bluuebunny/crossref_metadata_2025 huggingface dataset:
Lytic infection ofEscherichia colibiofilms by bacteriophage T4
Authors: Doolittle M. M. , Cooney J. J. , Caldwell D. E.
Published: October 2009

Escherichia coli 3000 XIII formed biofilms on the surface of polyvinylchloride coupons in a modified Robbins device. Bacteriophage T4D+infected cells in the biofilm and replicated. It is commonly held that bacteriophage cannot infect surface-attached bacteria (biofilms) because such bacteria are protected by an exopolymeric matrix that binds macromolecules and prevents their diffusion into the biofilm. To our knowledge this is the first observation that a bacteriophage can infect and multiply within cells growing as a biofilm.Key words: bacteriophage, biofilm, modified Robbins device, Escherichia coli, MRD.
Tryptophan 2,3-dioxygenase inSaccharomyces cerevisiae
Authors: Iwamoto Yoshiki , Lee In Sook Matsui , Kido Ryo , Tsubaki Motonari
Published: October 2009

The tryptophan pyrrole-ring cleavage enzyme (TPCE) was detected in the yeast Saccharomyces cerevisiae. TPCE activity existed constitutively and was markedly induced by culturing the cells in a medium containing 0.1% (w/v) L-tryptophan. We purified partially the enzyme from the L-tryptophan-induced cells by phospho-cellulose column chromatography. The partially purified enzyme was stimulated solely by L-ascorbic acid, a nonspecific reductant, suggesting that the yeast TPCE is not indoleamine 2,3-dioxygenase, but rather tryptophan 2,3-dioxygenase. The enzyme metabolized L-tryptophan preferentially, and D-tryptophan slightly. KCN and NaN3, exogenous ligands of heme, inhibited the enzyme activity drastically, indicating that yeast tryptophan 2,3-dioxygenase contains heme(s) in its active site. The optimal pH of the enzyme was 6.5. Upon two-dimensional polyacrylamide gel electrophoresis, a protein staining spot was identified that was induced by L-tryptophan and whose intensity changed in correlation with the tryptophan 2,3-dioxygenase activity after phospho-cellulose column chromatography. This protein, exhibiting a molecular weight of approximately 38 000 and an isoelectric point of approximately pH 8.0, may be identified as a subunit of yeast tryptophan 2,3-dioxygenase.Key words: tryptophan 2,3-dioxygenase, Saccharomyces cerevisiae, partial purification, heme.
Contact-dependent acquisition of transferrin-bound iron by two strains of Haemophilus parasuis
Authors: Charland N. , D'silva C. G. , Dumont R. A. , Niven D. F.
Published: October 2009

Two strains of Haemophilus parasuis, namely, the type strain (ATCC 19417) and strain E751, were investigated with respect to iron acquisition. Both strains produced iron-repressible outer membrane proteins and could acquire iron from porcine transferrin but not from porcine lactoferrin. Neither strain used bovine transferrin, and human transferrin was used to only a very limited extent, if at all. In all cases, iron acquisition from transferrin required direct contact between the organisms and the protein. An affinity isolation technique based on biotinylated porcine transferrin plus streptavidin-agarose, followed by SDS-PAGE, allowed the isolation and identification of two potential porcine transferrin binding polypeptides (94 and 60 kDa) from total membranes derived from the type strain grown under iron-restricted conditions but only one (96 kDa) from strain E751. Each of these polypeptides was iron repressible and was not isolated when biotinylated human or bovine transferrin was used instead of biotinylated porcine transferrin. It is concluded that both strains acquire transferrin-bound iron by means of siderophore-independent mechanisms and that the isolated polypeptides represent porcine transferrin receptor components.Key words: Haemophilus parasuis, iron, transferrin, receptors.
Cloning the gene for the heat shock response positiwe regulator (sigma 32 homolog) from Pseudomonas aeruginosa
Authors: Naczynski Zerlina M. , Kropinski Andrew M. , Mueller Chris
Published: October 2009

A 31 base pair synthetic oligonucleotide based on the genes for the Escherichia coli heat shock sigma factor (rpoH) and the Pseudomonas aeruginosa housekeeping sigma factor (rpoD) was employed in conjunction with the Tanaka et al. (K. Tanaka, T. Shiina, and H. Takahashi, 1988. Science (Washington, D.C.), 242: 1040–1042) RpoD box probe to identify the location of the rpoH gene in P. aeruginosa genomic digests. This gene was cloned into plasmid pGEM3Z(f+), sequenced, and found to share 67% nucleotide identity and 77% amino acid homology with the rpoH gene and its product (σ32) of E. coli. The plasmid containing the rpoH gene complemented the function of σ32 in an E. coli rpoH deletion mutant. Furthermore, this plasmid directed the synthesis of a 32-kDa protein in an E. coli S-30 in vitro transcription–translation system. Primer extension studies were used to identify the transcriptional start sites under control and heat-stressed (45 and 50 °C) conditions. Two promoter sites were identified having sequence homology to the E. coli σ70 and σ24 consensus sequences.Key words: heat shock, Pseudomonas aeruginosa, sigma factor, transcription, oligonucleotide probe.
Neither reduced uptake nor increased efflux is encoded by tellurite resistance determinants expressed inEscherichia coli
Authors: Turner Raymond J. , Weiner Joel H. , Taylor Diane E.
Published: October 2009

Rates of uptake of the TeO32−oxyanion were investigated in Escherichia coli cells containing tellurite resistance determinants from both plasmid (RK2Ter, R478, pMER610, MIP233, pHH1508a, pMUR) and chromosomal (tehAB) sources. The uptake was investigated to determine whether or not reduced uptake or increased efflux is involved in the tellurite resistance mechanism. Reduced TeO32−uptake generated by cultures harboring arsABC from the plasmid R773, which has been previously shown to be an oxyanion efflux transporter, was used as the standard. Uptake curves were found to be essentially identical among E. coli cultures harboring the tellurite resistance plasmids RK2Ter, pMER610, pHH1508a, and pMUR and cultures harboring tellurite-sensitive control plasmids. Cultures harboring clones of the tehAB operon from E. coli showed no change in the TeO32−accumulation. Cultures harboring R478 demonstrated reduced uptake. However, a subclone containing only the tellurite resistance determinant displayed no reduced uptake. This suggests that there may be another determinant on R478 other than the primary tellurite resistance determinant that gives rise to TeO32−efflux. These results demonstrate that neither reduced uptake nor increased efflux is responsible for the tellurite resistance in the resistance determinants investigated here.Key words: tellurite resistance, uptake, metal resistance, resistance.
Purification and partial characterization of two extracellular alkaline proteases from Streptomyces corchorusii ST36
Authors: El-Shanshoury Abd El-Raheem R. , El-Sayed Mostafa A. , Sammour Reda H. , El-Shouny Wageeh A.
Published: October 2009

Two distinct alkaline proteases were detected in the culture medium of Streptomyces corchorusii ST36 isolated from an Egyptian soil sample. These enzymes were purified by precipitation with chilled ethanol and gel filtration on carboxymethyl Sepharose and on Sephadex G-120. The enzymes were purified 6 and 6.5 fold to homogeneity. The purified enzymes had final specific activities on substrate of 3.6 and 3.9 U/mg. Protease 1 had a molecular mass of 31 kDa and protease 2 was composed of two subunits, of molecular masses 18.6 and 17.4 kDa. The optimal pH and temperature for catalytic activity of protease 1 were pH 11 and 70 °C, respectively, and for protease 2 they were pH 10 and 70 °C, respectively. Protease 1 was more thermostable than protease 2. Both enzymes showed substrate specificity to casein, serum albumin, and ovalbumin. Calcium, copper, and cobalt stimulated protease 2 but did not significantly affect protease 1. Mercury was the strongest inhibitor for protease 2. The proteolytic activities of both proteases were inhibited by 10 mM phenanthroline and 50 mM ethylenediaminetetraacetic acid (EDTA). The inhibitory effect of EDTA on both enzymes was reversed by the addition of 50 mM of copper, calcium, or cobalt. Both enzymes were more stable at −20 °C under alkaline conditions than under neutral or acidic conditions.Key words: Streptomyces corchorusii, alkaline proteases, partial characterization.
Antimicrobial mode of action of secretions from the metapleural gland ofMytmecia gulosa(Australian bull ant)
Authors: Mackintosh J. A. , Trimble J. E. , Beattie A. J. , Veal D. A. , Jones M. K. , Karuso P. H.
Published: October 2009

Secretions from exocrine metapleural glands of Myrmecia gulosa (Australian bull ant) exhibit broad-spectrum antimicrobial activity. Treatment of the yeast Candida albicans with metapleural secretion resulted in the rapid and total leakage of K+ions from cells within 10 min. Ultrastructural analysis of the bacteria Bacillus cereus, Escherichia coli, and Pseudomonas aeruginosa, and cells and protoplasts of Candida albicans demonstrated gross damage of the cell membrane and aggregation of the cytoplasmic matrix of treated cells. Degradation of membrane-bound organelles was also observed in Candida albicans. The antimicrobially active components of metapleural secretions were nonpolar and interacted with the phospholipid bilayer, causing damage to the structural integrity of liposomes and the release of carboxyfluorescein. The data suggest that the antimicrobial agents in metapleural secretion act primarily by disrupting the structure and function of the phospholipid bilayer of the cytoplasmic membrane.Key words: ant metapleural secretion, antimicrobial, Candida albicans, cytoplasmic membrane.
Induction of resistance to hydrogen peroxide and radiation inDeinococcus radiodurans
Authors: Wang Ping , Schellhorn Herb E.
Published: October 2009

Though bacteria of the radiation-resistant genus Deinococcus have a high resistance to the lethal and mutagenic effects of many DNA-damaging agents, the mechanisms involved in the response of these bacteria to oxidative stress are poorly understood. To investigate antioxidant enzyme responses in Deinococcus spp., the catalase activity produced by these bacteria was measured and the sensitivity of these bacteria to hydrogen peroxide was tested. Deinococcus spp. had higher levels of catalase and were more resistant to hydrogen peroxide than Escherichia coli K12. The high levels of catalase produced by Deinococcus radiodurans were, in part, regulated by growth phase. Cultures of D. radiodurans, when pretreated with sublethal levels of hydrogen peroxide, became relatively resistant to the lethal effects of hydrogen peroxide and exhibited higher levels of catalase than untreated control cultures. These pretreated cells were also resistant to lethality mediated by ultraviolet light and γ-rays. These results suggest that Deinococcus spp. possess inducible defense mechanism(s) against the deleterious effects of oxidants and ionizing and ultraviolet radiation.Key words: catalase, oxidative stress, radiation resistance, Deinococcus spp.
Isolation of endophytic fungi from eastern larch (Larix lancina) leaves from New Brunswick, Canada
Authors: Dobranic J. K. , Johnson J. A. , Alikhan Q. R.
Published: October 2009

Endophytic fungi were isolated from leaves of eastern larch (Larix laricina (Du Roi) K. Koch) collected in Saint John County, New Brunswick, Canada. Two thousand three hundred and six isolates were obtained from 1600 leaves (6400 leaf segments) from 40 trees. Isolates were obtained from 36.0% of the segments. These were distributed among 73.9% of the leaves. Multiple isolates (two or more) were obtained from 12.8% of the leaves but 26.1% of the leaves had no endophyte isolated whatsoever. The isolate designated RP31 accounted for 84.2% of all isolates and was the lone endophyte isolated from 46.5% of the leaves. Isolates RPB82, RPB65, and coelomycete 3 accounted for 10.2% of all isolates obtained. Of the 10 taxa isolated, RP31, RPB82, RPB65, and coelomycete 3 represented 98.1% of the total number of isolates. There was no significant (p > 0.05) difference in the number of isolations between leaf segments from the petiole to the tip when all isolates were considered together. However, if RP31 was excluded from the analysis, the remaining isolates were isolated significantly (p > 0.05) more frequently from the petiole segment.Key words: endophytic fungi, Larix laricina, leaves, deciduous.
Pyruvate:ferredoxin oxidoreductase in Campylobacter species
Authors: Daucher James A. , Krieg Noel R.
Published: October 2009

Twelve strains representing 11 Campylobacter species were tested by two methods to see whether pyruvate:ferredoxin oxidoreductase, which occurs mainly in anaerobes, was also present in these oxygen-respiring microaerophiles. Crude extracts exhibited a pyruvate-dependent reduction of benzyl viologen and, to a lesser extent, metronidazole. Addition of Clostridium pasteurianum ferredoxin enhanced the metronidazole-reducing activity. All strains were inhibited by metronidazole, with minimal inhibitory concentrations ranging from 3 to 25 μg/mL. The results suggest that pyruvate: ferredoxin oxidoreductase is characteristic of the genus.Key words: Campylobacter, microaerophilic, metronidazole, pyruvate.
Fructose transport byHaloferax volcanii
Authors: Takano Jin-Ichiro , Kaidoh Kouichi , Kamo Naoki
Published: October 2009

Uptake of fructose by intact cells of Haloferax volcanii, one of the sugar-utilizing halobacteria, was examined with the following results. (i) The fructose transporter was inducible, (ii) Kinetic analysis showed a Ktof 0.37 μM and a Vmaxof 4.61 nmol∙mg protein−1∙min−1. For a glucose transport system in this bacterium, Ktwas 12.5 μM, showing that the fructose transporter had a much larger affinity for a substrate than the glucose transporter, (iii) No uptake was observed by the envelope vesicles, (iv) Phloridzin and phloretin inhibited fructose transport, although a relatively higher concentration was required than that needed to inhibit glucose uptake, (v) The driving force for fructose transport was a Na+electrochemical potential gradient, (vi) Glucose and fructose were interchangeable and this conversion led to the expression of the glucose transporter even when fructose was used as the sole carbohydrate, and vice versa.Key words: glucose uptake, symport with Na+, phloridzin, phloretin, Haloferax volcanii.
Spore-forming bacteria that carboxylate phenol to benzoic acid under anaerobic conditions
Authors: Létouraeau L. , Bisaillon J.-G. , Lépine F. , Beaudet R.
Published: October 2009

A methanogenic consortium transforming phenol to benzoic acid was submitted to different treatments to characterize the carboxylating microorganisms and eventually to facilitate their isolation. Under aerobic conditions, phenol was not transformed by the consortium and no growth was observed on solid medium. The consortium from an inoculum that was treated with heat, or heat and ethanol, retained the ability to carboxylate phenol under strictly anaerobic conditions. Electron microscopic observations of the consortium from an inoculum that was heated for 15 min at 80 °C revealed only Gram-positive bacilli. In this culture, methane production was not detected and benzoic acid accumulated. Five colonies with distinct morphologies were isolated from this culture on solid medium. Four of these strains were identified as Clostridium spp. In contrast to the untreated culture, none of the strains isolated were able to carboxylate phenol in pure culture or in coculture, nor could they decarboxylate or dehydroxylate 4-hydroxybenzoic acid, or oxidize 2-hydroxybenzyl alcohol, or O-demethylate anisole or 2-methoxyphenol. Also, the consortium from a treated inoculum retained its ability to decarboxylate and dehydroxylate 4-hydroxybenzoic acid forming phenol and benzoic acid, respectively, but could not accomplish the other reactions. These results suggest that spore-forming microorganisms are involved in the carboxylation of phenol and in the decarboxylation and dehydroxylation of 4-hydroxybenzoic acid.Key words: spore-forming bacteria, phenol, benzoic acid, methanogenic conditions, carboxylation.
The removal of phages T1 and PP7, and poliovirus from fluids with hollow-fiber ultrafilters with molecular weight cut-offs of 50 000, 13 000, and 6000
Authors: Oshima Kevin H. , Evans-Strickfaden Tammy T. , Highsmith Anita K. , Ades Edwin W.
Published: October 2009

We tested the ability of hollow-fiber ultrafilters with molecular weight cut-offs (MWCOs) of 50 000, 13 000, and 6000 to remove and detect viral agents (phage T1, 50–150 nm; phage PP7, poliovirus, 28–30 nm) from ultrapure water, 0.85% saline with 1% trypticase soy broth, and Dulbecco's modified Eagle minimum essential medium with 10% fetal bovine serum (DMEM-10). Virus diluted in saline and DMEM-10 were tested to evaluate filter performance under conditions that minimize the adsorption of viral particles to the filter matrix. During filtration, the retentate was returned to the input reservoir, and the permeate was removed to a separate vessel. Thus, the virus concentration in the feed increased over the course of filtration. Filter performance was evaluated by comparing the concentration of infectious virus in the initial virus suspension with the virus concentration in the permeate and retentate. Very efficient removal of phages T1 and PP7 was observed with the filters with MWCOs of 13 000 and 6000 (titer reduction > 7 logs) for all three fluids tested. No poliovirus was detected in the permeate of the ultrafilters with MWCOs of 13 000 or 6000 (titer reduction > 6 logs). These results indicate that the ultrafilters with MWCOs of 13 000 and 6000 were very effective in removing small viral particles (25–30 nm) by size exclusion. The recovery efficiency of the virus in the retentate varied by fluid type. However, filtration with virus diluted in DMEM-10 resulted in consistent recovery of the viruses tested. The results suggest that these ultrafilters may have the dual potential of removing viral contaminants from fluids and concentrating virus in the retentate.Key words: virus removal, virus concentration, ultrafiltration membranes.
Viability and respiratory activity ofPseudomonas syringaecells starved in buffer
Authors: Cabral João P. S.
Published: October 2009

Pseudomonas syringae cells starved in buffer released orcinol-reactive molecules and materials that absorbed ultraviolet light. The number of cells culturable in nutrient medium decreased more rapidly than the number of intact particles determined by microscopy. The results suggested that starvation resulted in the lysis of an increasing number of cells, and that a fraction of the intact particles were not culturable. Starvation also resulted in a decrease in the rate of oxygen consumption with acetate, glycerol, and succinate, but at different levels. Whereas the respiration of acetate and glycerol decreased concomitantly with culturability, the respiration of succinate decreased to levels similar to the concentration of intact cells, suggesting that all intact particles respired the succinate, but only the culturable cells respired the acetate and glycerol. The results suggest that measuring the activity of the electron-transport system can overestimate the viability of starved bacterial cells, and that complex metabolic activities such as the respiration of acetate and glycerol are probably better suited for the evaluation of this parameter.Key words: Pseudomonas syringae, starvation, culturability, viability, respiration.
A reevaluation of the thermodynamics of growth of Saccharomyces cerevisiae on glucose, ethanol, and acetic acid
Authors: Battley Edwin H.
Published: October 2009

Statistically determined values for the free energy and enthalpy change per equivalent accompanying the combustion of organic substances have been used to test the hypothesis that the free energy, enthalpy, and entropy changes accompanying anabolism are zero or nearly so. It is found that while this can be true in some cases, it is not always so, because the free energy and entropy changes per available electron accompanying biological combustion are different for different small molecular weight substrates although they are constant for cellular biomass. If the average energy change per equivalent accompanying the biological combustion of the cells is markedly different from that of the substrate, there will be a corresponding change in energy accompanying anabolism that is determined by the difference between them. The efficiency of available electron conservation is proposed as being preferable to free energy, enthalpy, and entropy efficiencies in that the latter parameters all have different values, whereas the former is common to all three of the latter. Free energy, enthalpy, and entropy efficiencies can be calculated from available electron conservation efficiencies if the biological combustion energies of the substrate, cells, and products are known per electron transferred. The methods described are applicable equally to aerobic and anaerobic growth process systems.Key words: yeast growth thermodynamics, electron conservation.
Isolation of FC3-11, a bacteriophage specific for theKlebsiella pneumoniaeporin OmpK36, and its use for the isolation of porin-deficient mutants
Authors: Hernández-Allés Santiago , Albertí Sebastián , Rubires Xavier , Merino Susana , Tomás Juan M. , Benedí Vicente J.
Published: October 2009

FC3-11, a bacteriophage specific for the Klebsiella pneumoniae porin OmpK36, was isolated by its ability to infect Escherichia coli strains expressing the cloned OmpK36 porin. Porin OmpK36 was shown to be the receptor for phage FC3-11 by the observations that K. pneumoniae and E. coli strains that do not express OmpK36 were resistant to phage FC3-11, the purified porin inactivated the phage, and mutants selected for FC3-11 resistance had lost OmpK36. The outer membrane protein OmpK35 was isolated from a K. pneumoniae phage-resistant mutant by using porin isolation methods and was shown to contain an N-terminal sequence typical of enterobacterial porins. Bacteriophage FC3-11, alone or in combination with previously described lipopolysaccharide-specific phages, is a valuable tool to obtain OmpK36-porinless mutants.Key words: Klebsiella pneumoniae, porins, bacteriophage.
A glycosylation pattern that is unique to fimbriae from the taxon Microbotryales
Authors: Celerin Martina , Day Alan W. , Castle Alan J. , Laudenbach David E.
Published: October 2009

Eukaryotic fimbriae are extracellular structures that have been shown to play a key role in mating and pathogenesis. Previous work has established that the major component of the fimbriae of the anther smut fungus Microbotryum violaceum (Pers.) G. Deml & Oberw. (Ustilago violacea (Pers.) Rouss.) is a 74-kDa glycoprotein subunit. This study analyzes the carbohydrate moiety in the fimbrial subunit and reveals that it is composed of mannose and possibly some acetylated sugars that are anchored to an asparagine residue of the protein via two N-acetylglucosamine residues. The carbohydrate component comprises 35% of the fimbrial subunit and the aglycone has an apparent molecular mass of 47 kDa. Using polyclonal antibodies, we have examined the conservation of the two components of the fimbrial subunits (protein and carbohydrate) in four divisions of fungi. Antibodies raised against native intact fimbriae of M. violaceum detected only the unique glycosylation pattern of these fimbriae, whilst antibodies raised against denatured fimbrial subunits were able to detect protein epitopes. Our results indicate that the fimbrial subunits of M. violaceum are composed of a protein moiety that is highly conserved amongst fungal species and a carbohydrate moiety that has a pattern that is unique to members of the dicot-infecting smut fungi. The possible implications of a conserved glycosylation pattern and the limited host range of this phylogenetic line of smut fungi are discussed in relation to current attempts to reclassify these species into the taxon Microbotryales.Key words: fimbriae, Ustilago, Microbotryum, glycoprotein, antibodies, systematics, basidiomycetes.
Survival of and lacZ expression in recombainant Pseudomonas strains introduced into riwer water microcosms
Authors: Leung Kamtin , Trevors Jack T. , Lee Hung
Published: October 2009

The lacZY gene cassette inserted into the chromosome of Pseudomonas aeruginosa UG2Lr and Pseudomonas aureofaciens Ps3732RNL11 was used as a genetic marker to study the fate of the Pseudomonas strains in river water microcosms. Expression of the lacZ marker in UG2Lr and Ps3732RNL11 was detected in microcosms containing as few as 12 and 14 colony-forming units (cfu)/mL of river water, respectively, by fluorimetric measurement of the β-galactosidase activity against 4-methylumbelliferyl-β-D-galactoside as the substrate. The persistence of and lacZ expression in the Pseudomonas strains were monitored in sterile and nonsterile river water in the presence and absence of added nutrients by dilution plating and fluorimetry, respectively. After incubation for 10 d at 10 °C, culturable populations of strain UG2Lr in sterile water samples, with and without nutrient added, decreased from an initial density of 1.5 × 104 to 1.7 × 103 and 4.6 × 103 cfu/mL, respectively. Despite similar numbers of UG2Lr cells in the two treatments, expression of the lacZ marker in the surviving cells of the nutrient-supplemented sample was 24 times higher than in the cells of the unamended sample. In nonsterile water samples, the density of UG2Lr declined to less than 6 cfu/mL in 30 d regardless of the nutrient conditions. A nutrient supplement increased the growth of the native bacterial population but did not enhance growth of and lacZ expression in the bacteria. Polymerase chain reaction analysis showed a decrease in amplification signal indicating a genuine decline in viable UG2Lr cell density in the water samples. Ps3732RNL11 cells established themselves in both sterile and nonsterile water samples. In nonsterile water samples, Ps3732RNL11 maintained a density of 20–40% of the culturable bacterial population. However, lacZ expression in both UG2Lr and Ps3732RNL11 cells was undetectable in nonsterile water samples after 30 d.Key words: survival, genetic marker, lacZ expression, polymerase chain reaction, recombinant Pseudomonas strains.
Degradation of polycyclic aromatic hydrocarbons (PAHs) by a mixed culture and its component pure cultures, obtained from PAH-contaminated soil
Authors: Trzesicka-Mlynarz D. , Ward O. P.
Published: October 2009

A mixed culture, isolated from soil contaminated with polycyclic aromatic hydrocarbons (PAHs), grew on and degraded fluoranthene in aqueous media supplemented with glucose, yeast extract, and peptone. Increased complex nitrogen levels in the medium promoted bacterial growth and a greater extent of fluoranthene degradation. Amendment of the media with high glucose levels also diminished specific fluoranthene degradation. The mixed culture was capable of degrading a range of other PAHs, including benzo[a]pyrene, anthracene, phenanthrene, acenaphthene, and fluorene. The mixed culture contained four predominant isolates, all of which were Gram-negative rods, three of which were identified as Pseudomonas putida, Flavobacterium sp., and Pseudomonas aeruginosa. Better degradation of a defined PAH mixture was observed with the mixed culture than with individual isolates. A reconstituted culture, prepared by combining the four individual isolates, manifested a similar PAH biodegradation performance to the original mixed culture. When compared with the mixed culture, individual isolates exhibited a relatively good capacity to remove more water-soluble PAHs (acenaphthene, fluorene, phenanthrene, fluoranthene). In contrast, removal of less water-soluble PAHs (anthracene and pyrene) was low or negligible with isolated cultures compared with the mixed culture.Key words: polycyclic aromatic hydrocarbons, mixed culture, fluoranthene, Pseudomonas, Flavobacterium.
Developmentally regulated expression of heat shock genes in Leptosphaeria maculans
Authors: Patterson N. A. , Kapoor M.
Published: October 2009

To determine whether heat shock genes (hsp) were expressed in a stage-specific manner, total RNA was isolated from dormant pycnidiospores, germinating spores, and mature mycelium of representatives of two pathotypes of Leptosphaeria maculans: the weakly virulent (or avirulent) and the virulent. Northern blots prepared by using total RNA isolated from normally grown and heat-shocked samples were hybridized with DNA of hsp genes of Neurospora crassa, hsp70, hsp80, and the heat shock inducible manganese peroxidase cDNA of Phanerochaete chrysosporium. No hybridization signal was apparent in RNA from dormant spores in the absence of heat shock treatment, while heat shock treatment resulted in the induction of mRNA corresponding to these three hsp genes. In contrast, substantial amounts of hsp transcripts were observed in germinating spores, even in the complete absence of externally applied stress. The mature mycelium failed to show these transcripts under normal growth conditions, but following exposure to hyperthermal treatment, a characteristic set of heat shock specific transcripts was witnessed. The two strains exhibited a similar pattern. Expression of the fungal hsp genes was also detectable in the plant tissue, following infection by both the virulent and weakly virulent strain. These data suggest that products of stress-responsive genes may have a role in an early event during spore germination.Key words: fungal phytopathogen, canola, fungal gene expression.
Oxidation of galena by Thiobacillus ferrooxidans and Thiobacillus thiooxidans
Authors: Garcia Jr. Oswaldo , Tuovinen Olli H. , Bigham Jerry M.
Published: October 2009

The objective of this work was to determine solution- and solid-phase alterations associated with galena (PbS) oxidation by Thiobacillus ferrooxidans and Thiobacillus thiooxidans. In T. ferrooxidans experiments with 2.5–5% (w/v) galena, the pH remained almost constant at pH 2, whereas the pH increased in uninoculated controls. In T. thiooxidans cultures, the pH initially increased from 2 to 4. This initial increase was comparable to the pH change in an abiotic control, but the oxidation reaction in T. thiooxidans cultures subsequently became acid producing. Anglesite (PbSO4) was detected by X-ray diffraction as a solid-phase product of galena decomposition in both abiotic and inoculated experiments. When ferrous sulfate was added as a supplementary energy source for T. ferrooxidans, jarosite (MFe3(SO4)(OH)6) was detected as a new solid phase. Elemental S was not detected in the residues.Key words: anglesite, bioleaching of galena, galena oxidation, jarosite, lead sulfide oxidation, Thiobacillus ferrooxidans, Thiobacillus thiooxidans.
Effect of mutations in the general secretory pathway on outer membrane protein and surface layer assembly in Aeromonas spp.
Authors: Howard S Peter , Meiklejhon Heather G.
Published: October 2009

Mutations in the exeC-N operon of Aeromonas hydrophila, which block extracellular protein secretion, also result in large decreases in the level of the major outer membrane porin, protein II. Immunoblot analysis demonstrated that the porin missing from the outer membrane of the mutant was not accumulating elsewhere in the cell. Pulse-chase and immunoprecipitation analyses showed that the porin was as stable in the mutant as in the wild type, but that far less porin was synthesized in the exe mutants. The relationship between extracellular secretion involving the exe genes and the assembly of other outer membrane and surface proteins was also examined. Both the wild type and exeE mutants of A. hydrophila were capable of assembling the protein I and protein III porins under inducing conditions. Aeromonas sohria As9071, which contains a surface array protein, could secrete A. hydrophila aerolysin and required homologues of the A. hydrophila exe genes to do so; however, an exe− derivative of this bacteria was unaffected in its ability to assemble its surface array. These results demonstrate that the exe genes are not required for general outer membrane protein assembly in these bacteria, but that the synthesis of protein II is specifically downregulated in the exe mutants.Key words: extracellular secretion, outer membrane assembly, surface layer, Aeromonas.
Hydrogen production from continuous fermentation of xylose during growth of Clostridium sp. strain No. 2
Authors: Taguchi Fumiaki , Mizukami Naoki , Saito-Taki Tatsuo , Hasegawa Katsushige
Published: October 2009

Experimental conditions are presented for continuous fermentation of xylose and glucose to hydrogen (H2), using Clostridium sp. strain No. 2 growing in 300 mL of culture containing 0.3% substrate, either without pH control or with the pH controlled at 6.0. The H2 production rate increased in proportion to increasing dilution rate within the range of about 0.4 to 1.0 h−1. The maximal H2 production rates of 21.03 and 20.40 mmol∙h−1∙L−1 were obtained from xylose and glucose with dilution rates of 0.96 and 1.16 h−1, respectively, at pH 6.0. About 2.06 mol H2 with a dilution rate of 0.21 h−1 at pH 6.0 and 2.36 mol H2 with a dilution rate of 0.18 h−1 were formed per mole of xylose and glucose consumed, respectively, at uncontrolled pH.Key words: hydrogen production, continuous fermentation, xylose, Clostridium sp. strain no. 2.
Purification of sex pheromones specific for pMB1 and pMB2 plasmids ofEnterococcus faecalisS-48
Authors: Quirantes R. , Valdivia E. , Martín I. , Martínez-Bueno M. , Maqueda M. , Gálvez A. , Méndez E.
Published: October 2009

The strain Enterococcus faecalis S-48 carries two large conjugative plasmids (pMB1 and pMB2) encoding for antagonistic substances. The pheromone response of these two plasmids was established by purifying the corresponding sex pheromones, using conventional reversed-phase columns. Plasmid pMB1 responds to pheromone cCF10. Plasmid pMB2 responds to a pheromone with an amino acid sequence identical to that of cPD1 (Phe-Leu-Val-Met-Phe-Leu-Ser-Gly). The two pheromone-responding plasmids coexist in a stable fashion in the wild-type strain E. faecalis S-48.Key words: pheromone response, Enterococcus faecalis, conjugative plasmids.
Medium to study carbon utilization by Bradyrhizobium strains
Authors: Wagner Stephen C. , Skipper Horace D. , Hartel Peter G.
Published: October 2009

Literature on the utilization of C sources by cowpea Bradyrhizobium strains is difficult to interpret because the media employed often contained other sources of C in addition to the C source being tested. In addition, culture incubation periods varied widely. We modified a complex medium to contain a minimal amount of yeast extract (10 mg/L); the yeast extract provided adequate growth factors but was inadequate as a C source. Eight cowpea bradyrhizobia strains were inoculated into this modified medium containing 1 of 16 different C sources for incubation periods of 7, 10, and 14 days at 28 °C. After 14 days of incubation, most of the strains grew with six hexoses (fructose, galacturonic acid, gluconate, glucose, mannitol, and rhamnose), two pentoses (arabinose and xylose), and four other compounds (malate, γ-aminobutyric acid, glutamate, and yeast extract) as C sources; no strains were able to grow on two disaccharides (lactose and trehalose) and two other C sources (citrate and nicotinic acid). Large differences in growth were observed for fructose, γ-aminobutyric acid, malate, mannitol, and rhamnose between 7 and 14 days incubation. Because of the possibility of false negatives, our data suggest that the strains should be grown in a medium low in yeast extract over a long instead of a short incubation time.Key words: bradyrhizobia, metabolism, C source, growth.
Enrichment and isolation of a reductively debrominating bacterium from the burrow of a bromometabolite-producing marine hemichordate
Authors: Steward Charles C. , Dixon Terry C. , Chen Yung Pin , Lovell Charles R.
Published: October 2009

An anaerobic 2,4,6-tribromophenol debrominating bacterium, strain DSL-1, was isolated from enrichment cultures inoculated with sediment from the burrows of the bromoaromatic-producing marine hemichordates Balanoglossus aurantiacus and Saccoglossus kowalewskyi. DSL-1 preferentially removed ortho-position bromines, resulting in the transient appearance of 2,4-dibromophenol and accumulation of 4-bromophenol. Cell-free extracts and partially purified reductive debrominase preparations from DSL-1 also debrominated 2,4,6-tribromophenol, yielding 2,4-dibromophenol and 4-bromophenol. Both NADH and NADPH stimulated 2,4,6-tribromophenol reduction by partially purified debrominase. These data are consistent with a reductive debromination mechanism. The organic cosubstrate(s) and specific electron donors used by DSL-1 in vivo are currently unknown.Key words: dehalogenation, bromometabolites, bromophenols, hemichordates.
Biocontrol of Sclerotinia wilt of sunflower by Pseudomonas fluorescens and Pseudomonas putida strains
Authors: Expert J. M. , Digat B.
Published: October 2009

An increase in early injury to the roots and collar of sunflower plants caused by Sclerotinia sclerotiorum has been observed in France for several years. In vitro tests (inhibition of fungi and cyanide production by bacterial strains) and in situ tests (in nonsterile humus, in a growth chamber) were performed to screen the most efficient Pseudomonas fluorescens and Pseudomonas putida strains effective against this form of injury caused by S. sclerotiorum. Although there was no correlation between in vitro and in situ tests, a positive correlation between in situ tests and field experiments was obtained. At least 1 × 106 bacteria per seed were required for significant protection in in situ tests and field trials demonstrated that significant protection of sunflower was obtained by seed bacterization with selections of P. fluorescens and P. putida.Key words: biological control, Pseudomonas fluorescens, Pseudomonas putida, Sclerotinia sclerotiorum, sunflower, bacterization.
Interaction of sulfhydryl reactive reagents with components involved in (1,3)-β-glucan synthesis fromCandida albicans
Authors: Frost David J. , Brandt Kim , Kaufmann Tim , Goldman Robert
Published: October 2009

Glucan synthesis was sensitive to several sulfhydryl reacting compounds: mercurials, reversible disulfides, and an alkylating sulfhydryl reagent (IC503–45 μM). Thiol groups associated with glucan synthesis were hydrophilic in nature, since both hydrophilic and hydrophobic reagents were active. Glucan synthase complex consists of at least two components: a peripheral GTP-binding protein that can be solubilized with detergents (supernatant) and the catalytic membrane-bound component (pellet). A rapid separation technique was developed to study sulfhydryl interactions with the complex. The GTP-binding protein was solubilized with 0.6% 3-((3-cholamidopropyl)dimethylammonio)-1-propane sulfonate from isolated microsomes of Candida albicans cells grown at either 10 or 30 °C. The residual membranous fraction contained the core catalytic moiety of glucan synthase. Both fractions were devoid of glucan synthase activity until they were reconstituted by mixing the two fractions together. In reconstitution experiments, the pellet lost almost 50% activity when preincubated with 2.5 μM N-ethylmaleimide and combined with an untreated supernatant whereas only 10% activity was lost when the supernatant was treated with N-ethylmaleimide. The catalytic active site of glucan synthase was not protected with UDP-Glc when preincubated with 10 μM N-ethylmaleimide but the GTP-binding fraction was partially protected with GTPγS.Key words: Candida albicans, (1,3)-β-glucan synthase, GTP-binding proteins, solubilization, sulfhydryl reagents.
External and internal root colonization of lodgepole pine seedlings by two growth-promotingBacillusstrains originated from different root microsites
Authors: Shishido M. , Loeb B. M. , Chanway C. P.
Published: October 2009

Root colonization and in vitro carbon substrate utilization by two seedling growth-promoting Bacillus strains that originated from different root microsites were studied in greenhouse and growth chamber experiments. Strain L6, identified as Bacillus polymyxa, was previously isolated from rhizosphere soil containing roots of pasture plants, and Pw-2, tentatively identified also as B. polymyxa, was isolated from within surface-sterilized lodgepole pine (Pinus contorta var. latifolia (Dougl.) Engelm.) roots. Rifamycin-resistant strains derived spontaneously from wild-type strains L6 and Pw-2, designated strain L6-16R and Pw-2R, respectively, were used to monitor lodgepole pine root colonization in a closed tube assay system. Three-week-old pine seedlings were inoculated with 105colony-forming units (cfu) of strain Pw-2R or 106 cfu of strain L6-16R, and external and internal root colonization was assessed 2 and 4 weeks later. Strains L6-16R and Pw-2R were both recovered from pine rhizosphere samples with > 5 × 107 cfu/g fresh root tissue 2 weeks after inoculation, but neither strain was detected in the root interior. When root colonization was assessed 4 weeks after inoculation, the rhizosphere populations of both strains had declined slightly to between 5 × 106and 5 × 107 cfu/g fresh root tissue, but strain Pw-2R was also detected within root tissues with 105 cfu/g fresh root tissue. Lateral root formation was abundant 4 weeks after inoculation and may have facilitated colonization of internal root tissues by strain Pw-2R. Both strains possessed pectolytic activity, although differences between the strains were detected in in vitro substrate utilization capabilities using BIOLOG assays. These differences may be related to their abilities to colonize internal root tissues. On the basis of our results, we hypothesize that internal root colonization by Bacillus strains is not a random event and that root-endophytic Bacillus strains possess specific physiological and (or) biochemical characteristics that facilitate colonization of internal root tissues.Key words: Bacillus, PGPR, rhizosphere, endophytes, colonization.
A novel method for cloning DNA of plant-pathogenic mycoplasmalike organisms
Authors: Chen K. H. , Chen T. A.
Published: October 2009

A novel method was developed for cloning the DNA from a representative of plant-pathogenic mycoplasmalike organisms (MLOs). This procedure utilized random amplified polymorphic DNA (RAPD) and basic recombinant DNA techniques. It consisted of amplification of total DNA from diseased plants using one oligonucleotide primer with arbitrary sequence and separation of RAPD products in agarose gels. Unique RAPD band(s) of MLO origin was (were) then recovered from the gel and cloned into the specifically designed vector pCRTM II. With this method, a DNA fragment of the SA2 isolate of grapevine yellows MLO was cloned. Southern blot hybridizations revealed that most of the DNA in the unique RAPD band was derived from MLO. Results from dot-blot hybridizations used for screening showed that approximately 60% of transformants harbored MLO-specific recombinant plasmids. Our approach is relatively simple, quite efficient, and not limited by the amount of diseased material available. It does not depend on DNA sequence information for primer design and does not rely on restriction endonucleases for cloning. In addition, it can be used directly for disease diagnosis and for differentiation of closely related MLOs. Our system may serve as a model for cloning DNAs of other fastidious plant pathogens.Key words: mycoplasmalike organism, DNA, cloning, RAPD, detection.
Weak acid effects and fluoride inhibition of glycolysis byStreptococcus mutansGS-5
Authors: Belli W. A. , Buckley D. H. , Marquis R. E.
Published: October 2009

Fluoride and a variety of other weak acids acted to reduce reversibily the acid tolerance of glycolysis by intact cells of Streptococcus mutans GS-5 as shown by higher final pH values in acid-drop experiments with glucose in excess. The order of effectiveness was fluoride > indomethacin > ibuprofen > ketoprofen > salicylate > sorbate > cinnamate > p-hydroxybenzoate > benzoate > ascorbate. Only fluoride also acted as an inhibitor of the glycolytic enzyme enolase. However, enolase in permeabilized cells was also inhibited by acidification with a sharp drop-off in activity between pH 6 and 5. It was proposed that the weak acids, including fluoride, acted to reduce glycolytic acid tolerance by enhancing cytoplasmic acidification and thereby inhibiting enzymes such as enolase. The potencies of the acids could not be predicted accurately from knowledge of pKavalues, octanol–water partition coefficients, and molecular weights. It was concluded that their modes of action in acid sensitization involved perturbations of membrane function in addition to their acting as transmembrane carriers of protons. Methylparaben (methyl ester of p-hydroxybenzoate) was also a sensitizer but was less effective than the parent acid.Key words: fluoride, weak acids, enolase, Streptococcus mutans, glycolysis.
Distribution of the streptomycin-resistance transposon Tn5393among phylloplane and soil bacteria from managed agricultural habitats
Authors: Sundin George W. , Monks Dave E. , Bender Carol L.
Published: October 2009

The distribution of the strA–strB streptomycin-resistance (Smr) genes associated with Tn5393 was examined in bacteria isolated from the phylloplane and soil of ornamental pear and tomato. Two ornamental pear nurseries received previous foliar applications of streptomycin, whereas the tomato fields had no prior exposure to streptomycin bactericides. Although the recovery of culturable Smrbacteria was generally higher from soil, the highest occurrence of Smrwas observed in phylloplane bacteria of an ornamental pear nursery that received 15 annual applications of streptomycin during the previous 2 years. Twenty-two and 12% of 143 Gram-negative phylloplane and 163 Gram-negative soil isolates, respectively, contained sequences that hybridized to probes specific for the strA–strB Smrgenes and for the transposase and resolvase genes of Tn5393. These sequences were located on large plasmids (>60 kb) in 74% of the isolates. The 77 SmrGram-positive bacteria isolated in the present study showed no homology to the Tn5393-derived probes. Although the repeated use of a single antibiotic in clinical situations is known to favor the development of strains with resistance to other antibiotics, we found no evidence that intensive streptomycin usage in agricultural habitats favors the development of resistance to tetracycline, an antibiotic also registered for disease control on plants. The detection of Tn5393 in bacteria with no prior exposure to streptomycin suggests that this transposon is indigenous to both phylloplane and soil microbial communities.Key words: streptomycin, tetracycline, antibiotic resistance, phylloplane, transposon.