bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation of β-glucuronidase assay for the detection of Escherichia coli from environmental waters
Authors: Shadix Lois C. , Rice Eugene W.
Published: May 2010

The new United States Drinking Water Regulations state that water systems must analyze for Escherichia coli or fecal coliforms on any routine or repeat sample that is positive for total coliforms. The proposed methods for the detection of E. coli are based on β-glucuronidase activity, using the fluorogenic substrate 4-methylumbelliferyl β-D-glucuronide (MUG). This study was conducted to determine whether β-glucuronidase negative E. coli were present in significant numbers in environmental waters. Two hundred and forty E. coli cultures were isolated from 12 water samples collected from different environmental sources. β-glucuronidase activity was determined using lauryl tryptose broth with MUG, EC broth with MUG, and the Autoanalysis Colilert (AC) procedure. The isolates were also evaluated by the standard EC broth gas fermentation method for fecal coliforms. The results confirm that assaying for the enzyme β-glucuronidase utilizing the MUG substrate is an accurate method for the detection of E. coli in environmental waters. Key words: Escherichia coli, β-glucuronidase, 4-methylumbelliferyl β-D-glucuronide, water.
Influence of pH on endo- and exo-pectinase production by Aspergillus sp. CH-Y-1043
Authors: Aguilar Guillermo , Trejo Blanca A. , García Juan M. , Huitrón Carlos
Published: May 2010

Aspergillus sp. CH-Y-1043 grown on pectins with various degrees of esterification produces endo- and exo-pectinases at pH values as low as 2.5. Maximal production was attained at this pH, although fungal growth only approximated 50% of that obtained at higher pH values. Endopectinase was produced at pH 2.5–3.5 when the fungus was grown on low degree esterified pectin. With higher degree esterified pectin this enzyme was produced at all pH values analyzed. Exopectinase production was less affected by pH values. Still, maximal production was also reached at pH 2.5–3.5. Exopectinase was found to be associated to the cell and could be released after incubation at different pH values, whereas endo pectinase was not detected in the cellular fraction. Results confirmed by SDS–PAGE coupled with in situ activity assays in pectin–agarose gels allowed the identification of a protein band corresponding to endopectinase and a band with pectin esterase activity. Stability of Aspergillus sp. CH-Y-1043 pectinases at various pH values was also evaluated. Key words: Aspergillus sp. CH-Y-1043, extreme acidic pH pectinase production, in situ pectinase detection, cell-associated exopectinase.
Microbiological aspects of ammonia oxidation of swine waste
Authors: St-Arnaud S. , Bisaillon J. -G. , Beaudet R.
Published: May 2010

Ammonia-oxidizing bacteria were present at 102 MPN/mL (most probable number per millilitre) in swine waste, and they were outnumbered by a factor of 105 by the heterotrophs of the indigenous flora. To study these ammonia-oxidizing bacteria we attempted to isolate them in pure culture. We succeeded in increasing the concentration of these bacteria by successive transfers to an inorganic medium, but the heterotrophs were always dominant. To overcome this problem Nitrosomonas europaea ATCC 19718 was adapted to grow in stabilized swine waste. With this adapted strain it was shown that the number of cells inoculated into swine waste rapidly decreased both under the aerobic conditions used to oxidize the organic matter of swine waste and under the anaerobic conditions found in stored swine waste. Ammonia oxidation was delayed when adapted N. europaea was inoculated into a partially stabilized swine waste as compared with results in a completely stabilized waste. A biofilm of 107 MPN/cm2 of N. europaea was developed after 114 days of incubation at 29 °C on polyvinyl chloride discs covered with geotextile in a rotating biological contactor using an inorganic medium. This biofilm was gradually adapted to stabilized swine waste and the rate of disappearance of ammonia reached 270 mg∙L−1∙day−1 in the compartment of the reactor containing 2.5 L of waste. Key words: amonia-oxidizing bacteria, swine waste, ammonia oxidation, biofilm, most probable number.
Antifungal activity of naftifine against various yeasts and dermatophytes: determination of minimal inhibitory concentrations and revelation of cell wall alterations by scanning electron microscopy
Authors: Mallié M. , Butty P. , Montès B. , Jouvert S. , Bastide J.-M.
Published: May 2010

The in vitro antifungal activity of naftifine, an allylamine derivative, was compared with that of econazole and clotrimazole, imidazole derivatives. The minimal inhibitory concentration (MIC) values were determined against 136 strains of yeasts (Candida albicans, Candida guilliermondii, Candida krusei, Candida parapsilosis, Candida pseudotropicalis, Candida tropicalis, Torulopsis glabrata, Malassezia furfur) and 45 strains of dermatophytes (Microsporum canis, Microsporum gypseum, Microsporum langeronii, Trichophyton mentagrophytes, Trichophyton rubrum, Epidermophyton floccosum). The fungistatic activity was evaluated by the agar dilution method, using various media: YNB (pH 6) for Candida and Torulopsis, Dixon (pH 5.6) for Malassezia, and Sabouraud (pH 6.8) for dermatophytes. The MIC was evaluated at 28–30 °C after incubation for 24–48 h for Candida and Torulopsis and for 3 days for the dermatophytes. Malassezia were incubated for 3 days at 37 °C. Naftifine was highly active against all the dermatophytes tested and moderately or weakly active against the yeasts. Candida parapsilosis was the most sensitive of all the Candida species tested. The effects of naftifine on C. parapsilosis, Mi. canis, and Tr. mentagrophytes were studied by scanning electron microscopy. Naftifine acted on C. parapsilosis causing formation of convolutions and wrinkles and defective separation between mother and daughter yeast cells. Naftifine caused alterations of the dermatophyte cell wall and particularly the hyphal filaments. These alterations were dose and time dependent. The most prominent changes seen by scanning electron microscopy were swelling, ballooning of the hyphae, and bulbiform blunt hyphae ends. Key words: naftifine, Candida, Torulopsis, Malassezia, dermatophytes.
Identification of bacteria isolated from roots of sunflower (Helianthus annuus) cultivated in a French soil
Authors: Fages Jacques , Lux Benoit
Published: May 2010

The bacterial microflora of the rhizoplane and roots of sunflower (Helianthus annuus L.) grown in a French soil was examined. Gram-negative, oxidative isolates were dominant, among which Agrobacterium radiobacter and Pseudomonas vesicularis were the most common. Only 4 of the 45 isolates were nitrogen fixers (acetylene reducers), and they all belonged to the Azospirillum lipoferum species. Key words: sunflower, rhizoplane, Gram-negative isolates.
In vitro production of struvite by Bacillus pumilus
Authors: Nelson Berlin , Struble James , McCarthy Gregory
Published: May 2010

A strain of Bacillus pumilus, isolated from the soilborne sclerotia of a fungus, produced crystals of the mineral struvite (magnesium ammonium phosphate hexahydrate) on Difco nutrient agar and a yeast extract agar containing magnesium sulphate and potassium phosphate. Crystals were macroscopically observed after 6–20 days of bacterial growth and reached a maximum size of 3 × 0.5 mm. Crystals were identified by X-ray powder diffractometry. These findings support the hypothesis that bacteria are involved in the biogenic formation of struvite in nature. Key words: struvite, magnesium ammonium phosphate hexahydrate, Bacillus pumilus, biomineral.
Effect of the method of preparing monochloramine upon inactivation of MS2 coliphage, Escherichia coli, and Klebsiella pneumoniae
Authors: Berman Donald , Sullivan Robert , Hurst Christon J.
Published: May 2010

Monochloramine prepared in situ by first adding chlorine to a suspension of microorganisms, followed by subsequent addition of ammonia, inactivated the MS2 coliphage more rapidly than did exposure of phage to monochloramine prepared either by adding chlorine to ammonia or by adding chlorine and ammonia simultaneously. The rapid viral inactivation was apparently due to the exposure of MS2 to free chlorine before the addition of ammonia. The average 99% CT value of MS2 when exposed to free chlorine was 1.3 and 1.1 at 5 and 15 °C, respectively. The average 99% CT values of MS2 briefly exposed to the combined action of free chlorine followed by the addition of ammonia to form monochloramine in situ were 19.3 and 1.5 at 5 and 15 °C, respectively. No 99% CT values were calculated for the inactivation of MS2 with preformed monochloramine because less than 1 log (90%) of inactivation occurred during a 4-h contact time. Inactivation of MS2 by monochloramine was more rapid at 15 than at 5 °C and when the chlorine to nitrogen weight ratio was 5:1 compared with 3:1. Monochloramine was a more efficient inactivating agent for the coliforms Escherichia coli and Klebsiella pneumoniae than it was for the MS2 coliphage. Key words: chlorine, monochloramine, virus, bacteria, disinfection.
Bacteria that degrade p-chlorophenol isolated from a continuous culture system
Authors: Kramer Constance M. , Kory Martha M.
Published: May 2010

Two Gram-positive coryneform bacteria that degraded p-chlorophenol isolated from a continuous culture system are characterized. Isolate B (probably an Arthrobacter sp.) completely removed the p-chlorophenol from a medium with a concomitant increase in cell density within 16 h. Isolate F similarly removed the p-chlorophenol within 28 h but without an increase in cell density. Isolates B and F also removed the p-chlorophenol from a medium with p-chlorophenol as the sole carbon source within 32 and 48 h, respectively. The optimal temperature for p-chlorophenol degradation by both organisms was 25 to 30 °C. The optimal pH range for p-chlorophenol degradation was pH 7–9 for isolate B and pH 8–9 for isolate F. Since these native environmental bacteria can degrade p-chlorophenol, they may have an important application in waste water treatment processes. Key words: biodegradation, bioremediation, xenobiotics.
Role of spore coats in the germinative response of Bacillus cereus to adenosine and its analogues
Authors: Senesi Sonia , Freer Giulia , Batoni Giovanna , Barnini Simona , Capaccioli Anna , Cercignani Giovanni
Published: May 2010

Spores of the strain NCIB 8122 of Bacillus cereus have been depleted of coats by treatment with 0.1% sodium dodecyl sulfate – 200 mM 2-mercaptoethanol – 0.5 M NaCl (pH 9.6). The coat-depleted spores did not show any decrease in viability, heat resistance, refractility, dipicolinic acid content, or specific activities of several protoplastic enzymes. The germinative response of the coat-depleted spores to adenosine and several analogues thereof was found qualitatively similar to that obtained with intact spores. However, germination kinetics appeared to be affected by coat removal, since germination rate measured as loss of refractility was eight times slower even at inducer concentrations 10-fold higher than those required to promote optimal germination response of intact spores. Loss of heat resistance, on the other hand, was hardly affected by coat removal. These results suggest that, even though spore coats are not essential for the triggering reaction, they are required for a rapid evolution of the later events in the germination process. Key words: adenosine analogues, germination-triggering reaction, spore coats, coat-depleted spores, Bacillus cereus.
Compatibility of plant growth promoting rhizobacterial strains with agrichemicals applied to seed
Authors: Zablotowicz Robert M. , Press Caroline M. , Lyng Nicola , Brown Gerry L. , Kloepper Joseph W.
Published: May 2010

The compatibility of a select group of plant growth promoting rhizobacterial strains with chemicals commonly used as seed treatments was investigated. Strains in several genera (Serratia, Pseudomonas, and coryneform-like bacteria) were found to be tolerant to Vitavax RS (containing lindane, carboxin, and thiram), Epic (iprodione), and (or) captan tested in vitro at commercial rates. Six of 10 strains survived equally, and exhibited similar root colonization, on Vitavax RS treated and nontreated seed. Four of seven strains tested (Serratia spp. and P. fluorescens) were likewise found to be compatible with a captan seed treatment on supersweet corn, using the same criteria. Ability of bacteria to grow on pesticide-amended media did not always indicate compatibility with chemical seed treatments in vivo. A greenhouse study demonstrated that enhanced emergence occurred with the coryneform-like strain 44-9 on Vitavax RS treated canola seed grown under conditions favoring disease due to Rhizoctonia solani. The ability to combine plant growth promoting rhizobacterial strains with current agrichemicals for plant growth stimulation and disease control is indicated. Key words: pesticide compatibility, Pseudomonas, agrichemicals, Serratia, damping-off, plant growth promoting rhizobacteria.
Construction of a new shuttle vector for Lactobacillus
Authors: Chagnaud P. , Chion C. K. N. Chan Kwo , Duran R. , Naouri P. , Arnaud A. , Galzy P.
Published: May 2010

To clone the malolactic enzyme gene from Lactobacillus sp. 89, construction of a shuttle vector able to express itself in Lactobacillus sp. 89 and Escherichia coli was undertaken. The shuttle plasmid pLE16 resulted from the union of pBR328 and of the pLB10 plasmid extracted from Lactobacillus bulgaricus 10. The bacterial transformation in Lactobacillus sp. 89 was performed by electroporation, and the clones were selected on MRS medium with 30 μg∙mL−1 chloramphenicol added. Fifty percent of the clones from Lactobacillus sp. 89 lost their resistance to chloramphenicol following 28 generations when the selection pressure was not maintained. The restriction map of pLE16 (7600 bp) was established using several restriction enzymes. Key words: malolactic enzyme, shuttle plasmid, Escherichia coli, Lactobacillus, electroporation.
Separation and characterization of 61- and 57-kDa lipases from Geotrichum candidum ATCC 66592
Authors: Jacobsen Tomas , Poulsen Otto M.
Published: May 2010

Two lipolytic proteins (61 and 57 kDa) present in a Sephadex G-100 fraction of extracellular lipase from Geotrichum candidum ATCC 66592 were separated using high-performance liquid chromatography. Crossed electrofocusing immunoelectrophoresis was used to demonstrate that the 61-kDa lipase fraction contained two forms of lipase with pI 4.5 and 4.7. However, when deglycosylated with endoglycosidase H, the two forms gained an identical pI, 4.6. The 57-kDa lipase fraction contained one form of lipase with pI close to 4.5. Although the 61- and 57-kDa lipases were immunologically identical, the substrate specificity differed. Thus, the 61-kDa lipase hydrolysed palmitic acid methyl ester at an initial velocity of hydrolysis that was 60% of the initial velocity of hydrolysis of oleic acid methyl ester, whereas the 57-kDa lipase hydrolysed palmitic acid methyl ester at an initial velocity of hydrolysis that was only7% of the initial velocity of hydrolysis of oleic acid methyl ester. Key words: Geotrichum candidum, lipases, multiple forms, deglycosylation, substrate specificity.
Simultaneous detection of toxin A and toxin B genetic determinants of Clostridium difficile using the multiplex polymerase chain reaction
Authors: McMillin David E. , Muldrow Lycurgus L. , Laggette Shwanda J.
Published: May 2010

A multiplex polymerase chain reaction was developed to simultaneously detect the presence of toxin A and toxin B genes of Clostridium difficile. A 1050-bp fragment of the toxin B gene and a 1217-bp fragment of the toxin A gene were amplified from 42 toxic strains of C. difficile; however, from 10 nontoxic strains the toxin gene fragments were not amplified; these data demonstrate that this multiplex polymerase chain reaction procedure can be used to differentiate between toxic and nontoxic strains. This sensitive and specific multiplex polymerase chain reaction for C. difficile toxins may prove to be a valuable diagnostic procedure. Key words: Clostridium difficile, polymerase chain reaction, bacterial toxins.
O-Acetylated peptidoglycan: its occurrence, pathobiological significance, and biosynthesis
Authors: Clarke Anthony J. , Dupont Claude
Published: May 2010

Bacterial cell walls and their structural units, particularly peptidoglycan, induce a vast variety of biological effects in host organisms. The pathobiological effects of peptidoglycan are greatly enhanced by various modifications and substitutions to its basic composition and structure. One such modification is the presence of acetyl moieties at theC-6 hydroxyl group of N-acetylmuramyl residues, and to date, 11 species of eubacteria, including some important human pathogens, such as Neisseria gonorrhoeae, Proteus mirabilis, and Staphylococcus aureus, are known to possess O-acetylated peptidoglycan. This review addresses the influence of O-acetylation of peptidoglycan on its resistance to degradation both in vitro and in vivo, the clinical importance of the modification, and the currently held views on the pathway for its biosynthesis. Key words: peptidoglycan, murein, O-acetylation, lysozyme, arthritis.
Laboratory investigation of virulence among strains of Yersinia enterocolitica and related species isolated from pigs and pork products
Authors: Kwaga Jacob K. P. , Iversen John O.
Published: May 2010

Eighty strains of Yersinia enterocolitica and related species isolated from slaughtered pigs and pork products were tested for possession of virulence-associated phenotypes by employing 12 in vivo and in vitro assays. The isolates could be broadly divided into two groups: (i) strains belonging to pathogenic bioserotypes of Y. enterocolitica that displayed virulence-associated characteristics in most or all assays and (ii) strains belonging to Y. enterocolitica biotype 1A and to related species that were largely negative in these assays. No individual test was found as a single reliable measure of virulence. All strains belonging to Y. enterocolitica serotype O: 1,2,3 were pyrazinamidase positive (indicates avirulence) and autoagglutination negative but were positive in all other virulence assays. Salt aggregation was found to be a better indicator of virulence than latex particle agglutination, both of which measure surface hydrophobicity. Overall, tissue culture cell invasion provided the best selection of a subpopulation of yersiniae that are potentially virulent. However, crystal violet and Congo red binding assays among others provided good prediction of virulence at the time of testing. Our results provide further evidence that swine may constitute an important reservoir of human pathogenic strains. Key words: Yersinia enterocolitica, virulence phenotype, human pathogen.
Mode of antibacterial action of dodine (dodecylguanidine monoacetate) in Pseudomonas syringae
Authors: Cabral João P. S.
Published: May 2010

Treatment of Pseudomonas syringae cells with 50 μM dodecylguanidine monoacetate (dodine) resulted in the rapid degradation and release of RNA and cell lysis. Higher concentrations resulted in a progressive decrease in the intensity of these responses, and the appearance of extensive zones of coagulated cytoplasm, indicating that the decrease in RNA degradation probably resulted from an inhibition of the RNases, due to protein denaturation. Dodine also induced expansion of the outer membrane, with the formation of protuberances and intracellular myelin-like structures, which were already evident after 1 min of treatment, indicating that dodine is able to cross the outer and cytoplasmic membranes rather rapidly, and to form, alone or in combination with cell phospholipids and proteins, considerable amounts of triple-layered profiles. In P. syringae cells, saturation levels of dodine corresponded to more than five times the amount needed to form a close-packed monolayer of dodine on the cell surface. The different membranous structures formed in dodine-treated cells, and the coagulation of the cytoplasm, seem to be responsible for the uptake of such high amounts of dodine. The uptake isotherm was essentially Langmuirian. The results presented in this and previous reports indicate that the antibacterial activity of dodine on P. syringae is mainly the result of the action of micelles of the surfactant. Key words: dodine, fungicide, surfactant, Pseudomonas syringae.
Ultrastructure of natural and induced myxospores of Archangium gephyra
Authors: Galván A. , Marcotegui M. A. , Castro F. de
Published: May 2010

The ultrastructure of natural myxospores and glycerol-induced myxospores has been studied in four Archangium gephyra strains (AG3, AG5, AG9, and AG10). The induced myxospores show an oval shape, characteristic of the genus Archangium, and an electron-dense coat surrounds the cell wall. The cytoplasm contains abundant electron-lucent inclusions. Sometimes, a membranous structure appears between the coat and the outer membrane. The fruiting-body myxospores show an oval shape similar to that observed in induced myxospores, but with a slightly thicker outer coat. In many of the natural myxospores, some membranous, lamellar or vesicular structures appear. They are located between the coat and the outer membrane, resembling those that appeared in induced myxospores. These have not been previously mentioned in papers on fruiting-body myxospores. These structures occasionally seem to be fused, giving rise to membranous sacs filled with an amorphous material of medium electron density. We propose that these vesicles are formed from the waved outer membrane and from the fruiting-body matrix. Large, dense, fusiform inclusions with an internal crystalline array are frequently found in the cytoplasm of natural myxospores, and cells during differentiation, in strains AG3, AG9, and AG10. Key words: Archangium gephyra, electron microscopy, myxospores, ultrastructure.
Effects of culture conditions on the in vitro infection of fibroblasts by Candida albicans
Authors: Merkel G. J.
Published: May 2010

The effects of yeast culture age, carbon source, growth temperature, and germ-tube inducers on adherence to primary fibroblast cultures was studied in conjunction with the determination of adherence-mediated mammalian cell damage by measuring chromium-51 release from fibroblast monolayers. The results indicated that yeast culture age affected adherence only when the yeasts were grown at 37 °C, not after growth at 28 °C. At 37 °C, quantitatively fewer exponential-phase, glucose- or galactose-grown yeasts adhered to fibroblasts than did yeasts that were in lag or stationary phases. The reduced adherence correlated with less chromium-51 release and reduced germ-tube formation. The addition of germ-tube inducers, such as N-acetyl-D-glucosamine or serum, to exponential-phase yeasts caused an increase in germ-tube formation with a concomitant increase in yeast adherence and release of chromium-51 from the monolayers. Exponential-phase galactose-grown yeasts were more responsive to serum-induced germ-tube formation, germ-tube elongation, and fibroblast adherence than were exponential-phase glucose-grown yeasts. In addition, exponential-phase galactose-grown yeasts caused more chromium-51 release from monolayers in the presence of serum than did glucose-grown yeasts. Overall, conditions that enhanced germ-tube formation and elongation resulted in greatest adherence-mediated damage to the monolayers. Key words: Candida albicans, fibroblasts, morphogenesis.
Sulfide alleviation of acetylene inhibition of nitrous oxide reduction by Flexibacter Canadensis
Authors: Jones Alison M. , Knowles Roger
Published: May 2010

The role of sulfide in the relief of acetylene inhibition of nitrous oxide reduction by Flexibacter canadensis was studied. In this organism, the reversal of acetylene inhibition of nitrous oxide reduction is correlated with a 90% decrease in the dissolved sulfide concentration. The fate of this sulfide is not known, since there was no concomitant increase in acid-soluble sulfide and volatile sulfur compounds were not detectable by flame photometric gas chromatography. Of the other sulfur-containing compounds tested (sulfate, sulfite, thiosulfate, cysteine, methionine, dithionite, dithionate, and glutathione), only cysteine relieved the acetylene block of nitrous oxide reduction by F. canadensis. Under similar experimental conditions, other denitrifiers tested (Azospirillum brasilense, Pseudomonas stutzeri, and a Flavobacterium isolate) failed to reduce nitrous oxide in the presence of sulfide and an inhibitory concentration of acetylene. It is concluded that both biological and abiological factors contribute to the sulfide relief of acetylene inhibition of nitrous oxide by pure cultures of F. canadensis. Key words: denitrification, nitrous oxide, acetylene, sulfide, Flexibacter canadensis.
Localization of a 210-kDa microtubule-interacting protein in the yeast Saccharomyces cerevisiae
Authors: Hašek J. , Jochová J. , Dráber P. , Viklický V. , Streiblová E.
Published: May 2010

Using the monoclonal antibody MA-01, which recognizes a 210-kDa protein in cell-free extracts, spindle and cytoplasmic microtubules were visualized in budding yeast, Saccharomyces cerevisiae. In additional, a spot-like staining was found beneath the plasma membrane, revealing in part correlation with F-actin distribution. This pattern was common for cells of all cell-cycle stages. The interaction of the protein recognized by MA-01 with microtubules was confirmed in the double labeling with a polyclonal antitubulin antibody and by the sensitivity of intranuclear structures stained by MA-01 to the microtubule disrupting drug nocodazole. Key words: immunoblotting, immunofluorescence, microtubule-interacting protein, Saccharomyces cerevisiae.
Purification of an enterotoxin produced by Bacillus cereus by immunoaffinity chromatography using a monoclonal antibody
Authors: Shinagawa Kunihiro , Takechi Teimi , Matsusaka Naonori , Sugii Shunji
Published: May 2010

A murine monoclonal antibody (MAb) with high reactivity to an enterotoxin produced by Bacillus cereus was used to prepare an immunoadsorbent for purification of the enterotoxin. By immunoaffinity chromatography using the immunoadsorbent, approximately 25% of crude enterotoxin applied was recovered in the eluate. The purified enterotoxin was found to be electrophoretically and antigenically homogeneous. It also showed vascular permeability activity and mouse lethality, and caused fluid accumulation in mouse ligated intestinal loops, whereas it did not show any haemolytic and lecithinase activities. Thus, immunoaffinity chromatography proved useful in the purification of enterotoxin produced by B. cereus in terms of recovery, purity, and relative ease of performing the purification. Key words: purification, immunoaffinity chromatography, enterotoxin, monoclonal antibody, Bacillus cereus.
Cooperation between two Thiobacillus strains for heavy-metal removal from municipal sludge
Authors: Blais J.-F. , Auclair J. C. , Tyagi R. D.
Published: May 2010

A mixed culture of two fast-growing bacterial strains for heavy-metal solubilization of municipal sewage sludge has been developed. Strain VA-7 decreases the initial sludge pH (7–8.5) to a value between 4.0 and 4.5. Then, strain VA-4 begins growing and further reduces the pH to values below 2.0. The rapid decrease of sludge pH by a mixed culture through sulfur oxidation into sulfuric acid solubilizes the toxic metals (Cd 83–96%, Cr 16–54%, Cu 85–87%, Mn 91–94%, Ni 78–79%, Pb 28–46%, Zn 82–96%) to levels recommended for intensive use of residual sludge in agriculture. A study of the physiological and metabolic characteristics of these strains revealed that isolate VA-7 is a strain of Thiobacillus thioparus (ATCC 55127), while isolate VA-4 corresponds to a Thiobacillus thiooxidans (ATCC 55128). These bacterial strains possess distinctive physiological characteristics that allow them to easily grow and solubilize heavy metals in municipal sludge. Key words: heavy metals, sewage sludge, thiobacilli, bioleaching, elemental sulfur.
Nucleotide sequence analysis of the gene encoding the Caulobacter crescentus paracrystalline surface layer protein
Authors: Gilchrist Angus , Fisher James A. , Smit John
Published: May 2010

The entire nucleotide sequence of the rsaA gene, encoding the paracrystalline surface (S) layer protein (RsaA) of Caulobacter crescentus CB15A, was determined. The rsaA gene encoded a protein of 1026 amino acids, with a predicted molecular weight of 98 132. Protease cleavage of mature RsaA protein and amino acid sequencing of retrievable peptides yielded two peptides: one aligned with a region approximately two-thirds the way into the predicted amino acid sequence and the second peptide corresponded to the predicted carboxy terminus. Thus, no cleavage processing of the carboxy portion of the RsaA protein occurred during export, and with the exception of the removal of the initial methionine residue, the protein was not processed by cleavage to produce the mature protein. The predicted RsaA amino acid profile was unusual, with small neutral residues predominating. Excepting aspartate, charged amino acids were in relatively low proportion, resulting in an especially acidic protein, with a predicted pI of 3.46. As with most other sequenced S-layer proteins, RsaA contained no cysteine residues. A homology scan of the Swiss Protein Bank 17 produced no close matches to the predicted RsaA sequence. However, RsaA protein shared measurable homology with some exported proteins of other bacteria, including the hemolysins. Of particular interest was a specific region of the RsaA protein that was homologous to the repeat regions of glycine and aspartate residues found in several proteases and hemolysins. These repeats are implicated in the binding of calcium for proper structure and biological activity of these proteins. Those present in the RsaA protein may perform a similar function, since S-layer assembly and surface attachment requires calcium. RsaA protein also shared some homology with 10 other S-layer proteins, with the Campylobacter fetus S-layer protein scoring highest. Key words: Caulobacter crescentus, surface layer, nucleotide sequence, rsaA, calcium.
Fonsecaea pedrosoi: lipid composition and determination of susceptibility to amphotericin B
Authors: Gomes M. H. R. , Resende M. A.
Published: May 2010

Conidia and mycelial cells of Fonsecaea pedrosoi ATCC 46428 were obtained for analyses of lipid composition. Total lipids, phospholipids, sterols, and qualitative sterols and fatty acid composition were determined. A higher lipid content was detected in conidia than in mycelial cells of Fonsecaea pedrosoi, which could not be attributed to total sterols and phospholipids. In both forms of this fungus, ergosterol was the only sterol detected. The minimal inhibitory concentration of amphotericin B was lower for conidia than for mycelium. Key words: Fonsecaea pedrosoi, chromoblastomycosis, sterol, amphotericin B, lipid composition.
A succinate transport mutant of Bradyrhizobium japonicum forms ineffective nodules on soybeans
Authors: El-Din Abdel Kader Yousef Gamal
Published: May 2010

Biochemical evidence has shown that dicarboxylic acids actively support symbiotic nitrogen fixation by both fast- and slow-growing Rhizobium. Mutants defective in the active uptake of succinate have been previously described only in species of the fast-growing rhizobium. This article is a report on the isolation of mutants defective in dicarboxylate transport in a slow-growing species of rhizobium, Bradyrhizobium japonicum. One of these presumptive dicarboxylate transport mutants, GTS, was characterized further. Cultured GTS was unable to accumulate [14C]succinate above background levels but possessed normal rates of malate dehydrogenase, fumarase, and hydroxybutyrate dehydrogenase activities. When inoculated onto soybeans, GTS produced a Nod+, Fix− phenotype. The bacteroids isolated from these nodules failed to accumulate labelled succinate. Electron micrographs of nodules formed by inoculation with GTS appeared normal with the exceptions of more prominent peribacteroid spaces in the infected cells and the appearance of starch granules in the noninfected cells. The phenotypical and morphological changes observed for B. japonicum are similar to those previously reported for the fast-growing species. Key words: Fix−, mutant, Rhizobium, succinate, transport.
Isolation and biological activities of endotoxin from Leptospira interrogans
Authors: Souza L. de , Koury M. C.
Published: May 2010

Endotoxins extracted with ethylenediaminetetraacetate (EDTA) from Leptospira interrogans serovars icterohaemorrhagiae and canicola and Leptospira biflexa serovar patoc were tested for various biological activities characteristic of endotoxins. The presence of lipopolysaccharide biological activity was demonstrated by the Limulus amoebocyte lysate test, pyrogenicity in rabbits, complement interaction inhibiting the erythrocyte lysis, and chicken-embryo lethality. The lipopolysaccharides did not induce the local Shwartzman reaction. The lipopolysaccharides of serovars icterohaemorrhagiae and canicola were immunogenic in rabbits and were cytotoxic to chicken-embryo fibroblasts. Key words: Leptospira, endotoxin.
Occurrence of thermotolerant campylobacters in fresh vegetables sold at farmers' outdoor markets and supermarkets
Authors: Park C. E. , Sanders G. W.
Published: May 2010

A total of 1564 fresh samples of 10 vegetable types from two different retail levels (533 samples from farmers' outdoor markets and 1031 samples from supermarkets) were surveyed for the occurrence of thermotolerant campylobacters. In samples from the outdoor markets, campylobacters were detected on six types of vegetables; the detection rates were spinach, 3.3; lettuce, 3.1; radish, 2.7; green onions, 2.5; parsley, 2.4; and potatoes, 1.6%. Campylobacter jejuni was the predominant species (88%), with the remainder being C. lari (8%) and C. coli (4%). When the outdoor market samples were thoroughly washed with chlorinated water, all were negative for campylobacters. Of the samples from supermarkets, all were negative for campylobacters whether purchased in summer or winter. These results suggest that vegetables sold at farmers' outdoor markets are produced and (or) stored under less sanitary conditions than those sold at supermarkets, and they could constitute health hazards. Therefore, vegetables (e.g., potatoes and spinach) from farmers' markets must be decontaminated by washing with chlorinated water or cooked thoroughly before consumption. Key words: campylobacters, vegetables, decontamination.
Changes in selected enzyme activities during growth of pure and mixed cultures of the white-rot decay fungus Trametes versicolor and the potential biocontrol fungus Trichoderma harzianum
Authors: Freitag Michael , Morrel Jeffrey J.
Published: May 2010

Two filamentous fungi, the white-rot fungus Trametes versicolor and the soil fungus and potential biocontrol organismTrichoderma harzianum, have been grown in pure and mixed cultures on low-N (0.4 mM) and high-N (4 mM) defined synthetic media to determine the activities of selected wood-degrading enzymes such as cellobiase, cellulase, laccase,and peroxidases. Growth characteristics and enzyme activities were examined for potential correlations. Such correlations would allow the use of simple enzyme assays for measuring biomass development and would facilitate predictions about competitiveness of species in mixed fungal cultures. Our results show that while laccase and Poly Red-478 peroxidase activities indicate survival of the decay fungus, none of the monitored extracellular enzymes can serve as a quantitative indicator for biomass accumulation. As expected, the level of available nitrogen affected the production of the enzymes monitored: in low-N media, specific cellobiase, specific cellulase, and peroxidase activities were enhanced, while laccase activities were reduced. Most importantly, laccase activities of Trametes versicolor, and to a smaller extent, cellobiase activities of both fungi, were significantly induced in mixed cultures of Trametes versicolor and Trichoderma harzianum. Key words: biocontrol, wood decay, laccase, peroxidases.
Effect of substitution at C-6 on the susceptibility of pullulan to pullulanases.Enzymatic degradation of modified pullulans
Authors: Ball Derek H. , Wiley Bonnie J. , Reese Elwyn T.
Published: May 2010

Pullulan, with all of the primary hydroxyl groups modified, is an excellent substrate for defining the effect of degree of substitution on biodegradability because of the uniform distribution of substituents on the polysaccharide. 6-Chloro-6-deoxypullulan and 3,6-anhydropullulan are highly resistant to hydrolysis by the four different types of pullulanase. 6-Azido-6-deoxypullulan is resistant to three types but susceptible to hydrolysis by the fourth, isopullulanase.Neopullulanase is strongly inhibited by 6-chloro-6-deoxypullulan and 6-azido-6-deoxypullulan, the other pullulanases much less so. Key words: 3,6-anhydropullulan, 6-azido-6-deoxypullulan, biodegradability, 6-chloro-6-deoxypullulan, pullulan, pullulanases.
Cytosolic and membrane-bound chitinases of two mucoraceous fungi: a comparative study
Authors: Balasubramanian R. , Manocha M. S.
Published: May 2010

Chitinases isolated from membrane and cytosolic fractions of two mucoraceous fungi, Choanephora cucurbitarum and Phascolomyces articulosus, were investigated. The membrane-bound chitinase was isolated by Bio-Gel P-100 and DEAE Bio-Gel A chromatographic techniques. On SDS–PAGE the chitinase from both fungi migrated as a single band of Mr 66 kDa. The cytosolic chitinase from the mycelial extracts of these fungi was separated by heat treatment, ammonium sulphate precipitation, and by affinity chromatography with regenerated chitin. SDS–PAGE showed two bands for each fungus with Mr of 69.5 and 55 kDa in C. cucurbitarum and Mr 69.5 and 53 kDa in Ph. articulosus. Chitinases, membrane bound or cytosolic, hydrolyzed regenerated chitin, colloidal chitin, glycol chitin, N,N′-diacetylchitobiose, and N,N′,N′ ′-triacetylchitotriose. Heavy metals, inhibitors, and N-acetylglucosamine inhibited chitinase activity, whereas trypsin and an acid protease enhanced its activity. Chitinase preparations showed lysozyme activity that was inhibited by histamine but not by N-acetylglucosamine. There was no N-acetylglucosamanidase activity, but β-1,3 glucanase activity was found in cytosolic preparations only. Despite slight differences in their molecular mass, both the membrane-bound and cytosolic chitinases showed similarities in substrate utilization, response to inhibitors, and activation by trypsin and acid protease; pH and temperature optima also were similar. Key words: chitinase, membrane-bound chitinase, cytosolic chitinase, Choanephora cucurbitarum, Phascolomyces articulosus.
Correlation between calmodulin-like protein, phospholipids, and growth inglucose-grown Mycobacterium phlei
Authors: Reddy P. Hemalata , Burra Sastry S. , Murthy P. Suryanarayana
Published: May 2010

In Mycobacterium phlei TMC 1548 supplementation of growth medium containing 2% v/v glycerol with glucose(up to 5% w/v) resulted in an increase in growth (yield of cells), in amount of total phospholipids, and in each of the individual phospholipids (cardiolipin, phosphatidylethanolamine, phosphatidylinositol and its mannosides, and phosphatidylglycerol). However, when the medium was supplemented with a higher concentration (7.5% w/v) of glucose, both growth and phospholipid levels decreased to near control values (2% v/v glycerol alone). Cyclic AMP levels, which decreased at all concentrations of glucose, had no relation to phospholipid content or growth. The presence of a protein that possesses the property of stimulating c-AMP phosphodiesterase activity was recently demonstrated in Mycobacterium smegmatis (Falah et al. 1988. FEMS Microbiol. Lett. 56: 89–93). In M. phlei the level of this calmodulin-like protein (assayed by radioimmunoassay) changed with different concentrations of glucose in the growth medium in a manner identical with that of phospholipids. We suggest that in mycobacteria (i) intracellular calmodulin-like protein levels are affected by glucose concentration in the growth medium and (ii) there is a positive correlation between the levels of calmodulin-like protein, total and individual phospholipids, and growth (yield of cells) in glucose-grown M. phlei. Key words: Mycobacterium phlei, calmodulin-like protein, phospholipids, growth.
Influence of metribuzin on the Rhizobium leguminosarum–lentil (Lens culinaris) symbiosis
Authors: Sprout Sharon L. , Nelson Louise M. , Germida James J.
Published: May 2010

The effects of the triazine herbicide metribuzin (Sencor) on the lentil (Lens culinaris Medic.) - Rhizobium leguminosarum biovar viciae symbiosis were studied in Leonard jars and growth pouches. Lentils inoculated with Rhizobium leguminosarum strain 128C54 or 128C84, and noninoculated lentils grown in plant nutrient solution supplemented with 5 mM KNO3, had metribuzin applied to the plants at either 8 or 13 days after planting. When sprayed at 8 days, metribuzin had a significant (p ≤ 0.05) negative effect on plant weight, number of nodules, taproot growth, and acetylene reduction activity. Five to 10 days after spraying, the plants began to recover from the inhibitory effects. When spraying was delayed to 13 days after planting, metribuzin had little effect on plant growth. The R. leguminosarum strain used as inoculant affected the degree of inhibition of lentil growth and the rate of plant recovery. Less than 0.2% of foliarly applied metribuzin was translocated to the root. Thus the detrimental effects of metribuzin application to lentils were mainly due to direct effects on the plant, which resulted in indirect effects on nodulation and nitrogen fixation. Key words: Rhizobium leguminosarum, lentil, metribuzin.
A novel shuttle vector for Streptomyces spp. and Escherichia coli as a tool in site-directed mutagenesis
Authors: Moreau A. , Paradis F. W. , Morosoli R. , Shareck F. , Kluepfel D.
Published: May 2010

This paper describes the construction and utilization of a novel shuttle vector for Streptomyces spp. and Escherichia coli as a useful vector in site-directed mutagenesis. The shuttle vector pIAFS20 (6.7 kb) has the following features: a replicon for Streptomyces spp., isolated from plasmid pIJ702; the thiostrepton-resistance gene as a selective marker in Streptomyces; the ColE1 origin, allowing replication in E. coli; and the ampicillin-resistance gene as a selective markerin E. coli. Vector pIAFS20 also contains the phage fl intergenic region, which permits production of single-stranded DNA in E. coli after superinfection with helper phage M13K07. Moreover, the lac promoter is located in front of the multiple cloning sites cassette, allowing eventual expression of the cloned genes in E. coli. After mutagenesis and screeningof the mutants in E. coli, the plasmids can be readily used to transform Streptomyces spp. As a demonstration, a 3.2-kb DNA fragment containing the gene encoding the xylanase A from Streptomyces lividans 1326 was inserted into pIAFS20, and the promoter region of this gene served as a target for site-directed mutagenesis. The two deletions reported here confirm the efficiency of this new vector as a tool in mutagenesis. Key words: shuttle vector, single-stranded DNA, site-directed mutagenesis, Streptomyces spp., Escherichia coli.
Population biology and biological control of nematodes
Authors: Jaffee B. A.
Published: May 2010

We studied the population biology of the nematophagous fungus Hirsutella rhossiliensis to understand its potential as a biological control agent. Because the fungus is an infectious and transmissible parasite, we framed our study within an epidemiological context. Field observations, theory, and experiments demonstrated that (i) parasitism of nematodes by H. rhossiliensis is dependent on nematode density, (ii) local populations of the fungus will go extinct unless supplied with some minimum number of nematodes (the host threshold density), and (iii) natural epidemics of this fungus in populations of nematodes develop slowly and only after long periods of high host density. Additional in-depth research on population biology is needed to explain other biological control systems and to guide future research. The most effective research will combine field observation, theory, and experimentation. Key words: density-dependent parasitism, host-parasite dynamics, modeling, nematophagous fungi.
Solid-phase polymerase chain reaction: applications for direct detection of enteric pathogens in waters
Authors: Toranzos Gary A. , Alvarez Abdiel J.
Published: May 2010

The techniques in current use for detection of pathogens in environmental samples are restricted to those organisms whose replication in either culture media or cell culture is feasible. These methods lack the selectivity and sensitivity necessary for their unequivocal detection and identification. We have developed an assay for the detection of bacterial cells in large volumes of water. Low concentrations of cells containing target sequences were concentrated on membrane filters and were subjected to amplification directly using a stepwise polymerase chain reaction. This procedure, together with nucleic acid probes, has enhanced the limit of detection to the level of a single bacterial cell. This technique could be used for the detection of any bacteria or virus in water or air. Key words: polymerase chain reaction, waterborne pathogens, water.
Ultrastructural localization of carbohydrate in cell walls of the entomogenous hyphomycete Nomuraea rileyi
Authors: Pendland J. C. , Boucias D. G.
Published: May 2010

Several probes were used in this ultrastructural study to localize polysaccharides in cell walls on conidial germ tubes, hyphal bodies, and mycelia of the entomogenous hyphomycete Nomuraea rileyi. With the exception of galactose, labelling patterns did not vary from one morphological stage to another. Galactose, which was localized by using a monoclonal antibody to a galactose-specific lectin purified from insect larval hemolymph, was absent from cell walls of hyphal bodies and conidia but was present on germ-tube and mycelial surfaces. Chitin (N-acetylglucosamine), labelled with a wheat-germ agglutinin-ferritin conjugate, was present in the middle regions of lateral walls and septa, and β1-4 glucans were located in the middle and inner regions, as indicated by binding of a cellulase-gold conjugate. An anti-laminaribiose antibody was used to label β1-3 glucans present in the outer wall areas and inner regions near the plasmalemma. The location of mannose residues as indicated by concanavalin A - ferritin binding was similar to that of the β1-3 glucans; vesicle-like structures were also labelled. None of the probes labelled the outer conidial pellicle or exocellular sheath surrounding germ tubes, and labelling of mycelial sheath was inconsistent. The absence of galactose from Nomuraea hyphal body walls is discussed in terms of host-parasite interaction. Key words: Nomuraea rileyi, entomopathogenic fungi, glycoconjugates, lectins, monoclonal antibodies.
Cavity disease of Agaricus bitorquis caused by Pseudomonas cepacia
Authors: Gill W. M. , Cole A. L. J.
Published: May 2010

A new bacterial disease of a cultivated mushroom, Agaricus bitorquis, is reported. Symptoms exhibited by affected sporocarps range from mild blotching to deep pitting, where large pervasive cavities extending from the cap surface to the stipe may form. Sporocarps often show a characteristic eroded appearance where the tissues are completely degraded. Some areas of diseased sporocarps fail to show any external symptoms, but on cutting the cap, massive breakdown of the underlying tissue is evident, resulting in a hollow cap. The causal bacterium, confirmed by application of Koch's postulates, is a nonfluorescent pseudomonad identified as Pseudomonas cepacia. Key words: Pseudomonas cepacia, Agaricus bitorquis, tissue degradation, cavities.