 40 °C.Key words: pyruvate, microaerophile, Spirillum volutans, heat shock.
Application of an optimized electroporation procedure for replacement of the polyhydroxyalkanoate synthase I gene inNocardia corallina
Authors: Valentin Henry E. , Dennis Douglas
Published: February 2010

To develop a system for gene replacement in Nocardia corallina, a protocol for electroporation was optimized by systematic alterations of growth conditions, field strength, time constant and the electroporation buffer. Transformation efficiencies of 0.5 × 106–3 × 106transformants/μg plasmid DNA were obtained routinely. The gene encoding the polyhydroxyalkanoate (PHA) synthase I of N. corallina was cloned and interrupted by insertion of a kanamycin-resistance gene. The resulting plasmid was introduced into N. corallina by electroporation to inactivate the wild-type gene by homologous recombination. Kanamycin-resistant clones were screened by Southern hybridization for the absence of the wild-type gene and analyzed for PHA accumulation.Key words: gene replacement, Nocardia, polyhydroxyalkanoates (PHAs).
Cold shock proteins and cold acclimation proteins in the psychrotrophic bacteriumPseudomonas putidaQ5 and its transconjugant
Authors: Gumley Andrew W. , Inniss William E.
Published: February 2010

The production of cold shock proteins (csps) and cold acclimation proteins (caps) was characterized in the psychrotrophic bacterium Pseudomonas putida Q5 and its transconjugant P. putida Q5T which contains the toluene-degradative TOL (pWWO) plasmid, using two-dimensional gel electrophoresis and computing scanning laser densitometry. Similar growth rates for the psychrotrophic bacterium P. putida Q5 and the transconjugant were found at temperatures ranging from 30 to 0 °C. Sixteen proteins were quantified and compared in P. putida Q5 and P. putida Q5T following a 25 to 5 °C cold shock or constant growth at 5 °C. During constant growth at 25 °C, a decrease in the synthesis of various proteins occurred in the transconjugant. Following cold shock to 5 °C or constant growth at 5 °C, csps and caps were produced with a greater number occurring in the transconjugant. This may suggest an additional stress response in the transconjugant owing to metabolic load exerted by the TOL plasmid. Growth of P. putida Q5T with toluate produced seven proteins that appeared to be TOL-plasmid mediated and of which some were also designated as caps.Key words: cold shock proteins, cold acclimation proteins, TOL pWWO plasmid, psychrotrophic bacterium.
Purification and characterization of the esterases involved in aflatoxin biosynthesis in Aspergillus parasiticus
Authors: Kusumoto Ken-Ichi , Hsieh Dennis P. H.
Published: February 2010

The esterases from the cell-free extracts (CFEs) of Aspergillus parasiticus ATCC15517, an aflatoxin-producing strain, catalyzing the hydrolytic conversion of versiconal hemiacetal acetate (VHA) to versiconal was biochemically studied. The specific activity of the enzymes increased 2.5-fold during incubation of mycelia through 40–55 h. No metal ions were required for enzyme stability, but EDTA at 1 mM and dithiothreitol at 0.5–5 mM increased its stability. Three peaks of VHA esterase activity were resolved when the proteins in the CFEs prepared from the mycelia of different ages were separated by anion-exchange column chromatography, suggesting that at least three VHA esterases were present in the eluate of this purification step. One of these esterases extracted from the mycelia of a 55-h culture was partially purified in five steps by means of preparative chromatography and fast protein liquid chromatogaphy. The partially purified enzyme when reacted with [14C]diisopropylfluorophosphate followed by sodium dodecyl sulfate – polyacrylamide gel electrophoresis gave a single radiolabelled band, which corresponded to a protein of 32 kDa. The molecular mass of the partially purified VHA esterase determined with gel filtration was around 60 kDa. The results suggested that the enzyme consists of two isomeric subunits.Key words: aflatoxin biosynthesis, esterase, versiconal hemiacetal acetate, Aspergillus parasiticus.
Identification of two core types in lipopolysaccharides ofActinobacillus pleuropneumoniaerepresenting serotypes 1 to 12
Authors: Jacques Mario , Rioux Stéphane , Paradis Sonia-Élaine , Bégin Caroline , Gottschalk Marcelo
Published: February 2010

Lipopolysaccharides (LPS) of Actinobacillus pleuropneumoniae were separated by Tricine–SDS–polyacrylamide gel electrophoresis, which has been shown to improve resolution of low-molecular-mass fast migrating bands. Strains representing the 12 serotypes of A. pleuropneumoniae can be divided in two groups according to the gel mobility of the core–lipid A region of their LPS. The first electromorphic core type (core type I), found in serotypes 1, 6, 9, and 11, had a migration slower than Salmonella typhimurium Ra LPS. The second electromorphic core type (core type II), found in the remaining serotypes (i.e., 2, 3, 4, 5, 7, 8, 10, and 12) had a migration similar to S. typhimurium Ra LPS. Furthermore, we observed that these two core types were antigenically different. Western blot analyses indicated that core – lipid A region of LPS from electromorphic core type I strains reacted when probed with serum from a pig experimentally infected with a core type I strain but not when probed with serum from a pig experimentally infected with a core type II strain. Conversely, core – lipid A region of LPS from electromorphic core type II strains reacted only when probed with serum from a pig experimentally infected with a core type II strain. Our results, based on both electrophoretic mobility and antigenicity, suggest the presence of two LPS core types in A. pleuropneumoniae.Key words: Actinobacillus pleuropneumoniae, lipopolysaccharides, core.
Genetic and biochemical analysis of the cysteinyl residues of isopenicillin N synthase from Streptomyces clavuligerus
Authors: Durairaj Micheal , Leskiw Brenda K. , Jensen Susan E.
Published: February 2010

Isopenicillin N synthase (IPNS) from Streptomyces clavuligerus catalyses the oxidative cyclization of the acyclic tripeptide δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine into isopenicillin N. All four of the cysteine residues found in this enzyme were mutated individually into serine residues, either by the polymerase chain reaction or by single-strand site-directed mutagenesis. Functional analysis of these single mutants showed that the C104S mutant lost more than 96% of its activity, while the remaining C37S, C142S, and C251S mutants each lost 30–50% of their activity. Treatment with the thiol-group-specific reagent N-ethylmaleimide confirmed the importance of the cysteine 104 residue. Activity analysis of an IPNS triple mutant (C37S, C142S, and C251S), prepared by recombining fragments of the IPNS-encoding pcbC gene from each of the three single mutants, showed that it had lost more than 90% of its activity. Conformational analysis by circular dichroism spectroscopy indicated that the IPNS triple mutant was structurally different from the wild type, suggesting that the loss of activity may be due to conformational changes rather than active site modifications.Key words: penicillin, Streptomyces, site-directed mutagenesis, polymerase chain reaction, thiol groups.
A moderately halophilic Vibrio from a Spanish saltern and its lytic bacteriophage
Authors: Goel Usha , Kauri Tiiu , Kushner Donn J. , Ackermann Hans-W.
Published: February 2010

A number of bacteria and their phages were isolated from a saltern near Alicante, Spain. One isolate, Vibrio B1, a moderate halophile that is probably a strain of Vibrio costicola, was host to a lytic phage, UTAK. Studies of the host bacterium included the effects of salt concentrations on the action of a number of inhibitory agents. Phage UTAK has a head, a tail, and a baseplate. It contains 80 kbp of double-stranded DNA with no unusual bases. It was stable for long periods in the absence of high salt concentrations and even in distilled water. Salt concentrations had little effect on adsorption of UTAK to its host but resulted in considerable changes in burst size. It appears that phages of halophilic and salt-tolerant eubacteria, and also of some marine bacteria, have much lower salt requirements for stability than the phages of halophilic archaebacteria. Our results suggest that ionic controls of phage replication in these eubacteria may differ from those of growth.Key words: halophiles, Vibrio sp., bacteriophage, salt responses.
Water and temperature relations and microconidial germination of Fusarium moniliforme and Fusarium proliferatum from maize
Authors: Marín Sonia , Sanchis V. , Teixido A. , Saenz R. , Ramos A. J. , Vinas I. , Magan N.
Published: February 2010

The effects of water activity (aw, 0.994–0.85 = 0.4–21.0 (−)MPa water potential), temperature (5–42 °C), and their interactions on microconidial germination of three isolates each of Fusarium moniliforme and Fusarium proliferatum were determined in vitro on a maize meal extract medium. Temporal germination rates of microconidia of isolates of both species were significantly influenced by both aw and temperature. Germination was very rapid at >0.94 aw with an almost linear increase with time. Germination rates of microconidia of F. moniliforme were slower than those of F. proliferatum isolates at marginal aw levels and 5–25 °C, while at higher temperature (30–37 °C), the former germinated more rapidly than the latter. The aw minima for germination of isolates of both species was 0.88, with none occurring at 0.85 aw over a 40-day incubation period. At 37 °C, isolates of F. moniliforme had slightly lower aw minima than those of F. proliferatum. The narrowest range of aw for germination was at 5 °C, and none occurred at 42 °C. The effect of aw × temperature interactions on the lag phases (h) prior to germination and the germination rates (h−1) were estimated using the Gompertz model and the Zwietering equation. This showed that lag phases were shorter at 25–30 °C and 0.994–0.98 aw, and were increased to 10–500 h at marginal temperatures (5–10 °C) for F. proliferatum and longer for F. moniliforme. At marginal aw levels (0.92–0.90), lag times were increased to >250 h. Germination rates (h−1) were different for the two species. Microconidia of F. moniliforme germinated optimally at 25–37 °C and 0.96–0.98 aw, but this changed to 30 °C at 0.90–0.94 aw, while germination of microconidia of F. proliferatum remained optimum at 30 °C, regardless of aw. There were statistically significant (P < 0.01) effects of aw, temperature, isolate, and two- and three-way interactions for F. proliferatum, but there were no intraisolate effects for F. moniliforme. The ecological significance of these data for understanding colonization patterns of these important fumonisin-producing fungi are discussed.Key words: water activity, temperature, germination, fumonisin producing, Fusarium spp.
Assimilation of oxalate, acetate, and CO2byOxalobacter formigenes
Authors: Cornick N. A. , Allison M. J.
Published: February 2010

Oxalobacterformigenes is the only well-documented oxalate-degrading bacterium isolated from the gastrointestinal tract of animals. The production of ATP by Oxalobacter formigenes is centered around oxalate metabolism and oxalate is required for growth. A small amount of acetate (0.5 mM) is also required. Oxalate is decarboxylated to formate plus CO2in nearly equimolar amounts. Experiments were conducted to determine which potential carbon sources (oxalate, acetate, formate, CO2) were assimilated by Oxalobacter formigenes and which metabolic pathways were operative in carbon assimilation. Measurements of the specific activities of total cell carbon after growth with different14C-labeled precursors indicated that at least 54% of the total cell carbon was derived from oxalate and at least 7% was derived from acetate. Carbonate was also assimilated, but formate was not a significant source of cell carbon. Labeling patterns in amino acids from cells grown in [14C]oxalate or14CO3were different; however, in both cases14C was widely distributed into most cellular amino acids. Carbon from [14C]acetate was less widely distributed and detected mainly in those amino acids known to be derived from α-ketoglutarate, oxaloacetate, and pyruvate. Cell-free extracts contained citrate synthase, isocitrate dehydrogenase, and malate dehydrogenase activities. The labeling observed in amino acids derived from acetate is in agreement with the function of these enzymes in biosynthesis and indicates that the majority of acetate carbon entered into amino acid biosynthesis via well-known pathways.Key words: biosynthesis, carbon assimilation, metabolism.
Simultaneous but differential metabolism of glucose and cellobiose inFibrobacter succinogenescells, studied by in vivo13C-NMR
Authors: Matheron Christelle , Delort Anne-Marie , Gaudet Geneviève , Forano Evelyne
Published: February 2010

Kinetics of [1-13C]glucose utilization were monitored by in vivo NMR spectroscopy on resting cells of Fibrobacter succinogenes, in the presence of 32 mM [1-13C]glucose, 32 mM [1-13C]glucose and 64 mM unlabelled glucose, or 32 mM [1-13C]glucose and 32 mM unlabelled cellobiose. A similar production of acetate and succinate and a similar storage of glycogen were observed whatever the exogenous substrate. The presence of cellobiose or that of an equivalent amount of glucose did not reduce [1-13C]glucose incorporation to the same extent. Glucose seemed preferentially used for glycogen storage and energy production, while part of the cellobiose appeared to be used for cellodextrin synthesis. Both cellobiase and cellobiose phosphorylase activities were assayed in cell-free extracts. Finally, the intracellular concentration of glucose 6-phosphate was increased by over threefold when cells metabolized cellobiose (alone or in parallel to glucose) as compared with the metabolism of glucose alone.Key words: Fibrobacter succinogenes, rumen, glucose 6-phosphate, cellobiose, NMR.
Genus- and species-specific detection ofListeria monocytogenesusing polymerase chain reaction assays targeting the 16S/23S intergenic spacer region of the rRNA operon
Authors: Graham Tom , Golsteyn-Thomas Elizabeth J. , Gannon Victor P. J. , Thomas James E.
Published: February 2010

In this study, the 16S/23S rRNA intergenic spacer (IGS) regions of six Listeria species were examined. DNA bands of 590 and 340 bp were observed following polymerase chain reaction (PCR) amplification of DNA from Listeria monocytogenes, Listeria innocua, Listeria seeligeri, Listeria welshimeri, and Listeria ivanovii strains with generic rRNA IGS oligonucleotide primers. For strains of Listeria grayi subspp. grayi and murrayi, DNA band sizes of 550 and 340 bp were observed with this primer pair. DNA bands of these sizes were not observed for other Gram-negative or -positive bacteria in this PCR assay. Four RsaI digestion profiles were noted for the Listeria PCR products. Listeria monocytogenes strains had one profile; L. innocua strains had a second; L. seeligeri, L. welshimeri, and L. ivanovii strains had a third; and L. grayi strains had a fourth. The small and large 16S/23S rRNA IGSs of L. monocytogenes ATCC 15313 were identical in the first 58 5′ and the last 169 3′ nucleotides. However, the large rRNA IGS contained a central 267-bp region with tRNAIleand tRNAAlagenes. Large rRNA 16S/23S IGS nucleotide sequence data has not been previously reported. This data was used to develop novel Listeria genus-specific and L. monocytogenes species-specific PCR assays.Key words: polymerase chain reaction, 16S/23S rRNA intergenic spacer region, Listeria monocytogenes.
Sterols and fatty acids in Peziza muralis
Authors: Křen Vladimír , Řezanka Tomáš
Published: February 2010

Composition of sterols and fatty acids in fruiting bodies of fungus Peziza muralis were determined by gas chromatography – mass spectometry. Sterol distribution (e.g., brassicasterol occurrence) confirms phylogenetic relations to the order Tuberales. Nontypical lipid composition reflects extreme conditions (e.g., sunny, dry, and mineral-rich niches) where this fungus occurs.Key words: Peziza muralis, lipids, sterols, brassicasterol, fatty acids.
Characterization of partial anaerobic metabolic pathway for 2,4,6-trinitrotoluene degradation by a sulfate-reducing bacterial consortium
Authors: Boopathy R. , Manning J. F.
Published: February 2010

The anaerobic degradative pathway for metabolism of 2,4,6-trinitrotoluene (TNT) by a consortium of Desulfovibrio spp. isolated from a creek sediment was studied. This consortium has the metabolic capability to degrade TNT to fatty acids. The growth of the consortium and the metabolism of TNT were greatly enhanced in the presence of an additional carbon source like pyruvate. The optimal concentration of pyruvate for the maximum rate of TNT degradation was 15–20 mM. Various intermediates of TNT metabolism were identified. The first step in the pathway was reduction of TNT to 4-amino-2,6-dinitrotoluene and 2-amino-4,6-dinitrotoluene, which were further reduced to 2,4-diamino,6-nitrotoluene. The next intermediate to appear in the culture medium was nitrobenzoic acid, followed by cyclohexanone, 2-methyl pentanoic acid, butyric acid, and acetic acid. A study using radiolabeled TNT showed that no CO2was produced from TNT during metabolism. The mass balance of the radiolabeled study showed that 49.6% of the TNT was converted to acetic acid, 28% was assimilated into biomass as trichloroacetic acid precipitable materials, and the rest was distributed as various TNT intermediates. Most Desulfovibrio spp. are incomplete oxidizers that are unable to carry out the terminal oxidation of organic substrates. The major end product of TNT metabolism was acetic acid. The bacteria grew on all the TNT intermediates tested as sole source of carbon, except on acetic acid, confirming that the Desulfovibrio spp. have the enzymes necessary for complete degradation of TNT to acetate.Key words: TNT, bioremediation, sulfate reducers, anaerobic process, butyric acid, Desulfovibrio, spp.
Mineralization of [14C]octacosane by Acinetobacter calcoaceticus S30
Authors: Lal Banwari , Khanna Sunil
Published: February 2010

Acinetobacter calcoaceticus S30 could grow (doubling time, 7 h) on octacosane (C28) and degraded about 70% of the substrate during growth. Octacosanol, octacosanoic acid, and other lower carboxylic acids were identified during degradation of octacosane. Acinetobacter calcoaceticus S30 could also grow on intermediate metabolites, namely octacosanol and octacosanoic acid, although the doubling time was greater on octacosanoic acid (72 h on octacosanol and 120 h on octacosanoic acid). Whole cells of A. calcoaceticus S30 using [18-14C]octacosane mineralized 65% of the octacosane to 14CO2 and 30% of the radiolabel was retained in the cell biomass in 24 h. Acinetobacter calcoaceticus S30 converts octacosane to octacosanol through an oxidation step, which is then oxidized to octacosanoic acid and then β-oxidized to CO2. Among several metabolic inhibitors, those of the sulphydryl group greatly inhibited the uptake of octacosanol and octacosanoic acid at much lower concentrations. The electron transport inhibitors were potent inhibitors of octacosane, octacosanol, and octacosanoic acid uptake, suggesting that the oxidation of these substrates is an energy-dependent process.Key words: Acinetobacter calcoaceticus, mineralization, octacosane, octacosanol, octacosanoic acid.
Relationships amongFusariumspp. estimated by comparing restriction fragment length polymorphisms in polymerase chain reaction-amplified nuclear rDNA
Authors: Bateman G. L. , Ward E. , Kwaśna H.
Published: February 2010

Nuclear rDNA from 120 isolates of 34 Fusarium spp. and Microdochium nivale was compared by restriction fragment length polymorphism (RFLP) analysis after polymerase chain reaction amplification. The RFLPs allowed differentiation between species or groups of species. The presence or absence of each of 75 DNA bands was also used to compile a similarity matrix for cluster analysis to show estimated phylogenetic relationships. There was mostly little diversity between isolates of the same species. However, there were at least two distinct genetic types among isolates that conformed morphologically to each of the species F. avenaceum, F. sambucinum, F. flocciferum, and F. proliferatum. Most relationships were consistent with current understanding of Fusarium taxonomy. The division into taxonomic sections based on morphological characteristics was generally not supported.Key words: Fusarium, rDNA, phylogeny.
A dual cofactor-specific isocitrate dehydrogenase fromPythium ultimum
Authors: Kim Hakryul , Mozaffar Zahid , Weete John D.
Published: February 2010

Isocitrate dehydrogenase is considered to be one of the key regulatory enzymes in the conversion of glucose into fatty acids by oleaginous microorganisms. A dual coenzyme-specific isocitrate dehydrogenase (EC 1.1.1.41) (IDH) was isolated from the primitive fungus Pythium ultimum and purified by 211-fold by sequential ion-exchange, affinity, and gel filtration chromatographies. Specific activity of the partially purified enzyme was 76.2 μmol/(min∙mg protein) with NAD+and 40% less active with NADP+. Optimum pH for activity was 8.5–9.5. Kmvalues for threo-D-isocitrate and NAD+were 0.031 and 0.55 mM, respectively. The estimated molecular mass of the IDH was 96 kDa under nondenaturing conditions and 48 kDa under denaturing conditions, suggesting that the enzyme is composed of two subunits of the same size. The enzyme was relatively stable up to 55 °C, but no activity was detected after exposure to 65 °C for 15 min. Mg2+or Mn2+were required for activity.Key words: isocitric dehydrogenase, Pythium ultimum, dual cofactor specific, oleaginicity.
Pantoea agglomerans is the etiological agent for black spot necrosis on beach peas
Authors: Khetmalas Madhukar B. , Bal Arya K. , Noble Lisa D. , Gow John A.
Published: February 2010

Pantoea agglomerans was isolated from necrotic spots in the leaves of a beach pea (Lathyrus maritimus L.) that grew on the shorelines of Newfoundland, Canada. The bacterium produced cellulase and amylase and was shown to be a wound parasite. Ultrastructural studies of infected leaves showed bacterial aggregates surrounded by pellicles, within intercellular spaces of the necrotic tissue. The bacterium was adapted to temperate climatic conditions. On a culture medium it grew at 5–37 °C, with optimal growth observed at 10–25 °C. Under natural environmental conditions beach peas may be exposed to seawater. When the bacterium was tested for growth tolerance to NaCl, the NaCl was inhibitory, most noticeably at concentrations above 250 mM. Beyond contributing to mechanical injury of plant tissue and keeping the plant surface moist, it is likely that seawater would hinder, rather than enhance, the survival of the bacterium during the phase of the infection process when it was outside the host plant.Key words: beach pea, host–pathogen, Lathyrus maritimus, Pantoea agglomerans.