bluuebunny/crossref_metadata_2025 huggingface dataset:
Modification of reagents in the EnviroAmp™ kit to increase recovery of Legionella organisms in water
Authors: Oshiro Robin K. , Picone Teresa , Olson Betty H.
Published: February 2010

Organisms of the bacterial genus Legionella, commonly found in aqueous reservoirs, have been associated with Legionnaires' disease (legionella pneumonia, caused by Legionella pneumophila) and Pontiac fever (nonpneumonic legionellosis). EnviroAmp™ Legionella sample preparation, polymerase chain reaction amplification, and detection kits (Perkin-Elmer Corp.) were developed for rapid detection of DNA from organisms of the genus Legionella and the species L. pneumophila from environmental water samples. The kits are based on molecular techniques incorporating polymerase chain reaction amplification and detection by reverse dot blot hybridization to particular genus and species probes. The manufacturer states that the EnviroAmp™ Legionella sample preparation, polymerase chain reaction amplification, and detection kits can detect approximately 100 Legionella organisms/mL (10 000 organisms/100 mL) in the original water sample. The sensitivity of the kits was increased to 0.1 colony-forming units/mL (10 colony-forming units/100 mL), at least for cultured organisms, by modifying the EnviroAmp™ Legionella sample preparation kit protocol. Data obtained in this study indicated that sample volume could be increased from 100 to 1000 mL (in the absence of interfering substances such as humic acid) and DNA extraction volume could be decreased from 2 to 0.5 mL to increase the ability of the kit to detect lower numbers of Legionella spp. or L. pneumophila per volume.Key words: Legionella, environment, water, EnviroAmp™ kit.
Phytoalexin production by wounded white bean (Phaseolus vulgaris) cotyledons and hypocotyls in response to inoculation with rhizobacteria
Authors: Hynes R. K. , Hill J. , Reddy M. S. , Lazarovits G.
Published: February 2010

Fifteen rhizobacteria were tested for their ability to induce a hypersensitive response and phytoalexin production by wounded white bean (Phaseolus vulgaris) cotyledons and hypocotyls. Of these, nine strains of the genus Pseudomonas and two Serratia strains induced a hypersensitive response and the production of three antifungal compounds by cotyledons. Two of the antifungal compounds shared uv spectra, retention time in high-performance liquid chromatography, and Rf by thin-layer chromatography that were identical to those of thephytoalexins kievitone and phaseollinisoflavan. The hypersensitive response was rarely observed and phytoalexins were not detected in extracts from cotyledons treated with water, bacterial culture fluids, Bacillus sp., Bacillus subtilis, Corynebacterium sp., Azospirillum brasilense sp. strain 7, or the pathogen Pseudomonas syringae pv. phaseolicola. The white bean root rot pathogen Fusarium solani f.sp. phaseoli induced production of five phytoalexins by cotyledons. Three were identical in their chemical properties to phytoalexins induced by the rhizobacteria, but only F. solani induced phaseollin production. Kievitone levels from cotyledons treated with various rhizobacteria varied from 83 to 333 μg/g fresh mass, whereas in cotyledons treated with F. solani the kievitone levels averaged 850 μg/g fresh mass. In similar studies with wounded hypocotyls, only the two Serratia spp. and F. solani induced production of phytoalexins other than phaseollin.Key words: rhizobacteria, plant defense mechanisms, phytoalexins, hypersensitive response, Phaseolus vulgaris.
The proximity of mini-Mu termini can affect the products of DNA transposition
Authors: DuBow Michael S. , Lalumière Manon
Published: February 2010

Temperate bacteriophage Mu's 37 kilobase pair (kb) linear, double-stranded DNA requires DNA sequences at both termini in cis to transpose in a duplicative manner and thus propagate its genome during the lytic cycle. To define any spatial or orientation parameters between these sequences, we constructed "isomer" plasmids pMD186 and pMD861 containing identical Mu termini in close (or inverse) orientation 407 base pairs apart, or separated by 9.3 kb of plasmid pSC 101 DNA, respectively. Both mini-Mu plasmids transposed at equal frequencies onto an F′ episome in vivo, in the presence of an induced helper Mu prophage. However, while the vast majority of pMD861 mini-Mu transposition and maturation events were found to be the result of duplicative transposition (cointegrates), virtually all pMD186 transpositions yielded simple linear insertions of the mini-Mu plasmid without concomitant duplication of the mini-Mu. However, the construction of pMD186 mini-Mu plasmid derivatives, containing increased lengths of DNA sequences cloned between the ends of the Mu genome, yielded increasing frequencies of duplicative transposition products. These results suggest that the distance between the Mu termini can affect the final outcome of the transposition reaction.Key words: DNA transposition, phage Mu, cointegrates.
Expression of recombinant DNA introduced into Chlamydia trachomatis by electroporation
Authors: Tam Jeffrey E. , Davis Carolyn H. , Wyrick Priscilla B.
Published: February 2010

Electroporation was used to introduce DNA into the elementary bodies of the obligate parasitic bacterium Chlamydia trachomatis. The source of DNA for these experiments was the chimeric plasmid pPBW100, which was constructed from the well-characterized 7.5-kb plasmid of C. trachomatis and the Escherichia coli plasmid pBGS9. To select directly for C. trachomatis carrying pPBW100, an in-frame gene fusion between the chlamydial promoter P7248 and a promoterless chloramphenicol acetyltransferase (cat) cassette was incorporated into the plasmid. After infection of McCoy cells with electroporated elementary bodies containing pPBW100, the following were observed: (i) the plasmid DNA was detected inside the chloramphenicol-resistant chlamydial inclusions by in situ and Southern hybridization analyses; (ii) both physical and biochemical evidence showed that chloramphenicol acetyltransferase was synthesized by the electroporated C. trachomatis; (iii) expression of P7248::cat was developmentally regulated and occurred during the early stages of chlamydial reticulate body development; and (iv) although the expression from P7248::cat was mainly transient, there were rare instances where chloramphenicol-resistant C. trachomatis were observed after four passages.Key words: chlamydia, electroporation, chimeric plasmid, expression.
Features of the cellodextrinase gene from Fibrobacter succinogenes S85
Authors: Iyo Abiye H. , Forsberg Cecil W.
Published: February 2010

The nucleotide sequence of a 2.3-kb DNA fragment containing a cellodextrinase gene (cedA) from the ruminal anaerobe Fibrobacter succinogenes S85 was determined. Activity was expressed from this fragment when it was cloned in both orientations in pBluescript KS+ and SK−, indicating a functional F. succinogenes promoter in Escherichia coli. Promoter sequences (TTGAACA and AATAA) were identified upstream of the ATG initiation codon preceded by a putative ribosome binding site. The cedA open reading frame of 1071 base pairs encoded a protein of 357 amino acid residues with a calculated molecular mass of 41.9 kDa, similar to the40-kDa size of the native protein as determined by gel filtration chromatography. CedA is proposed to belong to family 5 (family A) of the glycosyl hydrolases. The primary structure of the cellodextrinase showed over 40% similarity with endoglucanase 3 from F. succinogenes S85. Short regions of similarity were also demonstrated with endoglucanase C from Clostridium thermocellum, CelA from Ruminococcus flavefaciens, and two exoglucanases from yeast.Key words: Fibrobacter succinogenes, cedA, cellodextrinase, sequence, rumen, gene.
Purification and some properties of aspartate aminotransferase of Amycolatopsis methanolica
Authors: Abou-Zeid A. M.
Published: February 2010

Aspartate aminotransferase (EC 2.6.1.1) from Amycolatopsis methanolica was purified and characterized. It is a dimeric protein with an overall molecular weight of approximately 90 000 with maximal activity at 70 °C and at pH 8.5. The enzyme was specific for L-aspartate as amino donor and 2-ketoglutarate as amino acceptor: the Km values for L-aspartate and 2-ketoglutarate were 1.27 and 0.38 mM, respectively.Key words: Amycolatopsis methanolica, aspartate aminotransferase, L-aspartate, 2-ketoglutarate.
Mitochondrial DNA restriction fragment length polymorphisms in field isolates of the Aspergillus niger aggregate
Authors: Varga János , Kevei Ferenc , Vriesema Aldwin , Debets Fons , Kozakiewicz Zofia , Croft James H.
Published: February 2010

The mitochondrial DNAs (mtDNAs) and the ribosomal repeat unit (ribosomal DNA, rDNA) of black Aspergillus isolates collected in various parts of the world were examined. Wide-ranging mtDNA variation was observed in natural populations of the Aspergillus niger aggregate. Most isolates were classifiable as A. niger or Aspergillus tubingensis according to their rDNA and mtDNA patterns. The mtDNA variation was distributed unevenly in the populations studied. The mtDNAs of most of the isolates collected in Australia were of the A. tubingensis type, with an unexpectedly high degree of variation, while the rDNA of these isolates exhibited the same A. tubingensis pattern as that of isolates from other locations. Some other local populations displayed very little polymorphism in their mtDNA and rDNA. Hybridization experiments in which cloned A. niger and Aspergillus nidulans mtDNA fragments were used revealed that the two main mtDNA groups corresponding to A. niger and A. tubingensis are more distantly related than concluded earlier. Six of the 13 Brazilian isolates examined exhibited mtDNA and rDNA types different from those of all the other strains and could not be classified into the above species. Classical taxonomic examination of these strains is in progress.Key words: Aspergillus niger aggregate, mitochondrial DNA, RFLP.
Physiological consequences of the S-layer of Aeromonas salmonicida in relation to growth, temperature, and outer membrane permeation
Authors: Garduño Rafael A. , Phipps Barry M. , Kay William W.
Published: February 2010

S-layers are paracrystalline protein multimers that cover the entire cell surface of many bacterial species. The presence of an S-layer in Aeromonas salmonicida (also known as A-layer) predisposed this bacterium to apparently unrelated physiological consequences: inhibition of growth at 30 °C, enhanced cell filamentation at 37 °C, and enhanced uptake of the hydrophobic antibiotics streptonigrin and chloramphenicol. Growth inhibition or enhanced filamentation was not observed when the native A-layer was missing or its arrangement altered, as in Ca2+-limited or Ca2+- and Mg2+-limited cells, in A-layer-negative (A−) cells with an artificially reconstituted A-layer, or in mutants unable to correctly assemble this layer. A-layer-positive cells (A+) were far more sensitive to the intracellularly acting antibiotics streptonigrin and chloramphenicol than were A− cells, and streptonigrin-resistant mutants were predominantly A−. Hemin, a compound known to specifically bind to the A-layer, alleviated streptonigrin toxicity to A+, but not A−, cells. As well, Ca2+- and Mg2+-limited cells, or mutants harboring A-layer defects had a reduced sensitivity to streptonigrin, and A− cells with reconstituted A-layers remained resistant to streptonigrin and chloramphenicol. Thus, the presence of a native A-layer arrangement on the cell surface, and not the mere presence of A-layer protein subunits, predisposed A. salmonicida toward the aforementioned physiological consequences. The A-layer is suggested to specifically effect these consequences, in particular the permeation of streptonigrin or chloramphenicol, by a specific interaction of A-layer subunits with the outer membrane.Key words: Aeromonas salmonicida, S-layers, physiological S-layer effects.
Cloning, molecular characterization, and expression of the genes encoding the lytic functions of lactococcal bacteriophage ϕLCE: a dual lysis system of modular design
Authors: Birkeland Nils-Kåre
Published: February 2010

The genes encoding the lysis proteins of Lactococcus lactis bacteriophage [Formula: see text] were cloned, sequenced, and expressed in Escherichia coli. The [Formula: see text] lysis genes, lysA and lysB, encode a membrane-disrupting protein (LysA) of 88 amino acids, and a cell wall degrading protein (LysB) of 429 amino acids, which shares significant sequence similarity with lysins from the Streptococcus pneumoniae phages Cp-1, Cp-7, and Cp-9, and Lactobacillus delbrueckii phage mv 1. Both LysA and LysB function in E. coli, as judged by lysis of the E. coli host cells and by lytic activity against lactococcal cells when the cloned lysA and lysB genes are expressed. The LysA protein possesses two putative transmembrane helices and highly charged N- and C-termini, and is structurally similar to phage holins that are known to induce lesions in the inner membrane through which phage endolysin can be released to its cell wall substrate. The C-terminal end of LysB contains two highly homologous sequence repeats of 43 amino acids. The LysB repeats show strong sequence similarity to repeats found in lytic enzymes from other Gram-positive bacteria and from Bacillus subtilis phage [Formula: see text] and PZA, as well as in some functionally unrelated proteins, and they are possibly involved in binding of the enzyme to the cell wall substrate. The organization of the dual [Formula: see text] lysis system supports earlier suggestions that exchange of modular units is an important principle in protein evolution.Key words: amino acid sequence repeats, bacteriophage [Formula: see text], holin, Lactococcus lactis, lysin, lytic enzyme.
Characterization of a Tn551-mutant of Staphylococcus aureus defective in the production of several exoproteins
Authors: Giraudo Ana T. , Raspanti Claudia G. , Calzolari Aldo , Nagel Rosa
Published: February 2010

A Tn551 insertional pleiotropic mutant defective in the production of several exoproteins was isolated from Staphylococcus aureus 196E and characterized. The pleiotropism of the mutant was due to a single insertion of the transposon as evidenced by Southern blot hybridization and by the transfer of its phenotype by transduction to S. aureus ISP479. The mutants showed diminished or null levels of α- and β-hemolysins, DNase, coagulase, and protein A in the supernatants of broth cultures. Production of proteases, lipase, staphylokinase, or enterotoxin A was not modified. The mutants did synthesize the cell-bound form of protein A and also the extracellular form of this protein coded by pRIT11, which lacks the COOH-terminal segment of the molecule. These observations suggest that the sae locus does not involve a positive regulatory gene acting at the transcriptional level. The phenotype of the mutant was different from that of other insertional mutants affecting exoprotein synthesis, such as agr, xpr, or sar. This new mutation has been designated sae (for S. aureus exoprotein expression).Key words: S. aureus, transpositional mutant, exoprotein production, pleiotropism, protein A.
Binding of germanium and lead to Pseudomonas putida lipopolysaccharides
Authors: Kłapcińska Barbara
Published: February 2010

The binding of germanium and lead ions to lipopolysaccharide isolated from Pseudomonas putida ATCC 33015 was studied by high-resolution 31P nuclear magnetic resonance spectroscopy. Chemical shift changes toward higher field as well as broadening of all phosphorus resonances were observed with added lead. Addition of germanium to the lipopolysaccharide sample resulted in more pronounced chemical shift changes toward lower field. The effects of binding of germanium and lead ions on the 31P nuclear magnetic resonance spectrum of lipopolysaccharide imply that the glycosidic monophosphate joined to the reducing end of N-acetylglucosamine (C-1 position of α-D-G1cNI) as well as β-phosphate groups of glycosidically linked diphosphates of the same residue of the lipid A moiety provide high-affinity metal-binding sites.Key words: germanium, lead, lipopolisaccharide (LPS), binding, Pseudomonas putida.
Hypersensitivity of Rhodobacter sphaeroides ribosomes to protein synthesis inhibitors: structural and functional implications
Authors: Sánchez E. , Teixidó J. , Guerrero R. , Amils R.
Published: February 2010

The elongation cycle of protein synthesis systems of purple nonsulfur photosynthetic bacteria Rhodobacter sphaeroides, grown both phototrophically and chemotrophically, was studied using 33 inhibitors with different chemical structures and functional and domain specificities. No functional differences between phototrophic and chemotrophic ribosomal systems were detected. Rhodobacter sphaeroides ribosomes exhibited strong hypersensitivity to nine functional inhibitors when compared with Escherichia coli ribosomes. Most of the R. sphaeroides ribosomal hypersensitivities corresponded to peptidyltransferase inhibitors, implying that this important functional neighborhood must be somehow different in the two organisms.Key words: protein synthesis inhibitors, ribosomal function, peptidyltransferase, photosynthetic bacteria.
Motility behavior of Azospirillum species in response to aromatic compounds
Authors: Lopez-de-Victoria Geralyne , Fielder Donald R. , Zimmer-Faust Richard K. , Lovell Charles R.
Published: February 2010

The motility of Azospirillum lipoferum Sp59b and Azospirillum brasilense Sp7 in the presence of the aromatic compounds benzoate, protocatechuate, 4-hydroxybenzoic acid, and catechol was studied using computer-assisted video motion analysis. Increases in swimming speed with concomitant decreases in the rate of change in direction and increases in path linearity are indicative of bacterial chemoattraction. With the exception of 4-hydroxybenzoic acid, the aromatic compounds tested altered the motility patterns of both species, consistent with a chemoattraction mechanism. 4-Hydroxybenzoic acid elicited significant behavioral changes only in A. brasilense. The highest speeds were recorded for most compounds at concentrations between 1 and 10 mM for A. lipoferum and at lower concentrations for A. brasilense. Catechol, supplied at a 10 mM concentration, reduced the swimming speed of A. brasilense but not of A. lipoferum. The distinct differences in swimming behavior observed between these two species of Azospirillum in response to aromatic compounds may be of ecological significance in soils.Key words: Azospirillum spp., motility, video motion analysis, aromatic compounds.
Effects of environmental factors present in the St. Lawrence Estuary (Quebec, Canada) on experimental survival of Salmonella salamae as determined by flow cytometry
Authors: Monfort Patrick , Baleux Bernard
Published: February 2010

Survival of Salmonella salamae in the St. Lawrence Estuary was studied experimentally during an oceanographic cruise using in situ exposure diffusion chambers. The abundance distribution (colony-forming units) of culturable S. salamae on media was compared with the distribution of cells enumerated by flow cytometry. Flow cytometry was also used to characterize the size distribution and DNA content of cells exposed to various environmental factors. Solar radiation, starvation, and a gradual increase in salinity led to an abrupt loss of the ability of S. salamae cells to form cultures and to a gradual reduction in the cell size and DNA content. Conversely, starvation combined with a gradual increase in salinity in the absence of sunlight led to a gradual loss of the cells' ability to form cultures and an abrupt reduction in cell size and DNA content (i.e., a rapid increase in cell damage). Mortality (i.e., a decrease in total cell count) of S. salamae placed in darkness began at a lower salinity (11.4‰) than did the mortality of cells exposed to sunlight (23.1‰). Therefore, the S. salamae cells exposed to sunlight seemed to be more resistant to gradual salinity stress than the cells that were not subjected to sunlight.Key words: flow cytometry, survival, Salmonella, St. Lawrence Estuary.
Metabolism of trinitrobenzene by a Pseudomonas consortium
Authors: Boopathy Ramaraj , Manning John , Montemagno Carlo , Rimkus Kris
Published: February 2010

The metabolism of trinitrobenzene by a Pseudomonas consortium was studied. The Pseudomonas consortium used trinitrobenzene as a sole source of nitrogen, but not as a sole source of carbon. Trinitrobenzene was metabolized within 60 h of incubation. The main intermediates produced were dinitroaniline, 1,5-dinitrobenzene, nitroaniline, 5-nitrobenzene, and ammonia. The ammonia concentration in the culture medium increased during the course of incubation. Nearly stoichiometric amounts of 1,5-dinitrobenzene and 5-nitrobenzene were produced by the consortium. During trinitrobenzene metabolism by this bacterial consortium, the trinitrobenzene was first reduced to an amino compound, dinitroaniline. This intermediate was reductively deaminated with the release of ammonia into the culture medium and production of 1,5-dinitrobenzene. By the same mechanism, 1,5-dinitrobenzene was further converted to 5-nitrobenzene, which was not metabolized further, even after 60 days of incubation. This pathway is believed to be novel in that an aerobic bacterial consortium uses the nitroaromatic compound as its nitrogen source but leaves the ring intact. The bacterial consortium studied could be used in a syntrophic culture system with other 5-nitrobenzene-degrading bacteria to remove trinitrobenzene completely from soil and water at contaminated sites.Key words: trinitrobenzene, aniline, nitrobenzene, biodegradation, transformation.
The osmotic lysis of spiroplasma cells and its use in enzyme studies
Authors: Chen Jianchi , Chang C. J.
Published: February 2010

A simple and efficient osmotic lysis method was developed for enzyme studies in spiroplasmas. Log phase cells in R2 medium were harvested by centrifugation (19 600 × g for 30 min). Wash buffer supplemented with 0.23 M sucrose maintained the helicity of spiroplasma cells during washing. Osmotic lysis of spiroplasmas was achieved in H buffer that contained no sucrose. Sucrose at concentrations as low as 0.004 M dramatically increased the resistance of the spiroplasmas to osmotic lysis. NADH oxidase, lactate dehydrogenase, and malate dehydrogenase were detected in cell lysates of Spiroplasma floricola (23-6), Spiroplasma citri (R8A2), Spiroplasma apis (SR 3), and Spiroplasma melliferum (AS 576). Citrate synthase, aconitase, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, succinyl coenzyme A synthetase, succinate dehydrogenase, and fumarase were not detected in cell lysates of S. floricola (23-6). NADH oxidase and malate dehydrogenase were found in the cytosol whereas lactate dehydrogenase was loosely associated with the cytomembrane.Key words: spiroplasmas, osmotic lysis, tricarboxylic acid cycle, enzymes.
Mineralization of alachlor by lignin-degrading fungi
Authors: Ferrey Mark L. , Koskinen William C. , Blanchette Robert A. , Burnes Todd A.
Published: February 2010

White rot fungi were able to mineralize the aromatic ring carbon of alachlor to CO2. After 122 days, 14 and 12% of the alachlor that was initially present in malt extract cultures supplemented with a wood substrate was mineralized at room temperature by Ceriporiopsis subvermispora and Phlebia tremellosa, respectively. Although Phanerochaete chrysosporium mineralized alachlor at 25 °C, it did so more slowly than the other two white rot fungi. The brown rot fungus Fomitopsis pinicola did not mineralize alachlor.Key words: alachlor, mineralization, white rot fungi, pesticide.
Characterization of Aeromonas genomic species by using quinone, polyamine, and fatty acid patterns
Authors: Kämpfer Peter , Blasczyk Klaus , Auling Georg
Published: February 2010

A chemotaxonomic study was carried out on representative strains of 13 Aeromonas genomic species. Quinone, polyamine, and fatty acid patterns were found to be very useful for an improved characterization of the genus and an improved differentiation from members of the families Enterobacteriaceae and Vibrionaceae. The Q-8-benzoquinone was the predominant ubiquinone, and putrescine and diaminopropane were the major poly amines of the genus. The fatty acid patterns of 181 strains, all characterized by DNA–DNA hybridization, showed a great homogeneity within the genus, with major amounts of hexadecanoic acid (16:0), hexadecenoic acid (16:1), and octadecenoic acid (18:1), and minor amounts of the hydroxylated fatty acids (3-OH 13:0, 2-OH 14:0, 3-OH 14:0) in addition to some iso and anteiso branched fatty acids (i-13:0, i-17:1, i-17:0, and a-17:0). Although some differences in fatty acid profiles between the genomic species could be observed, a clearcut differentiation of all species was not possible.Key words: Aeromonas, polyamines, quinones, fatty acids, differentiation.
Effect of cultural and nutritional conditions on the control of flocculation expression in Saccharomyces cerevisiae
Authors: Soares Eduardo V. , Teixeira José A. , Mota Manuel
Published: February 2010

The effect of cultural (temperature and pH) and nutritional conditions (nitrogen and carbon source) on the flocculation expression of three strains was studied. The strains' flocculation ability was determined by placing the cells in a stationary phase of growth in standard flocculation conditions. The flocculation ability of strain NCYC 1195, recently classified in the literature as the NewFlo phenotype, was more sensitive to growth temperature than Flo1 phenotype strains (NCYC 869 and NRRL Y265). The initial pH of the culture medium did not affect the flocculation ability of Flo1 phenotype strains but in the case of strain NCYC 1195 flocculation was repressed when the initial pH of the culture medium was below 3.5. Flocculation in strain NCYC 1195 was also repressed in defined culture medium; this inhibition was not related to a deficiency in any particular nitrogen source, but rather to the poor buffering capacity of the defined medium. All strains showed strong flocculation when grown in glucose, but were nonflocculent in glycerol. It was clearly demonstrated that the phenotypic expression of flocculation could be induced or repressed by changing cultural and nutritional conditions. Two distinct behaviours were also displayed with regard to the effect of the cultural conditions upon flocculation, namely the effect of pH. These different behaviours can be used to distinguish the two flocculation phenotypes.Key words: Saccharomyces cerevisiae, flocculation, growth temperature, pH, nitrogen source.
Presqualene metabolism in two species of Lagenidium
Authors: Gallagher Patricia A. , Warner Steven A. , Domnas Aristotle J.
Published: February 2010

The fate of precursors of the isoprenoid pathway was studied in the sterol auxotroph Lagenidium giganteum and in the positive control organism Lagenidium callinectes. Acetate derived from glucose and mevalonic acid was converted to sterols and fatty acids in L. callinectes. Lagenidium giganteum converted mevalonic acid to sterols and fatty acids, but glucose-derived acetate was not utilized for sterol synthesis. The results showed that the defect in the isoprenoid pathway of L. giganteum occurs at the level of the β-hydroxy-β-methylglutarylcoenzyme A reductase–synthase complex. Various aspects of this defect are discussed relative to metabolism of the organism.Key words: Lagenidium giganteum, Lagenidium callinectes, glucose, mevalonic acid utilization, fatty acids, sterol production.
Cryptococcus neoformans infection can elicit protective antibodies in mice
Authors: Mukherjee Jean , Scharff Matthew D. , Casadevall Arturo
Published: February 2010

An IgG1 monoclonal antibody generated from a mouse infected with Cryptococcus neoformans modified the course of intravenous cryptococcal infection in A/J mice by prolonging survival and reducing lung fungal burden, brain mass, and serum polysaccharide levels. The results demonstrate that C. neoformans infection can elicit useful antibodies.Key words: Cryptococcus neoformans, monoclonal antibody, antibody immunity.
L-Rhamnose metabolism in Pichia stipitis and Debaryomyces polymorphus
Authors: Twerdochlib A. L. , Pedrosa F. O. , Funayama S. , Rigo L. U.
Published: February 2010

The pathway for the breakdown of L-rhamnose by the yeast Pichia stipitis NRC 5568 was shown to involve nonphosphorylated intermediates and to produce pyruvate and L-lactaldehyde. The activities of the enzymes and the nature of several intermediates were determined. The enzymes involved are L-rhamnose dehydrogenase, L-rhamnonate dehydratase, and 2-keto-3-deoxy-L-rhamnonate aldolase. This pathway was found to be inducible by L-rhamnose and repressed by D-glucose. These enzymes were also present in a mutant of P. stipitis (PR1) resistant to catabolite repression and in Debaryomyces polymorphus 1747. Cell-free extracts of P. stipitis and D. polymorphus grown in L-rhamnose as sole carbon source were found to contain NAD+-dependent aldehyde dehydrogenase activities.Key words: Pichia stipitis, enzyme induction, oxidative pathway, catabolite repression.
The effect of the nitrogen source and ntrC on the adenylylation of glutamine synthetase I in Rhizobium etli
Authors: Noguez Raúl , Moreno Soledad , Gúzman Josefina , Espín Guadalupe
Published: February 2010

Regulation of the adenylylation of glutamine synthetase I in Rhizobium etli was found to be different from that in enteric bacteria. Adenylylation was high in cells growing in low concentrations of ammonia and was not affected by an excess of ammonia. Adenylylation was dependent on NtrC, since strain CFN2012 carrying a ntrC::Tn5 insertion mutation showed low levels of adenylylation. This effect was not due to the lack of glutamine synthetase II caused by the ntrC mutation, because strain SM88, which lacks glutamine synthetase II activity owing to a glnII mutation, showed levels of adenylylation similar to those of the wild type.Key words: Rhizobium glutamine synthetase, nitrogen regulation, adenylylation.
Detection of soluble methane monooxygenase producing Methylosinus trichosporium OB3b by polymerase chain reaction
Authors: Mohan K. S. , Walia Satish K.
Published: February 2010

The soluble methane monooxygenase (sMMO) enzyme complex of methanotrophs cometabolizes haloaliphatic compounds such as trichloroethylene. Two 18-mer oligonucleotides as primary primers and a nested primer of the same length were selected to amplify specific DNA sequences of the sMMO gene cluster using polymerase chain reaction (PCR). Two DNA fragments of sizes 270 and 400 base pairs were obtained when purified DNA from the methanotroph Methylosinus trichosporium OB3b was used as template. The primers were specific for sMMO sequences of M. trichosporium, since none of the 13 bacterial isolates screened yielded the expected length of PCR-amplified DNA fragments. The detection limit of the PCR method was 5 × 102 cells of M. trichosporium. The sMMO sequences were successfully amplified in groundwater (containing native microbial population) when seeded with M. trichosporium, FP1 sense (5′-ATGTCCAGCGCTCATAAC-3′), RP1 antisense (5′-TCAGATGTCGGTCAGGGC-3′), FP2 sense nested (5′GCCATCATCGGTCAGGGC-3′), and FP2 sense nested (5′-GCCATCATCGAGGACATC-3′).Key words: detection, soluble methane monooxygenase, polymerase chain reaction, Methylosinus trichosporium OB3b.
The survival and recovery of Pseudomonas aeruginosa and its effect upon salmonellae in water:methodology to test bottled water in Canada
Authors: Warburton Donald W. , Bowen Bruce , Konkle Anne
Published: February 2010

Methodology used to support changes to the Regulations for bottled water in the Food and Drugs Act of Canada, which include criteria for Pseudomonas aeruginosa (0 colony-forming units/100 mL of water), was used to assess the survival of P. aeruginosa in inoculated bottled water. The effects of P. aeruginosa on the survival of Salmonella spp. in bottled water were also investigated. The methodology used in the isolation included the use of hydrophobic grid membrane filters, a resuscitation step on tryptic soy agar, and selective plating on P. aeruginosa selective agar for P. aeruginosa and on xylose lysine desoxycholate agar for salmonellae. Pseudomonas aeruginosa and salmonellae proliferated and survived in inoculated water for up to 100days or longer. Pseudomonas aeruginosa had a synergistic effect on the survival of salmonellae, enabling them to survive for more than 140 days in double distilled water.Key words: Pseudomonas aeruginosa, salmonellae, bottled water, methodology.
Production of multiple δ-endotoxins by Bacillus thuringiensis: δ-endotoxins produced by strains of the subspecies galleriae and wuhanensis
Authors: Chestukhina Galina G. , Kostina Lyubov I. , Zalunin Igor A. , Revina Lyudmila P. , Mikhailova Alla L. , Stepanov Valentin M.
Published: February 2010

A method was developed to assess the number of δ-endotoxins contained in Bacillus thuringiensis entomocidal crystals. It utilized proteolytic conversion of 130-kDa protoxin into 60- to 65-kDa "true" toxin via limited proteolysis with trypsin and separation of stable N-terminal domains by fast-performance liquid chromatography. Immunodiffusion experiments and N-terminal sequence determination (applied to the major component isolated by SDS-PAGE) completed the analysis of the crystal protein composition. The application of this approach to crystals produced by cells of B. thuringiensis subsp. galleriae and wuhanensis allowed us to identify at least seven and eight different δ-endotoxins, respectively. Among those δ-endotoxins assigned to previously described families, CryIA, CryID, Cry IF, and CryIG were found, as well as crystal proteins, which possess N-terminal amino acid sequences very different from those of all known δ-endotoxins. Possible functional consequences of δ-endotoxin multiplicity are discussed.Key words: Bacillus thuringiensis, δ-endotoxins, multiplicity of crystal proteins, separation of true toxins.
Cloning of promoter-like sequences from Lactobacillus paracasei subsp. paracasei CG11 and their expression in Escherichia coli, Lactococcus lactis, and Lactobacillus reuteri
Authors: Djordjevic Gordana , Bojovic Bojana , Banina Ana , Topisirovic Ljubisa
Published: February 2010

Fragments of chromosomal DNA from Lactobacillus paracasei subsp. paracasei CG11 (formerly Lactobacillus casei CG11) capable of functioning as promoters were isolated using the broad host range, promoter-probe vector pGKV210. Five such fragments designated P61, P79, P80, P116, and P144 were completely sequenced and analyzed. Fragment P61 had the highest transcriptional efficiency in Escherichia coli and Lactobacillus reuteri whereas P80 was the most active in Lactococcus lactis. In general, the orders of the transcriptional strengths were almost identical in E. coli and Lactobacillus reuteri but different from that in Lactococcus lactis. Mapping of the 5′ end of cat mRNA showed that different regions of fragments P79 and P144 were used as promoters in Lactococcus lactis than in E. coli and Lactobacillus reuteri. Analysis of these DNA sequences revealed that the putative −35 and −10 hexanucleotides resembled those of E. coli, Bacillus subtilis, and lactococci. The spacing between these two hexanucleotides and between the putative −10 hexanucleotide and the transcriptional start point (A residues predominated) ranged from 17 to 18 base pairs and from 5 to 7 base pairs, respectively. Each of the cloned Lactobacillus paracasei CG11 promoter-like fragments contained an AT-rich sequence upstream of the putative −35 region (from 60 to 73%).Key words: Lactobacillus, promoter-like sequences, transcriptional efficiency, translational efficiency.
On the thermodynamics of autotrophic and heterotrophic growth of Pseudomonas saccharophila
Authors: Battley Edwin H.
Published: February 2010

Calculations are made of the thermodynamic changes accompanying the growth of Pseudomonas saccharophila autotrophically on hydrogen and heterotrophically on acetic acid, pyruvic acid, lactic acid, glucose, and sucrose, for the purpose of determining the extent to which the energetics of autotrophic and heterotrophic growth are related. Growth with respect to each substrate is divided into equations representing the processes of anabolism and catabolism, the sum of which is an equation representing metabolism. Using appropriate values for the thermodynamic properties of the substances represented by the terms in the equations, it becomes possible to calculate the changes in free energy, enthalpy, and entropy accompanying these processes. The results suggest, but do not rigorously prove, that the quantity of available electrons conserved within the substance of the cells is linearly related to those potentially available from the electron donors, whether these latter are organic or inorganic. The efficiency of electron conservation can be used to calculate the changes in free energy, enthalpy, and entropy accompanying metabolism.Key words: bacterial growth thermodynamics, electron conservation.
Levels and identities of nonrhizobial microorganisms found in commercial legume inoculant made with nonsterile peat carrier
Authors: Olsen Perry E. , Rice Wendell A. , Bordeleau Lucien M. , Demidoff A. H. , Collins Mandy M.
Published: February 2010

Sixty samples of commercial North American legume inoculants manufactured for sale in 1994 using nonsterile peat as carrier were tested for Rhizobium (or Bradyrhizobium) content and nonRhizobium biological contaminant load. Products of three major producers of such inoculants for sale in Canada were examined. Viable Rhizobium content varied from 5.6 × 105 to 8.1 × 109 cells/g, while the contaminant load varied from 1.8 × 108 to 5.5 × 1010 cfu/g. Most of the inoculants contained more nonrhizobial organisms than they did rhizobia. Identifications were made of the most numerous nonrhizobial bacteria occurring in 100 samples of inoculants collected in 1993 and 1994. The most commonly identified contaminant was Xanthomonas maltophilia. Pseudomonas aeruginosa, Klebsiella pneumoniae, and Enterobacter cloacae were also found at high levels in some products. Contaminant organisms capable of inhibiting rhizobial growth in plate culture were found in the products of all three manufacturers.Key words: Rhizobium, contaminant, inoculant.
Azospirilluminoculation in pregerminating wheat seeds
Authors: Creus Cecilia M. , Sueldo Rolando J. , Barassi Carlos A.
Published: February 2010

Azospirillum cells were inoculated in pregerminating wheat during seed imbibition. Surface-sterilized seeds of Triticum aestivum cv. Buck Pucará were sequentially soaked for 3 h in water and 3 h in the inoculum of 3 × 108Azospirillum brasilense Sp 245 cells∙mL−1, to allow bacteria to enter during imbibition. Germination and seedling growth were accomplished in sterile distilled water at 20 °C, in the dark. To compare with more traditional methods based on plant–Azospirillum colonization after germination, seedlings from noninoculated seeds were inoculated in parallel by immersing roots in the same inoculum, for the same period of time. Autoclaved inocula were used as controls in all cases. We observed about 5 × 108Azospirillum cells∙g−1fresh weight in 11-day-old wheat seedlings inoculated before or after seed germination. However, roots from seed-inoculated seedlings had higher both bacterial concentration and length. On the other hand, seeds inoculated during imbibition and dried to 14% water content retained 3.7 × 106viable cells∙g−1dry weight up to 27 days. Moreover, seeds stored for 30 days were not only able to germinate but also to harbor over 106cells∙g−1fresh weight in roots after 7 days growth. Here we present the possibility of obtaining in a simple and inexpensive way, seeds containing high numbers of viable Azospirillum cells, which could avoid the use of external carriers or adhesives.Key words: Azospirillum, wheat, inoculation.
Assessment of the biodegradation potential of psychrotrophic microorganisms
Authors: Whyte Lyle G. , Greer Charles W. , Inniss William E.
Published: February 2010

Bioremediation of polluted temperate and cold temperature environments may require the activity of psychrotrophic bacteria, because their low temperature growth range parallels the ambient temperatures encountered in these environments. In the present study, 135 psychrotrophic microorganisms isolated from a variety of ecosystems in Canada were examined for their ability to mineralize14C-labelled toluene, naphthalene, dodecane, hexadecane, 2-chlorobiphenyl, and pentachlorophenol. A number of the psychrotrophic strains mineralized toluene, naphthalene, dodecane, and hexadecane. None of the psychrotrophs were capable of mineralizing 2-chlorobiphenyl or pentachlorophenol. Those strains demonstrating mineralization activity were subsequently screened by the polymerase chain reaction (PCR) and Southern hybridization of PCR products for the presence of catabolic genes (alkB, ndoB, todC1, and xylE) involved in known bacterial biodegradative pathways for these compounds. Some of the psychrotrophs able to mineralize toluene and naphthalene possessed catabolic genes that hybridized to xylE or todC1, and ndoB, respectively. The alkB PCR fragments obtained from the strains that mineralized dodecane and hexadecane did not hybridize to an alkB gene probe derived from Pseudomonas oleovorans. Psychrotrophic strain Q15, identified as a Rhodococcus sp., also mineralized the C28n-paraffin octacosane. A gene probe constructed from the "alkB" PCR fragment from strain Q15 did hybridize with the alkB PCR fragments from most of the psychrotrophic alkane biodegraders, indicating that the alkB primers may be amplifying another gene(s), perhaps with low homology to P. oleovorans alkB, which may be involved in the biodegradation of both short chain (dodecane) and longer chain alkanes (hexadecane, octacosane). All of the psychrotrophic biodegradative isolates examined were capable of mineralization activity at both 23 and 5 °C, indicating their potential for low temperature bioremediation of petroleum hydrocarbon contaminated sites.Key words: psychrotrophic microorganisms, biodegradation, catabolic gene probes, organic pollutants.
Mycoparasitic species ofTrichodermaproduce lectins
Authors: Neethling Diane , Nevalainen Helena
Published: February 2010

Culture filtrates and mycelial extracts of two mycoparasitic Trichoderma species were tested for the presence of lectins, by haemagglutination with human and marsupial erythrocytes. In Trichoderma viride, haemagglutinating activity was present in both mycelial extracts and culture filtrate. While secreted lectins were only detected after 6 days of growth, the presence of mycelium-associated lectins was first noted in 3-day-old cultures. Agglutinating activity was also demonstrated in the mycelium of 6-, 9- and 13-day-old cultures of Trichoderma harzianum. In this species, however, lectins were not secreted. In all instances, haemagglutination was inhibited by N-acetylgalactosamine and related sugars. This is the first report on the occurrence of lectins in Trichoderma spp.Key words: Trichoderma, lectins, mycoparasitism.
Isolation and modulation of growth of a colonization-impaired strain of Enterobacter cloacae in cucumber spermosphere
Authors: Roberts D. P , Marty A. M. , Dery P. D. , Hartung J. S.
Published: February 2010

Enterobacter cloacae A-46 was isolated for use in an environmental containment strategy for genetically modified derivative strains with enhanced biocontrol activity. The population of E. cloacae A-46, a transposon mutant of the plant-beneficial bacterium E. cloacae 501R3, increased 10-fold (significant increase at P ≤ 0.05) in cucumber spermosphere when applied to cucumber seeds along with casamino acids. In contrast, strain A-46 was incapable of proliferation in cucumber spermosphere in the absence of casamino acids. Populations of strain A-46 also failed to increase in corn, cowpea, sunflower, and wheat spermospheres in the absence of casamino acids, while populations of strain 501R3 increased 3162-, 512-, 1698-, and 93-fold, respectively. In addition, the persistence of strain A-46 in corn, cucumber, and sunflower rhizospheres and in natural soil was greatly reduced compared with the parental strain 501R3.Key words: Enterobacter cloacae, environmental containment, spermosphere, proliferation, biological control.
Bacterial biosynthesis of indole-3-acetic acid
Authors: Patten Cheryl L. , Glick Bernard R.
Published: February 2010

Production of the phytohormone indole-3-acetic acid (IAA) is widespread among bacteria that inhabit the rhizosphere of plants. Several different IAA biosynthesis pathways are used by these bacteria, with a single bacterial strain sometimes containing more than one pathway. The level of expression of IAA depends on the biosynthesis pathway; the location of the genes involved, either on chromosomal or plasmid DNA, and their regulatory sequences; and the presence of enzymes that can convert active, free IAA into an inactive, conjugated form. The role of bacterial IAA in the stimulation of plant growth and phytopathogenesis is considered.Key words: auxin, indoleacetic acid, microbial, biosynthesis, pathways.
An endo-β-1,4-glucanase gene (celA) from the rumen anaerobe Ruminococcus albus 8: cloning, sequencing, and transcriptional analysis
Authors: Attwood Graeme T. , Herrera Felicitas , Weissenstein Lee A. , White Bryan A.
Published: February 2010

A genomic library of Ruminococcus albus 8 DNA was constructed in Escherichia coli using bacteriophage λZapII. This library was screened for cellulase components and several Ostazin brilliant red/carboxymethyl cellulose positive clones were isolated. All of these clones contained a common 3.4-kb insert, which was recovered as a plasmid by helper phage excision. The carboxymethyl cellulase coding region was localized to a 1.4-kb region of DNA by nested deletions, and a clone containing the entire celA gene was sequenced. Analysis of the sequence revealed a 1231-bp open reading frame, coding for a protein of 411 amino acids with a predicted molecular weight of 45 747. This protein, designated CelA, showed extensive homology with family 5 endoglucanases by both primary amino acid sequence alignment and hydrophobic cluster analysis. Cell-free extracts of E. coli containing the celA clone demonstrated activity against carboxymethyl cellulose and acid swollen cellulose but not against any of the p-nitrophenol glycosides tested, indicating an endo-β-1,4-glucanase type of activity. In vitro transcription–translation experiments showed that three proteins of 48 000, 44 000, and 23 000 molecular weight were produced by clones containing the celA gene. Northern analysis of RNA extracted from R. albus 8 grown on cellulose indicated a celA transcript of approximately 2700 bases, whereas when R. albus 8 was grown on cellobiose, celA transcripts of approximately 3000 and 600 bases were detected. Primer extension analysis of these RNAs revealed different transcription initiation sites for the celA gene when cells were grown with cellulose or cellobiose as the carbon source. These two sites differed by 370 bases in distance. A model, based on transcription and sequence data, is proposed for celA regulation.Key words: Ruminococcus albus, endoglucanase, transcription, hydrophobic cluster analysis.