bluuebunny/crossref_metadata_2025 huggingface dataset:
An ''all-purpose" cellulase reporter for gene fusion studies and application to the paracrystalline surface (S)-layer protein of Caulobacter crescentus
Authors: Bingle Wade H. , Kurtz Jr. Harry D. , Smit John
Published: May 2010

The secreted endoglucanase (CenA) from the Gram-positive bacterium Cellulomonas fimi and a deletion derivative (ΔCenA) lacking the N-terminal leader peptide of native CenA were used to explore the potential of ΔCenA as a reporter molecule in Caulobacter crescentus. Expression of cenA in C. crescentus yielded extracellular endoglucanase activity, suggesting that the N-terminal leader peptide of CenA could direct the enzyme to the periplasm where it subsequently leaked into the medium. In contrast, expression of ΔcenA yielded only cell-associated endoglucanase activity; this suggested that the enzyme retained activity in the C. crescentus cytoplasm. Using the putative cytoplasmic and periplasmic forms of ΔCenA as markers, a simple assay for periplasmic ΔCenA hybrids was developed. This assay indicated that ΔCenA activity was largely independent of cellular location. To facilitate the use of ΔCenA as a reporter, a broad host range translational fusion vector (pEC215) incorporating ΔcenA was constructed. This vector was used to investigate factors important to the expression of the gene (rsaA) encoding the paracrystalline surface protein (S-layer) of the bacterium. It was found that altering the 5′ untranslated region of the rsaA mRNA reduced gene expression by 70%. One rsaA:ΔcenA gene fusion resulting from these experiments that incorporated only rsaA translation initiation information was further modified to serve as a general reporter for creating transcriptional gene fusions with other promoters. Gene fusions between alkaline phosphatase (phoA) and either cenA or lacZ were used to supplement information about RsaA secretion derived from rsaA:phoA gene fusions. It was found that linkage of the N-terminal leader peptide of CenA to PhoA yielded 50–100 times more cell-associated PhoA activity in C. crescentus than linkage of the RsaA N terminus. Taken together, these experiments indicated that ΔCenA was useful for tagging proteins localized to the cytoplasm, exported to the periplasm, or secreted from the cell, as well as for monitoring events in the cytoplasm such as examining factors important to the level of gene expression. Further, because ΔCenA was active in all cell compartments, it could be used to estimate the efficiency of hybrid protein export-secretion from enzyme activity measurements alone. In short, ΔCenA possessed many of the attributes of an "all-purpose" reporter.Key words: gene fusions, protein secretion, cellulase, alkaline phosphatase, Caulobacter crescentus.
Purification and some properties of β-glucosidase from the ectomycorrhizal fungus Pisolithus tinctorius strain SMF
Authors: Cao Weiguo , Crawford Don L.
Published: May 2010

A cell-associated β-glucosidase was purified 152-fold to homogeneity from the ectomycorrhizal fungus Pisolithus tinctorius strain SMF. The apparent molecular weight of the native protein, as determined by size exclusion chromatography, was approximately 450 000. A single band with a molecular weight of 150 000 was obtained after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). Thus, the native enzyme may consist of three monomers. The pI of the enzyme was determined to be 3.8 by isoelectric focusing. The enzyme had an optimal pH of 4.0 and an optimal temperature for activity of 65 °C. It showed a high substrate specificity toward aryl-β-glucosides, such as p-nitrophenyl β-D-glucopyranoside (PNPG), and β-1,6 glucosidic linkages. Cellobiose was hydrolyzed at about two-thirds the rate of PNPG. The Km for hydrolysis of PNPG was 0.87 mM. Strong inhibitors of the enzyme were aluminum, copper, ethylenediaminetetraacetic acid (EDTA), deoxynojirimycin, gluconic acid, and SDS. Calcium, manganese, and p-hydroxymercuribenzoic acid reduced the activity to a lesser extent. Potassium, mercury, cobalt, dithiothreitol, and glucosamine had no effect on activity. Enzyme activity was slightly increased to 112% in the presence of 1% glycerol. The enzyme was more stable under acidic conditions than under alkaline conditions.Key words: Pisolithus, ectomycorrhizal, β-glucosidase, purification.
Cloning of a xylanase gene from the ruminal fungus Neocallimastix patriciarum 27 and its expression in Escherichia coli
Authors: Lee J. M. Tamblyn , Hu Y. , Zhu H. , Cheng K. J. , Krell P. J. , Forsberg C. W.
Published: May 2010

An endo-β-1,4-xylanase gene was cloned from Neocallimastix patriciarum 27 in the bacteriophage vector λgtWESλB and was subcloned into the plasmid vectors pUC18 and pUC19 in which xylanase activity was expressed in both orientations. The xylanase was located in the periplasmic space of the host, Escherichia coli HB101. The pH and temperature optima for periplasmic xylanase activity were 6.2 and 40 °C, respectively, and the Km for oat spelt xylan hydrolysis was 0.89 mg∙mL−1. It also exhibited hydrolytic activity on carboxymethyl cellulose that was equivalent to 28% of the activity exhibited by the enzyme on xylan. It bound to crystalline cellulose, but lacked hydrolytic activity on amorphous cellulose. SDS-PAGE followed by zymogram analysis showed active bands of 68, 58, and 51 kDa. Isoelectric focusing in gels combined with zymogram analysis showed one band of xylanase activity with a pI of 3.6.Key words: Neocallimastix patriciarum, xylanase, gene.
Purification and partial characterization of a bacteriocin isolated from Bacteroides ovatus H47
Authors: Miranda C. M. S. , Farias L. M. , Carvalho M. A. R. , Damasceno C. A. V. , Totola A. H. , Tavares C. A. P. , Cisalpino E. O. , Vieira E. C.
Published: May 2010

A new intracellular bacteriocin isolated from a human fecal strain of Bacteroides ovatus was partially purified through ammonium sulfate precipitation, ion exchange chromatography, gel filtration, and preparative polyacrylamide gel electrophoresis (PAGE). The bacteriocin is stable at a pH range of 3–10, at 60 °C for 24 h, and at −70 °C for 6 months. It is inactivated by proteolytic enzymes. The molecular weight, estimated by sodium dodecyl sulfate – PAGE, is 78 kDa. Fifty strains of the Bacteroides fragilis group were isolated from fecal samples, and 41 of the isolates were shown to produce an antagonistic substance against at least 1 indicator strain. Iso-, auto-, and hetero-antagonisms were observed.Key words: Bacteroides ovatus, bacteriocin, human feces, bacterial growth inhibition.
Measurement of the intracellular pH of Propionibacterium acnes: comparison between the fluorescent probe BCECF and 31P-NMR spectroscopy
Authors: Futsaether C. M. , Kjeldstad B. , Johnsson A.
Published: May 2010

The intracellular pH of the Gram-positive skin bacterium Propionibacterium acnes was determined using the pH-sensitive fluorescent probe 2′, 7′ bis-(2-carboxyethyl)-5-(and-6)carboxyfluorescein (BCECF). The probe was introduced into the bacteria using the membrane-permeable acetoxymethyl ester BCECF-AM. The intracellular pH of the bacteria was determined by establishing a relation between the fluorescence ratio 505/450 and pH using the ionophore nigericin. To verify the intracellular pH determined using BCECF, the results were compared with those obtained using 31P-NMR spectroscopy. The effects of different external pH values and glucose addition upon the intracellular pH were examined using BCECF and 31P-NMR. Good correlation was obtained between the two techniques. Propionibacterium acnes maintained a pH gradient, inside alkaline, in the external pH range 5.0–7.4, which inverted when the pH was > 7.5. At external pH ≥ 8.5, the intracellular pH was close to the external pH. Glucose exposure did not affect the intracellular pH. Rapid, transient intracellular acidification and alkalinization brought about using NaHCO3 and NH4Cl, respectively, could be detected using BCECF. A limitation encountered when using BCECF was BCECF leakage, which could significantly affect the results if not taken into account.Key words: intracellular pH, BCECF fluorescence, 31P-NMR spectroscopy, Propionibacterium acnes.
Occurrence of cellulose and chitin in the hyphal walls of Pythium ultimum: a comparative study with other plant pathogenic fungi
Authors: Chérif Mohamed , Benhamou Nicole , Bélanger Richard R.
Published: May 2010

Exoglucanase, a β-1, 4-glucan cellobiohydrolase with specific affinity for β-1, 4-linked glucans, and wheat germ agglutinin, a lectin with N-acetylglucosamine-binding specifity, were used for localizing cellulosic β-1, 4-glucans and chitin in the cell walls of six plant pathogenic fungi. Both chitin and cellulose were found to occur in the cell walls of the oomycete fungus Pythium ultimum whereas only cellulose was present in those of Phythophthora parasitica var. nicotianae, another oomycete fungus. The two compounds were also simultaneously detected in the cell walls of two ascomycetes, Ophiostoma ulmi and Colletotrichum lindemuthianum. Finally, only chitin could be detected in the cell walls of the ascomycete Fusarium oxysporum f.sp. radicis-lycopersici (FORL) and the basidiomycete Rhizoctonia solani. The dual occurrence of chitin and cellulose in Pythium ultimum cell walls was further confirmed by enzymatic extraction performed on isolated cell walls. Treatment of Pythium ultimum cell walls with hen egg white lysozyme, an enzyme with strong chitinolitic activity, prior to labeling with wheat germ agglutinin - ovomucoid - gold complex, resulted in a near abolition of labeling. Similar results were obtained with FORL cell walls used as positive controls to assess the validity of the enzymatic extraction process. When cell walls of Pythium ultimum were treated with a mixture of hen egg white lysozyme and cellulase, both chitin and cellulose were hydrolysed as shown by the considerable reduction of labeling. Thus, these data provide evidence for the presence of chitin in Pythium ultimum cell walls and suggest that classification of Oomycetes as a cellulose-glucan group may be reconsidered.Key words: Pythium ultimum, chitin, cellulose.
Bacterial suppression of basal pod rot and end rot of dry peas caused by Sclerotinia sclerotiorum
Authors: Huang H. C. , Kokko E. G. , Yanke L. J. , Phillippe R. C.
Published: May 2010

Morphological and biochemical characteristics indicate that the two bacterial strains used in this study belong to Bacillus cereus Frankland and Frankland. Tests in vitro revealed that strains of B. cereus differ in their antagonistic activities on Sclerotinia sclerotiorum (Lib.) de Bary. Vegetative growth and ascospore germination of S. sclerotiorum were inhibited by diffusible metabolites induced by B. cereus strain alf-87A, but were unaffected by strain B43. In vivo studies showed that the antagonistic strain alf-87A, when sprayed onto pea plants (Pisum sativum L.) at the pod development stage, reduced the incidence of basal pod rot from infection by airborne ascospores of S. sclerotiorum by 39–55%. This treatment also significantly (P < 0.05) reduced the severity of basal pod rot by decreasing lesion size. Strain alf-87A significantly reduced the incidence of end pod rot. Spraying pea plants with strain B43 of B. cereus was not consistently effective in reducing basal and end pod rots. Scanning electron microscopic studies revealed that both strains of B. cereus could colonize senescing pea stamens but only the antagonistic strain alf-87A was consistently effective in controlling sclerotinia basal and end pod rots of dry peas.Key words: Sclerotinia sclerotiorum, Bacillus cereus, basal pod rot, end pod rot, stamens, ascospores, apothecia.
Physicochemical and D-galactose-mediated interactions in the attachment of Bradyrhizobium japonicum to roots of Glycine max
Authors: Smith Geoffrey B. , Wollum II A. G.
Published: May 2010

The roles of electrostatic, hydrophobic, and lectin-mediated interactions in the attachment of Bradyrhizobium japonicum to soybean (Glycine max [L.] Merr.) seedling roots were examined. Cell suspensions (104 cfu/mL) of B. japonicum strain USDA 110 were exposed to excised root segments during 2-h adhesion assays. The presence of the specific hapten for the soybean seed lectin, the sugar N-acetyl-D-galactosamine (GalNac), did not inhibit attachment, but D-galactose did (29% of untreated control). Some D-galactose inhibition (69% of control) was observed when the bacteria were preincubated in the presence of the sugar before the attachment assay but not when the roots were preincubated. Attachment was pH dependent, reaching maximum values between pH 6.0 and 6.5. The polycations DEAE-dextran and poly-L-lysine stimulated adhesion 12- and 5-fold to excised root segments, respectively; DEAE-dextran caused a 7-fold attachment stimulation to intact seedling roots. Segregation of bacterial cells into hydrophobic and nonhydrophobic fractions by preexposure to polystyrene did not affect attachment to roots, nor did the presence of two detergents, sodium dodecyl sulfate and Tween 80. These results indicate that hydrophobic interactions are not involved in the attachment, but anionic repulsion may limit attachment between B. japonicum cells and G. max roots (both surfaces are negatively charged at the assay medium pH of 6.3). The attachment is not via the GalNac-specific soybean lectin but apparently is mediated by a D-galactose-binding moiety located on the bacterial cell surface.Key words: bradyrhizobia, attachment, bacterial lectins, polycations
Analysis of phage resistance mechanisms encoded by lactococcal plasmid pAJ2074
Authors: Jarvis Audrey W.
Published: May 2010

Lactococcal plasmid pAJ2074 is a 74-kb plasmid that confers phage resistance at 30 °C against all lactococcal phages with prolate heads (referred to as prolate phage), and most small lactococcal phages with isometric heads (referred to as small isometric phage) that have been tested. The presence of pAJ2074 had no effect on phage adsorption or injection of phage DNA. Replication of prolate phage c2 DNA could not be detected in bacterial cells containing the plasmid up to 60 min after phage infection, whereas phage c2 DNA replication could be demonstrated at 20 min in the control strain. With pAJ2074 present there was no detectable growth of phage c2 and an 87% reduction in burst size for the small isometric phage sk1. Infective centres were reduced in the presence of pAJ2074 by 99% for phage c2 and by 93% for phage sk1. Plasmid pAJ2074 differed from pTR2030, in that the major effect of pAJ2074 was on prolate phage c2, rather than on the small isometric phage sk1, and no restriction and modification system could be detected. In addition, no DNA homology was detected between pAJ2074 and pTRK67 (derived from pTR2030). A recombinant plasmid pAJ88 containing an 8.4-kb insert from pAJ2074 conferred an intermediate level of phage resistance. The DNA region that encoded reduced phage sensitivity was further defined by the subcloning of a 5.6-kb EcoRV fragment that conferred resistance similar to pAJ88. The possibility of two phage-resistance mechanisms being encoded by pAJ2074 is discussed.Key words: phage resistance, lactococcal phages, lactococcal plasmids, lactococci.
Instability of the production of avermectins, sporulation, and pigmentation in Streptomyces avermitilis
Authors: Novák Jan , Kopecký Jan , Kofroňová Olga , Vaněk Zdenko
Published: May 2010

Production of avermectins, sporulation ability, and colony pigmentation were followed in Streptomyces avermitilis C-18/6 cultures during serial transfer (for eight subcultures) in four different liquid media. These phenotypes were found to be unstable and independent of each other. A procedure was established whereby depressed avermectin production could be reversed but the degree of reversion was dependent on the history of the isolate, i.e., the type of medium used. The presence of isoleucine as the sole N source caused an immediate loss of avermectin production and this could not be reversed.Key words: avermectins, instability, pigmentation, sporulation, Streptomyces avermitilis.
Comparison of exopolysaccharides from mucoid and nonmucoid strains of Clavibacter michiganensis subspecies sepedonicus
Authors: Henningson Paul J. , Gudmestad Neil C.
Published: May 2010

The exopolysaccharides produced by six strains of Clavibacter michiganensis ssp. sepedonicus were isolated and purified by liquid chromatography. Neutral sugar composition and molecular weights were determined for each polysaccharide fraction, using gas chromatography and high-performance size-exclusion chromatography. The serological reaction of each fraction was tested using enzyme-linked immunosorbent assay. Exopolysaccharide from nonmucoid strains contained only low molecular weight polysaccharides (1.5 × 103 to 1.1 × 104). Exopolysaccharide from mucoid and intermediate strains could be separated into low (4.0 × 103 to 1.1 × 104) molecular weight and high (5.0 × 105 to 1.6 × 106) molecular weight fractions. High molecular weight polysaccharides were composed almost exclusively of galactose, glucose, and fucose. The ratios of these sugars were highly variable among strains. Low molecular weight polysaccharides were primarily composed of galactose with significant and varying amounts of glucose, rhamnose, mannose, and ribose. All polysaccharide fractions except one, produced by a nonmucoid strain, reacted in the immunoassay test.Key words: exopolysaccharide, polysaccharide, Clavibacter, michiganensis, sepedonicus.
Does siderophore production influence the relative abundance of Rhizobium meliloti in two field populations?
Authors: Barran L. R. , Bromfield E. S. P.
Published: May 2010

Populations of indigenous Rhizobium meliloti isolated from nodules of alfalfa grown at sites A and B (soil pH, 7.0 and 6.1, respectively) were previously characterized on the basis of phage sensitivity and divided into 55 and 65 phage types. The available iron content of soil at site B was significantly higher than that at site A. Isolates representing the phage types comprising each of these populations were tested for the production of siderophores. The frequency of siderophore-producing (sid+) phage types of R. meliloti, estimated from the distribution of types in the two field populations, was significantly higher at site A (54%), where iron was less available, than at site B (18%). The distributions of frequency for sid+ and sid− phage types were similar at site A but differed (P < 0.005) at site B, where the soil was slightly acidic and contained more available iron. The apparent absence of a direct relationship between siderophore production and frequency of occurrence suggests that siderophore production may not influence the relative abundance of R. meliloti in these populations at sites differing in iron availability.Key words: siderophore, iron, Rhizobium meliloti, populations.
Molecular analysis of the plasmid-borne bed gene cluster from Pseudomonas putida ML2 and cloning of the cis-benzene dihydrodiol dehydrogenase gene
Authors: Tan Hai-Meng , Fong Karen P.-Y.
Published: May 2010

Pseudomonas putida ML2 contains a large catabolic plasmid, pHMT112, carrying genes that encode the dioxygenase and dehydrogenase involved in the catabolism of benzene via the ortho or β-ketoadipate pathway. pHMT112 was derived from a larger and less stable plasmid in P. putida ML2 following growth on succinate as carbon and energy source but was, however, stably maintained in P. putida even in the absence of selection for growth on benzene. Cleavage sites for the restriction endonucleases DraI, XbaI, and BamHI were mapped on the plasmid. A region of the plasmid, downstream of the benzene dioxygenase genes (bedC1C2BA), was found to encode the cis-benzene dihydrodiol dehydrogenase gene (bedD). Recombinant Escherichia coli containing bedC1C2BAD genes was found to express benzene dioxygenase and dehydrogenase activity, indicated by the production of catechol when incubated in the presence of benzene. Hybridization using benzene dioxygenase genes as probes was used to construct a restriction map of the 35.5-kb XhoI–DraI fragment on which the bed operon was located.Key words: Pseudomonas putida, catabolic plasmid, dioxygenase, dehydrogenase.
Temperature studies of iron-oxidizing autotrophs and acidophilic heterotrophs isolated from uranium mines
Authors: Berthelot Deborah , Leduc L. G. , Ferroni G. D.
Published: May 2010

Iron-oxidizing autotrophs and acidophilic heterotrophs were quantified at an incubation temperature of 18 °C in several samples obtained from the bioleaching areas of two uranium mines in Ontario, Canada. All samples were mine-water samples with temperatures in the range 13–18 °C. Iron-oxidizing autotrophs ranged from 2683 ± 377 to 245 000 ± 20 205 colony-forming units∙mL−1 and were always numerically superior to acidophilic heterotrophs, which ranged from 40 ± 8 to 9650 ± 161 colony-forming units∙mL−1. For each sample, approximately 20 isolates of each nutritional group were examined for the ability to grow at temperatures of 4, 18, 21, and 37 °C, respectively; overall, 559 isolates of iron-oxidizing bacteria (predominantly Thiobacillus ferrooxidans) and 252 acidophilic heterotrophic isolates were examined and categorized as 'broader temperature range psychrotrophs,' 'narrower temperature range psychrotrophs,' 'intermediates,' or mesophiles. Although psychrotrophic representatives of both groups were abundant, no psychrophiles were recovered from any of the samples. For the iron oxidizers, the temperature growth profiles of the isolates were similar from sample to sample. For the acidophilic heterotrophs, the temperature growth profiles varied considerably among samples.Key words: psychrotrophs; Thiobacillus ferrooxidans; uranium mines.
Rapid comparison of the cytochrome c3 gene from nine strains of Desulfovibrio vulgaris using polymerase chain reaction amplification
Authors: Kwoh Deborah Y. , Vedvick Thomas S. , McCue Ann F. , Gevertz Diane
Published: May 2010

Polymerase chain reaction amplification was used to compare different regions of the cytochrome c3 gene from nine strains of Desulfovibrio vulgaris, to examine homology within the species. Six 30-base polymerase chain reaction primers and three probes were synthesized on the basis of the published nucleic acid sequence of the cytochrome c3 gene from D. vulgaris, NCIMB 8303. Amplifications were performed on genomic DNA isolated from NCIMB 8303 as well as eight other strains. Six strains, NCIMB 8302, 8305, 8306, 8311, 11779, and DSM 2119, showed amplification products of equal size and quantity to those of strain 8303. Two other strains, NCIMB 8456 and DSM 1744, either showed reduced levels or no detectable amplification products. These results were confirmed by hybridization of amplified DNA to radiolabeled probes specific for each product. DNA sequencing of a 145-bp polymerase chain reaction fragment from strains NCIMB 8302, 8303, 11779, and DSM 2119 revealed complete sequence homology between these strains, whereas slight differences were seen with strain NCIMB 8456. Amino acid sequencing of the first 20 residues of cytochrome c3 purified from strains NCIMB 8456 and 8303 also showed differences in the two proteins. In contrast to the results obtained with strain NCIMB 8456, limited homology was observed between the first 20 amino acid residues of cytochrome c3 from strain DSM 1744 and strain NCIMB 8303.Key words: cytochrome c3, Desulfovibrio, polymerase chain reaction amplification, taxonomy.
Comparative analyses of Serratia spp. outer membrane porin proteins
Authors: Hutsul Joanne , Worobec Elizabeth , Parr Jr. Tom R. , Becker Gerald W.
Published: May 2010

Eight Serratia strains and several members of the Enterobacteriaceae family were used in immunoblot and Southern DNA hybridization experiments and probed with antibody and DNA probes specific for the 41-kDa Serratia marcescens porin, to determine the extent of homology between Gram-negative porins. Immunoblot analyses performed using porin-specific rabbit sera and cell envelope preparations from these strains revealed that all strains produced at least one cross-reactive protein in the 41-kDa molecular weight range. Chromosomal DNA from each of the same strains was used in Southern analyses, probed with a 20-base-length oligonucleotide probe deduced from the N-terminal amino acid sequence of the 41-kDa Serratia marcescens porin. The probe hybridized to DNA from all of the Serratia species and six of the nine other enteric bacteria. Putative porin proteins from all the Serratia species were subjected to N-terminal amino acid sequencing and porin functional analysis using the black lipid bilayer method. All amino acid sequences were identical, with one exception in which an asparagine was substituted for an aspartic acid in Serratia rubidaea. All porins had very similar porin function (single channel conductance ranging between 1.72 and 2.00 nS). The results from this study revealed that a strong conservation exists among the Serratia porins and those produced by other enteric bacteria.Key words: porins, Serratia marcescens, homology studies.
Microbial characteristics of Palau Jellyfish Lake
Authors: Venkateswaran K. , Shimada A. , Maruyama A. , Higashihara T. , Sakou H. , Maruyama T.
Published: May 2010

Microbiological examinations of total bacterial population, culturable aerobic heterotrophs, photosynthetic bacteria, and particulate DNA were carried out in Palau Jellyfish Lake. A 2 m thick bacterial plate layer at 13–15 m depth consisting of various components of microbes was observed in Jellyfish Lake. Photosynthetic bacteria, as seen by flow cytometry, were concentrated at 14–15 m depths with a maximal count of 2.2 × 105 cells∙mL−1 and microscopic analysis confirmed that these purple bacteria were Chromatium sp. Peaks in total bacterial counts (8.3 × 106 cells∙mL−1; 13 m), in the Synechococcus spp. population (2.4 × 106 cells∙mL−1; 13 m), in culturable heterotrophs (105 colony-forming units∙mL−1; 15 m), and in particulate DNA (17.8 μg∙L−1; 10 m) were observed either at the bacterial plate layer that was rich in nutrients or just above this layer in the oxic zone. Bacteriochlorophyll and chlorophyll a exhibited peaks at the photosynthetic bacterial plate (14–15 m). A high concentration of particulate organic carbon was also observed at 15 m. The particulate DNA showed a high degree of correlation with the total bacterial cell number. A low ratio of particulate DNA to particulate organic carbon (0.005) in the water column was found at 15 m and suggested that the particulate materials produced by photosynthetic bacteria would have influenced the concentration of particulate organic carbon. Culturable heterotrophs, clustered into nine different groups, were dominated by species of the genera Vibrio, Aeromonas, and Halomonas.Key words: Jellyfish Lake, microbiological characteristics, Chromatium, particulate DNA, heterotrophs.
Isolation of Brevibacterium sp. R312 mutants potentially useful for the enzymatic production of adipic acid
Authors: Moreau J. L. , Bernet N. , Arnaud A. , Galzy P.
Published: May 2010

Mutants of Brevibacterium sp. R312 were isolated for the production of adipic acid from adiponitrile. One mutant (Ad) with a modified cell wall showed activity against adipamide 3 times greater than the wild type. Another mutant (ACV2) derived from the Ad strain had 30 times more activity on cyano-5-valeric acid, and 7 times more on adipamide, than the wild type. The presence of an amidase acting on amide intermediates in the hydrolysis of dinitriles to organic acids was demonstrated in these mutants.Key words: Brevibacterium sp., adiponitrile, cyano-5-valeric acid, adipamide, adipic acid, mutant.
Carbon nutrition and hydrolytic and cellulolytic activities in the ectomycorrhizal fungus Pisolithus tinctorius
Authors: Cao Weiguo , Crawford Don L.
Published: May 2010

Four strains of an ectomycorrhizal fungus, Pisolithus tinctorius, were investigated for carbon nutrition, and for production of hydrolytic and cellulolytic enzymes. Glucose, mannose, and cellobiose supported rapid mycelial growth of all four strains. Fructose was utilized by two strains, SMF and S359. Of the 10 hydrolytic enzymes examined, acid phosphatase, acid α-galactosidase, acid esterase, and acid β-glucosidase were found in all four strains. β-Galactosidase was only observed in strain S359. α-Mannosidase, β-mannosidase, α-glucosidase, β-xylosidase, and proteinase were not detected in any of the four strains. Isozyme patterns of β-glucosidase and esterase in the four strains were compared by activity staining after native gradient gel electrophoresis. The isozyme pattern of β-glucosidase showed three major forms in all four strains. In addition, two more isoforms were found in strain S370. All strains shared two esterase bands, while strain S370 had three more isoforms. Study on strain SMF indicated that acid β-glucosidase was expressed constitutively, with increased activity in cellobiose-containing media. Under nitrogen-limiting conditions, a low level of endoglucanase and exoglucanase activity was observed in strains SMF and S359. Further study on S359 showed that high concentrations of nitrogen repressed the cellulolytic activity. When cellobiose served as carbon source, higher cellulolytic activity was observed. Cellulose did not induce higher activity.Key words: Pisolithus, ectomycorrhizal, β-glucosidase, hydrolytic enzymes, cellulolytic enzymes.
Nutrient effects on growth and the production of 5-hydroxy-4-oxonorvaline by Streptomyces akiyoshiensis
Authors: Glazebrook M. A. , Vining L. C. , White R. L. , Smith K. C. , Chedrawy E. G.
Published: May 2010

5-Hydroxy-4-oxonorvaline (HON) was produced optimally by Streptomyces akiyoshiensis in media containing starch and casein supplemented with high concentrations of phosphate and magnesium sulfate; cultures grown at initial pH values between 6.3 and 6.6 yielded HON titres of 13–14 mM. Physiological analysis of HON production provided evidence that the excess inorganic constituents in this medium played an important role in optimizing production. In simple defined media buffered at pH 5.5–6.0 and inoculated with either a spore suspension or washed vegetative mycelium, formation of HON was less than one-third the amount produced in the starch–casein–salts medium. Production was markedly affected by the carbon and nitrogen sources used; media containing starch and potassium nitrate or aspartate supported the highest yields of HON. In starch–nitrate media, production was suppressed by excess nitrogen source, and the HON titre decreased 3-fold as the phosphate content was increased from 0.5 to 5 mM; growth was not appreciably altered. The results suggest that the high level of HON production in the complex starch–casein–salts medium is associated with sequestration of metabolic ammonium and reduced availability of phosphate through formation of the poorly soluble magnesium ammonium phosphate.Key words: Streptomyces akiyoshiensis, 5-hydroxy-4-oxonorvaline, nitrogen catabolite regulation, phosphate suppression.
Zymoblot, a new microtechnique used to detect enzyme activity in spiroplasmas and bacteria
Authors: Wagih Elsayed E. , Fletcher Jacqueline
Published: May 2010

A new microtechnique that detects enzyme activity in prokaryotes is described. The technique, designated zymoblot, is based on the immobilization of negatively charged enzymes from an alkaline extract spotted onto a nitrocellulose membrane. The presence of specific enzyme activity in the extract is selectively assayed with a reaction mixture containing the corresponding substrate. The enzyme–substrate reaction produces an insoluble colored product that accumulates at the site. The zymoblot technique offers the advantages of simplicity, sensitivity, reproducibility, speed, and the use of microquantities of reactants. The protein in the spot can be visualized by a technique termed "proteinblot," in which the protein is stained with Coomassie blue. Esterase and tyrosinase were detected by the zymoblot method in six spiroplasmas including four strains of Spiroplasma citri, one of Spiroplasma kunkelii, and one of Spiroplasma melliferum, and two bacteria, Pseudomonas syringae pv. syringae B301D and Escherichia coli K-12. Acid phosphatase, alkaline phosphatase, alanine dehydrogenase, peroxidase, and 6-phosphogluconate dehydrogenase were not detected in any of the spiroplasmas, but were each detected in one or both of the walled bacteria.Key words: spiroplasma, enzyme, protein, zymoblot.
Metschnikowia agaveae sp.nov., a heterothallic haploid yeast from blue agave
Authors: Lachance Marc-André
Published: May 2010

Several strains of a new haploid, heterothallic species of Metschnikowia have been isolated from Agave tequilana var. azul in two agave-growing localities of Jalisco, Mexico. The new yeast species forms two aciculate ascospores per ascus after conjugation of enlarged and elongated compatible cells. Named after its host, Metschnikowia agaveae resembles superficially Metschnikowia hawaiiensis, but differs from the latter by some physiological and morphological characteristics. These two species exhibited no signs of sexual cross-reactivity. Strain UWO(PS)92-207.1 (h+, CBS 7744, ATCC 90148) is the type culture, and strain UWO(PS)92-210.1 (h−, CBS 7745, ATCC 90147) has been designated as isotype.Key words: agave, yeast, Metschnikowia, new species.
An inexpensive medium for mass fermentation production of Entomophaga aulicae hyphal bodies competent to form conidia
Authors: Nolan Richard A.
Published: May 2010

A mass fermentation medium for growth and morphogenesis of the entomopathogenic fungus Entomophaga aulicae was developed. This fungus is a major pathogen of larval eastern hemlock looper and spruce budworm. The medium consists of a basal medium plus 0.8% tryptic soy broth and 0.4% calcium caseinate. This medium is a major breakthrough in that (i) the E. aulicae developmental sequence from protoplast inoculum to hyphal bodies competent to form conidia can be carried out in a single medium without adjustment, (ii) by examining the fermentation product it can be determined if conidia can be produced prior to engaging in costly field spraying, (iii) this medium supports the growth of E. aulicae isolates from different geographical areas, (iv) the medium is relatively inexpensive, (v) the hyphal bodies are easily separated from the spent growth medium, and (vi) the hyphal body yield is high.Key words: Entomophaga aulicae, mass fermentation medium, hyphal bodies, conidia, insect biocontrol.
The role of physiological state in osmotolerance of the salt-tolerant yeast Debaryomyces hansenii
Authors: Larsson Christer , Gustafsson Lena
Published: May 2010

Great variations in tolerance to osmotic dehydration during batch growth of the salt-tolerant yeast Debaryomyces hansenii have been demonstrated. The variations in osmotolerance were dependent on the cultivation conditions. The cells developed osmosensitivity at the beginning of the growth period. In medium in which the nitrogen source of the cells was changed from ammonium to urea during the growth cycle, the resulting transition state induced a marked increase in the osmotolerance of the cells. However, when the pH was kept constant at 6.0, the osmosensitive period disappeared almost completely. Various possible osmotolerance factors, such as the intracellular polyol and ion concentrations, the "G3P shuttle," and the cells' total activity measured by means of microcalorimetry, have been studied. Only small variations in the intracellular pools of polyols and ions were observed during different cultivation conditions and phases of growth. No single factor seems to be the determinant for osmotolerance and the overlap between osmoregulatory and osmotolerance factors is limited.Key words: osmotolerance, stress tolerance, calorimetry, heat production, Debaryomyces hansenii.
Physiological and morphological aspects of interactions between Rhizobium meliloti and alfalfa (Medicago sativa) in association with Azospirillum brasilense
Authors: Itzigsohn R. , Kapulnik Y. , Okon Y. , Dovrat A.
Published: May 2010

In a 50-L pot experiment with Medicago sativa grown under nonsterile conditions, a combined treatment of Azospirillum and Rhizobium was measured against soil inoculated with Rhizobium or Azospirillum alone or a control with a low background level of autochthonous rhizobia. The combined treatment significantly increased the shoot length and weight at 6 weeks and the regrowth shoot weight at 14 weeks when compared with the treatment with Rhizobium alone. In 1.5-L pots in which gnotobiotic conditions were maintained, the combined treatment led to more nodules on the main root at intermediate Rhizobium concentrations, and a greater root surface area at intermediate and high Rhizobium concentrations after 2 weeks but not after 4 weeks. In pouch-grown seedlings, plants were inoculated with either Rhizobium alone or in combination with Azospirillum or applied together with a flavonoid, luteolin (a nodulation gene inducer), or with a cytokinin, benzyl adenine. Luteolin had similar effects to those of Azospirillum in increasing the main root nodule number and the total nodule number. With Fahraeus slides, a significant increase was observed in the number of root hairs and the root diameter in the presence of Azospirillum as compared with the control and Rhizobium alone. There was no increase in the total number of infection threads; however, the combined treatment caused a significant decrease in the percentage of infected root hairs.Key words: Rhizobium, Azospirillum, Medicago, flavonoid, inoculation.
Genetic mapping of bfmA mutation causing fatty acid deficiency in Bacillus subtilis
Authors: Akamatsu Takashi , Moriyama Hideaki , Kaneda Toshi
Published: May 2010

Bacillus subtilis strain 626, defective in the bfmA gene, is a derivative of B. subtilis strain 168 and requires branched short-chain carboxylic acids for growth. Branched-chain 2-keto acid decarboxylase activity and fatty acid synthesis in B. subtilis strain 626 were 14 and 7%, respectively, of their levels in strain 626-2R, a spontaneously reverted strain. These results indicate that the bfmA mutation is in either the branched-chain 2-keto acid decarboxylase gene itself or its controlling gene. Thus, primer synthesis from the 2-keto acid substrate in strain 626 is defective, causing a deficiency in fatty acid synthesis. A bfmA mutation was transferred to a suitable genetic background and analysed. The bfmA strain, CACl, was competent and motile, and required 50 μM CaCl2 or 5 μM FeCl3 for growth on glucose minimal agar plates. The bfmA gene was mapped between glyC (320°) and ctrA (325°) and estimated to be at 320° by protoplast fusion and PBS1 phage transduction.Key words: Bacillus subtilis, 2-keto acid decarboxylase, bfmA mutation, chromosome location, protoplast transformation, PBI phage transduction.
Metabolism of elemental sulphur (S°) during fungal spore germination
Authors: Beffa Trello
Published: May 2010

35S-labelled elemental sulphur (S°) at nontoxic levels (3 μM) was incorporated into sulphur amino acids and glutathione by the spores of the fungus Phomopsis viticola. Incorporation studies were performed with 3 μM 35S° during the pregermination period of P. viticola spores. The free sulphur amino acids and protein sulphur amino acids were purified by column and thin-layer chromatography. During the first minutes of the pregermination process of the spores, the S° was essentially metabolized into free cysteine and glutathione. Detectable concentrations of methionine and homocysteine were measured after 1 h of incubation. Azide (2 mM final concentration), an inhibitor of the mitochondrial respiratory chain at the level of cytochrome oxidase, strongly inhibited the incorporation of S°. In contrast, the uncoupler 2,4-dinitrophenol (15 μM final concentration) and sulphate did not affect the incorporation of S°. The metabolism of 35S° into the protein sulphur amino acids cysteine and methionine during the germination of spores of P. viticola and conidia of Neurospora crassa is also discussed. It appears that S° does not necessarily have to be oxidized to sulphate prior to its incorporation into sulphur amino acids, but could be directly reduced to sulphide at the level of the respiratory chain.Key words: elemental sulphur, assimilation, sulphur amino acids, Phomopsis viticola, Neurospora crassa.
Sodium ion dependent active transport of leucine in Methanosphaera stadtmanae
Authors: Sparling Richard , Holth Laurel Thorlacius , Lin Zhaosheng
Published: May 2010

A Na+-dependent active transport system for leucine has been observed in Methanosphaera stadtmanae. The Km for leucine as determined was 20 μM with a Vmax of 3.2 nmol∙min−1∙mg protein−1. A minimum of 5 mM Na+ was required for optimal uptake rates. After correction for unspecific binding and incorporation into trichloroacetic acid precipitable materials, [14C]leucine was accumulated inside the cell to concentrations > 100 times higher than in the medium. The uptake of leucine into active cells was inhibited by the protonophore 3,3′,4′5-tetrachlorosalicylanilide but stimulated by the synthetic Na+–H+ antiporter monensin. A 500 mM NaCl pulse was able to drive leucine into resting cells. Of the amino acids tested, only valine and isoleucine competed effectively with leucine for transport.Key words: archaea, archaebacteria, methanogen, leucine transport, bioenergetics, sodium motive force.
Appearance of the arginine phenotype in Lactococcus lactis subsp. cremoris 2204 following phage transduction
Authors: Davey Graham P. , Heap Howard A.
Published: May 2010

An induced phage 2204-T from an arginine-positive Lactococcus lactis subsp. lactis 2204 L strain was used in transduction experiments with Lactococcus lactis subsp. cremoris 2204 (an arginine-negative strain containing the chloramphenicol resistance plasmid pFX3). Arginine-positive transductants were isolated at a low frequency (< 10−10 arginine-positive isolates per initial recipient cell). Hybridization experiments with labelled DNA from phage 2204-T showed the presence of the temperate phage (2204-T) genome in total DNA of the transductant 2204 LT strains. No hybridization was observed with DNA from the arginine-negative strain 2204. A mitomycin C treated derivative of 2204 LT that was arginine negative also showed no hybridization, indicating that the prophage had been eliminated. The presence of the chloramphenicol resistance plasmid pFX3 in both transductants and mitomycin C cured derivatives was demonstrated by hybridization, confirming the origin of the arginine-positive isolates as derivatives of 2204.Key words: arginine metabolism, Lactococcus cremoris, Lactococcus lactis, induced phage.
Chick embryo, a model to study the lethal activity of pertussis toxin, infectivity of Bordetella pertussis, and their neutralization by immune sera
Authors: Calver G. A. , Burton W. W. , Gardell C. Y. R.
Published: May 2010

The toxin activity of Bordetella strains and acellular pertussis components was evaluated in chick embryos. Eleven-day-old embryos were found to be most suitable for determination of LD50 values. Eight of eight Bordetella pertussis strains possessed LD50 values of 104 to 106 colony-forming units per dose of 100 μL. Embryos were resistant to Bordetella parapertussis and Bordetella bronchiseptica at doses of 1010 colony-forming units. Bacterial growth did not occur in the bloodstream or tissues of the heart, kidney, brain, liver, or lungs. Pertussis toxin activity, determined by clustering of Chinese hamster ovary cells, was found in the allantoic fluid following inoculation of virulent bacteria. Purified pertussis toxin had an LD50 of 1–2 μg protein/dose. Striking pathological effects of liver necrosis and oedema of the head and neck were similar following bacterial or toxin inoculation. Widespread changes occurred in the liver at the perivascular level, suggesting that after allantoic inoculation of virulent Bordetella strains, toxin is secreted, carried in the bloodstream, and causes the resultant pathology. Purified B. pertussis lipopolysaccharide was nontoxic for embryos at doses containing 30 μg; filamentous haemagglutinin was nontoxic at doses up to 10 μg. A correlation exists between the ability of serum to neutralize killing of chick embryos by toxin and to neutralize clustering of Chinese hamster ovary cells by toxin. Although the chick embryo serum neutralization studies confirm observations of mouse intracerebral infection studies that antibody to pertussis toxin is not an absolute requirement for prevention of infection with B. pertussis, evidence is provided that anti-toxin will eliminate toxic effects owing to secretion of pertussis toxin.Key words: Bordetella, pertussis toxin, chick embryo.
Activity of microbial peptidases, oxidases, and esterases in lake waters of varying trophic status
Authors: Morgan J. Alun W. , Pickup Roger W.
Published: May 2010

The range and activities of microbial enzymes present in lake water were assessed directly in cells concentrated by tangential flow filtration. A total of 108 enzymes were assayed in this study, which included tests for 60 peptidases, 20 oxidases, and 10 esterases, and 18 miscellaneous tests. In general, no trends in the range of enzymes were associated with trophic status of the lakes. However, one lake that was hypereutrophic had a greater range of enzymes than the other lakes tested. An increase in total enzyme activity (activity/mL) was recorded with an increase in trophic status of the water. The relationship between the physical and chemical attributes of each lake and microbial enzyme activities was investigated by principal component analysis. Quantitative changes between lakes in 11 of the 21 variables were shown to be closely related to changes in the enzyme activities of the lakes; total organic carbon, particulate carbon, particulate nitrogen, pH, and chlorophyll a showed the closest relationships.Key words: lake water, enzyme activity, trophic status.
Glycosidase profiles of Bacillus spp. and their value for species differentiation
Authors: Kämpfer Peter , Keuch Richard , Dott Wolfgang
Published: May 2010

A total of 268 strains of Bacillus spp. were investigated for their ability to hydrolyze 14 different fluorogenic 4-methylumbelliferyl-linked glycosidase substrates within 6 and 24 h of incubation. Glycosidase profiles were heterogeneous within the genus and some of the tests showed high separation index values; the profiles can therefore improve the differentiation of Bacillus spp. However, glycosidase profiles are not sufficient for unambiguous identification and should be used in combination with other biochemical tests.Key words: Bacillus, glycosidases, fluorogenic substrates, differentiation.
Anaerobic degradation of 2-methylbenzoic acid by a methanogenic consortium
Authors: Londry Kathleen L. , Fedorak Phillip M.
Published: May 2010

2-Methylbenzoic has been detected as a metabolite in anaerobic consortia that degrade m-cresol and it was found to accumulate in cultures incubated under methanogenic conditions. This study was initiated to determine whether 2-methylbenzoic acid could be degraded under methanogenic conditions. Near stoichiometric amounts of methane were obtained from 2-methylbenzoic acid in enrichment cultures inoculated with 25% (v/v) domestic anaerobic sewage sludge. However, lag times of 12–36 weeks typically were required before 2-methylbenzoic acid degradation began, and it was difficult to maintain the activity of the cultures. A transient metabolite was detected but not identified. It appeared to be the product of 2-methylbenzoic acid hydroxylation because its trimethylsilyl derivative had a molecular weight of 296, but it was not 4-hydroxy-2-methylbenzoic acid, the intermediate of m-cresol metabolism from which 2-methylbenzoic acid likely originates.Key words: anaerobic, methane, 2-methylbenzoic acid.