bluuebunny/crossref_metadata_2025 huggingface dataset:
Staphylococcus aureusisogenic mutant, deficient in toxic shock syndrome toxin-1 but not staphylococcal enterotoxin A production, exhibits attenuated virulence in a tampon-associated vaginal infection model of toxic shock syndrome
Authors: De Boer Monica L , Kum Winnie WS , Chow Anthony W
Published: April 2011

Since menstrual toxic shock syndrome (MTSS) is associated with a predominant clone of Staphylococcus aureus which produces both toxic shock syndrome toxin-1 (TSST-1) and staphylococcal enterotoxin A (SEA), we sought to clarify the role of TSST-1 in a tampon-associated vaginal infection model in New Zealand White (NZW) rabbits, using isogenic tst+/sea+S. aureus mutants in which tst was inactivated by allelic replacement. Rabbits infected with the tst-/sea+strain became ill within 3 days, with fever, weight loss, conjunctival hyperemia, and lethargy. Mortality was significantly higher with the tst+/sea+strain compared to its tst-/sea+isogenic derivative (4/13 vs. 0/14; p < 0.05, Fisher's exact test, 2-tailed). Mean fever index was higher (p < 0.005; t test, 2-tailed) and weight loss more sustained among survivors in the tst+/sea+group. Furthermore, culture filtrates from the tst+/sea+strain induced a significantly greater response in mitogenesis and TNFalpha secretion from rabbit splenocytes in vitro compared to the tst-/sea+isogenic derivative. Thus, regardless of the role of SEA, TSST-1 significantly contributed to both morbidity and mortality in this tampon-associated vaginal infection model in NZW rabbits. This is the first demonstration of the potential role of TSST-1 and SEA in the pathogenesis of MTSS with a MTSS-associated clinical S. aureus strain in a relevant animal model.Key words: toxic-shock syndrome toxin-1, superantigens, rabbit model.
Microbial mineralization of diisopropanolamine
Authors: Gieg Lisa M , Coy Debora L , Fedorak Phillip M
Published: April 2011

Diisopropanolamine (DIPA) is a "sweetening agent" used to remove hydrogen sulfide from sour natural gas, and it is a contaminant at some sour gas treatment facilities in western Canada. To investigate the biodegradation of this alkanolamine,14C-DIPA was used in anaerobic and aerobic mineralization studies. Between 3 and 78% of the radioactivity from this compound was released as14CO2in sediment-enrichment cultures incubated under nitrate-reducing conditions. Similarly, 12-78% of the label was converted to14CO2in sediment-enrichment cultures incubated under Mn(IV)-reducing conditions. These activities were observed at 8°C, a typical groundwater temperature in western Canada, and at 28°C. In contrast, DIPA-degrading activity was difficult to sustain under Fe(III)-reducing conditions, and <25% of the radioactive label from14C-DIPA was liberated as14CO2. Two mixed cultures and two isolates (both irregular, non-sporeforming, Gram-positive rods) were used to assess aerobic mineralization of14C-DIPA. The aerobic mixed cultures released 73 and 79% of the radioactive label as14CO2, whereas the pure cultures liberated only 39 and 47% as14CO2. Between one-third and one-half of the nitrogen from DIPA was found as ammonium-N in aerobic batch cultures. These results clearly demonstrate that DIPA is mineralized under a variety of incubation conditions.Key words: alkanolamine, biodegradation, diisopropanolamine, mineralization, natural gas.
Cometabolic oxidation of phenanthrene to phenanthrenetrans-9,10-dihydrodiol byMycobacteriumstrain S1 growing on anthracene in the presence of phenanthrene
Authors: Tongpim Saowanit , Pickard Michael A
Published: April 2011

Mycobacterium strain S1, originally described as Rhodococcus strain S1 by chemotaxonomic criteria, was isolated by growth on anthracene, and is unable to use any of nine other polycyclic aromatic compounds as carbon source. Metabolism of phenanthrene during growth on anthracene as sole carbon source results in the accumulation of traces of a dihydrodiol metabolite in the growth medium, which, by comparison with authentic standards, has been tentatively identified as phenanthrene trans-9,10-dihydrodiol. Anthracene metabolites were ruled out on the basis of comparisons with authentic anthracene dihydrodiols from Pseudomonas fluorescens D1 and chemically synthesized anthrols. The original source of phenanthrene for dihydrodiol production was phenanthrene present as a <1% contaminant in the anthracene used as carbon source. However, addition of further phenanthrene to the anthracene growth medium increased the level of phenanthrene trans-9,10-dihydrodiol formed. Mycobacterium strain S1 also produced phenanthrene trans-9,10-dihydrodiol when grown in a glucose-salts medium in the presence of phenanthrene. This dihydrodiol is a dead-end metabolite, and neither it nor its parent hydrocarbon are able to support the growth of Mycobacterium strain S1. Studies with metyrapone and ancimidol, which did not inhibit growth on anthracene but did inhibit formation of phenanthrene trans-9,10-dihydrodiol, suggest it is likely the product of a cytochrome P450 monooxygenase-like activity.Key words: phenanthrene trans-9,10-dihydrodiol, Mycobacterium.
Clues to the origin of high external invertase activity in immobilized growing yeast: prolongedSUC2transcription and less susceptibility of the enzyme to endogenous proteolysis
Authors: Alteriis Elisabetta de , Alepuz Paula M , Estruch Francisco , Parascandola Palma
Published: April 2011

Expression of the SUC2 gene encoding invertase was studied using free and gelatin-immobilized yeast cells to try to explain the high activity of this enzyme exhibited by immobilized cells when allowed to grow in a nutrient medium. The results indicated that at least two factors are probably responsible for the accumulation of invertase in immobilized cells. First, the expression of the SUC2 gene was maintained throughout growth in immobilized cells, whereas its expression was only transient in free cells. Second, invertase of immobilized cells was shown to be less susceptible to endogenous proteolytic attack than that of the corresponding free cells. These results have been interpreted, respectively, in terms of diffusional limitations and changes in the pattern of invertase glycosylation due to growth of yeast in an immobilized state.Key words: immobilization, invertase, yeast, proteases.
Characterization ofBacillus thuringiensissubsp.kurstakistrain S93 effective against the fall armywormSpodoptera frugiperda)
Authors: Silva-Werneck Joseilde O , De-Souza Marlene T , de S. Dias José MC , Ribeiro Bergmann M
Published: April 2011

A Brazilian strain of Bacillus thuringiensis subsp. kurstaki, designated S93, was analyzed regarding its cry gene and protein contents and activity against the fall armyworm (Spodoptera frugiperda, Smith 1797). Bioassays using lyophilized powders of S93 or HD-1 and third instar larvae of S. frugiperda showed a 12.3-fold lower LC50for the S93 strain when compared with the standard HD-1 strain. The spore-crystal mixture, analyzed by SDS-PAGE, showed two major polypeptides of 130 and 65 kDa, corresponding to Cry1 and Cry2 toxins, respectively. Western blot analysis showed that these proteins were immunologically related to the Cry1A protein from B. thuringiensis subsp. kurstaki HD-73. The polymerase chain reaction technique (PCR) using total DNA from the S93 strain and specific primers showed the presence of cry1Aa, cry1Ab, and cry1Ac genes, and a cry1A-type gene was localized in a plasmid of about 44 MDa. A cry1Ab gene was isolated from a S93 plasmid DNA library and completely sequenced. Computer analysis showed that the gene sequence (GenBank acession number AF059670) is identical to cry1Ab1 and has 91.6 and 85.9% identity with cry1Aa1 and cry1Ac1 genes, respectively. The deduced amino-acid sequence showed a high degree of similarity with the amino-acid sequences of the Cry1Ab1 (100%), Cry1Aa1 (93.8%), and Cry1Ac1 (90.6%) proteins.Key words: Bacillus thuringiensis, Spodoptera frugiperda, biological control, crystal protein, cry genes.
Nutrient uptake byCandida albicans: the influence of cell surface mannoproteins
Authors: Braun Phyllis C
Published: April 2011

Numerous ultrastructural and biochemical analyses have been performed to characterize the cell wall composition and structure of Candida albicans. However, little investigation has focused on how subtle differences in cell wall structure influence the intracellular transport of amino acids and monosaccharides. In this study C. albicans 4918 and ATCC 10231 were grown in culture conditions capable of modifying surface mannoproteins and induced surface hydrophobic or hydrophilic yeast cell wall states. Subcultures of these hydrophobic and hydrophilic yeasts were subsequently incubated with one of seven L-[3H] amino acids: glycine, leucine, proline, serine, aspartic acid, lysine, or arginine. The transport of [3H] mannose and [3H] N-acetyl-D-glucosamine were also investigated. This study revealed significant strain differences (P [Formula: see text] 0.05) between hydrophilic and hydrophobic yeast transport of these nutrients throughout a 2 h incubation. Hydrophilic cultures of 4918 and ATCC 10231 transported nearly two times more (pmol mg-1dry weight) proline, mannose, and N-acetyl-D-glucosamine than hydrophobic yeast. Hydrophobic cultures preferentially incorporated serine and aspartic acid in both these strains. Strain variation was indicated with the transport of leucine, lysine, and arginine, as follows: experiments showed that hydrophilic 4918 cultures selectively transported leucine, lysine, and arginine, whereas, the hydrophobic ATCC 10231 cultures incorporated these amino acids.Key words: Candida albicans, mannoproteins, amino acid transport.
Conjugative transposition of Tn916 and Tn925 in Bacillus popilliae
Authors: Dingman Douglas W
Published: April 2011

Interspecies transfer of the conjugative transposons Tn916 and Tn925 into B. popilliae Pj1 occurred using Enterococcus faecalis and Bacillus subtilis CU4049 as transposon donors. Tn916 was stably maintained in B. popilliae Pj1 following growth without selective pressure and was successfully introduced into the plasmid-containing B. popilliae strains NRRL B-2524, Ch1, and KLN4 using E. faecalis CG110. In B. popilliae, expression of the tetracycline resistant determinants on Tn916 and Tn925 provided resistance to 25 μg/mL and 50 μg/mL tetracycline, respectively. An erythromycin resistant determinant, present in Tn916ΔE, was also functional in B. popilliae Pj1 and provided resistance to 1 mg/mL erythromycin. Transfer of Tn916 into E. faecalis, B. subtilis, and between B. popilliae strains was accomplished using a transposon-containing strain of B. popilliae as donor. Efforts to transfer Tn916 between E. coli and B. popilliae were unsuccessful. Key words: Bacillus popilliae, milky disease, Tn916, conjugative transposon.
Improved establishment and development of three cactus species inoculated withAzospirillum brasilensetransplanted into disturbed urban desert soil
Authors: Bashan Yoav , Rojas Adriana , Puente M Esther
Published: April 2011

Survival and development of cactus transplants in urban, disturbed areas of the desert near La Paz, Baja California Sur, Mexico, was monitored. Young plants of three species of pachycereid cacti (Pachycereus pringlei, Stenocereus thurberi, and Lophocereus schottii) inoculated with the plant growth promoting bacterium Azospirillum brasilense in an eroded area (a dirt road) had a high survival rate and developed more rapidly compared with uninoculated control plants during a 3.5-year period after transplantation. Soil erosion in the inoculated experimental area diminished. Small, but significant soil accumulated in association with the growth of cactus roots into the wind-deposited dust. One demonstrated mechanism for stabilizing dust was by the upward growth of small roots during the rainy season into the deposited dust. Azospirillum brasilense survived well in the rhizospheres of these cacti for 2 years, but not in root-free soil. This study demonstrated the feasibility of using bacterial inoculation of cacti to enhance their establishment in disturbed areas, with the potential to stabilize soil.Key words: Azospirillum, beneficial bacteria, cactus, plant inoculation, plant growth promoting bacteria, PGPR, soil erosion, soil reclamation.
Fine structure of the extracellular sheath and cell walls inOphiostoma novo-ulmigrowing on various substrates
Authors: Ouellette G B , Chamberland H , Goulet A , Lachapelle M , Lafontaine J -G
Published: April 2011

The presence of microfilamentous-like structures of tubular appearance (MFS) in cell walls and extracellular sheath material (ES) in a number of isolates of Ophiostoma novo-ulmi Brasier grown on various substrates and following various treatments is reported. Standard fixation or high-pressure freezing methods were used, and cytochemical tests were carried out to detect fungal and host wall components and, in some cases, fungal DNA. In some cases, serial 0.2-μm-thick sections were examined at 120 kV and tilted to obtain stereoscopic images. Whether the fungal cell walls were thick and composed of an outer opaque and inner more electron-lucent layers, or thin and barely perceptible, MFS were observed to extend from the cell cytoplasm as parallel structures across the walls into the surrounding medium, including host cell components in infected elm tissues. MFS were associated (in samples from inoculated trees) with cleavage and desquamation of fungal walls. ES and MFS did not label for cellulose or chitin, but generally labelled slightly for β-(1-3)-glucan and mannose, and strongly for galactose. Only the lucent, inner fungal wall layer labelled for chitin and cellulose. DNA labelling was confined to nuclei and mitochondria in fungal cells from cultures on agar medium; in cells from cultures on millipore membranes, it was pronounced over imprecisely delimited cell regions. The possible ontogeny of MFS components and their importance are discussed. Key words: chitin, Dutch elm disease, fungal fimbriae, fungal walls, gold-complexed probes, microfilamentous structures (MFS).
Biosynthesis of trehalose from maltooligosaccharides in Rhizobia
Authors: Streeter John G , Bhagwat Arvind
Published: April 2011

Previously, the enzymes for trehalose synthesis that are present in Escherichia coli were demonstrated in Bradyrhizobium japonicum and B. elkanii. An alternative mechanism recently reported for the synthesis of trehalose from maltooligosaccharides was considered based on the fact that high concentrations of sugars in liquid culture stimulated the accumulation of trehalose. An assay for the synthesis of trehalose from maltooligosaccharides using crude, gel-filtered protein preparations was developed. Analysis of a variety of the Rhizobiaceae indicates that the "maltooligosaccharide mechanism" is present in B. japonicum, B. elkanii, Rhizobium sp. NGR234, Sinorhizobium meliloti, R. tropici A, R. leguminosarum bv viciae, R. l. bv trifolii, and Azorhizobium caulinodans. Synthesis of trehalose from maltooligosaccharide could not be detected in R. tropici B or R. etli. With these two exceptions, it is suggested that rhizobia have two mechanisms for the biosynthesis of trehalose.Key words: maltooligosyl trehalose synthase, maltooligosyl trehalose trehalohydrolase, symbiotic nitrogen fixation.
Insertional inactivation of theListeria monocytogenescheYA operon abolishes response to oxygen gradients and reduces the number of flagella
Authors: Flanary Paul L , Allen Richard D , Dons Lone , Kathariou Sophia
Published: April 2011

The nucleotide sequence of a region downstream of the Listeria monocytogenes flagellin gene, flaA, revealed two putative chemotaxis genes, cheY and cheA. These genes have been shown to be transcribed as a bicistronic unit. In this study Tn916ΔE mutagenesis was used to generate two mutants, PF10 and PF16, which contain transposon inserts in the promoter region of this operon. These mutants were motile in liquid, but had reduced flagellin expression and were unable to burrow or swarm on soft agar plates. Complementation of the single transposon-copy mutant PF16 with cloned cheY and cheA in trans partially restored microaerotaxis and swarming on soft agar. The complemented strain did not exhibit any increase in flagellin production. Both PF10 and PF16 appear deficient in their ability to attach to the mouse fibroblast cell line 3T3.Key words: Listeria, motility, flagella, chemotaxis.
Salmonella landau as a live vaccine against Escherichia coli O157:H7 investigated in a mouse model of intestinal colonization
Authors: Conlan J Wayne , KuoLee Rhonda , Webb Ann , Perry Malcolm B
Published: April 2011

The present study was performed to assess the potential of a humoral mucosal immune response directed against the O157 antigen of Escherichia coli O157:H7 to prevent intestinal colonization by the pathogen. To this end, mice were gavaged with inocula of Salmonella landau, a Salmonella strain that naturally expresses the O157 antigen. Salmonella landau was avirulent for mice. Despite this, mice exposed to S. landau developed high titres of serum and coproantibodies against the O157 antigen. These mice, compared with controls, demonstrated some ability to resist transient intestinal colonization by an oral inoculum of an isolate of E. coli O157:H7. These findings suggest that a local immune response directed against the O157 antigen might increase host resistance to this pathogen.Key words: Salmonella landau, Escherichia coli O157:H7, mucosal immunity, mice.
Bacterial surface antigen-specific monoclonal antibodies used to detect beer spoilage pediococci
Authors: Whiting Michael S , Ingledew W M , Lee Sun Y , Ziola Barry
Published: April 2011

Fourteen monoclonal antibodies (Mabs) were isolated that react with surface antigens of Pediococcus beer spoilage organisms, including P. damnosus, P. pentosaceous, P. acidilactici, and unspeciated isolates. Immunoblotting, enzyme immunoassays (EIAs) of protease- and neuraminidase-treated surface antigen extracts, carbohydrate competition EIAs, and cardiolipin EIAs were used to characterize the bacterial antigens involved in Mab binding. Antigen stability in situ was tested by protease treatment or surface antigen extraction of washed bacteria. In most cases, the Mabs bind to Pediococcus surface antigens that appear to be covalently bound cell wall polymers resistant to alteration or removal from the bacterial surface. These bacterial surface antigen reactive Mabs show good potential for rapid, sensitive, and specific immunoassay detection of Pediococcus beer spoilage organisms.Key words: beer spoilage organism, immunoassay, monoclonal antibodies, Pediococcus, surface antigens.
Application of the GUS marker gene technique to high-throughput screening of rhizobial competition
Authors: Wilson Kate J , Parra Adriana , Botero Lina
Published: April 2011

The GUS marker gene system has been developed for the study of bacterial ecology, particularly rhizobial competition. For high-throughput field screening of rhizobial competition, the technique must be robust and reliable under diverse conditions, with diverse cultivars and strains. Here we demonstrate its applicability to the evaluation of competition on five different Phaseolus vulgaris cultivars with 10 different Rhizobium strains. We describe refinements of the GUS assay, which make it more affordable and applicable to field-based studies, and use the assay to examine the effect of sample size on the accuracy of nodule occupancy measurements.Key words: GUS gene, Rhizobium, rhizobial competition, microbial ecology.
Survival ofCryptosporidium parvumoocysts in source separated human urine
Authors: Höglund Caroline E , Stenström Thor Axel B
Published: April 2011

The survival of Cryptosporidium parvum in source separated urine was investigated as part of a broader study on microbial risks associated with the reuse of human urine for sustainable agriculture. A dye permeability assay and in vitro excystation were the primary methods used to assess viability. In the collected urine most of the nitrogen is present as ammonia and the pH is generally around 9. Parallel investigations were made in buffers to compare possible toxic effects of urine to actual pH effects. Oocysts in the untreated urine were inactivated below the detection limit (1/300) within 63 days. This inactivation rate was significantly higher (P < 0.01) than in urine adjusted to pH 5 or 7 according to the dye permeability assay. The corresponding difference between different pH values was not seen in buffers, suggesting that the antiprotozoan effect of urine was mediated by other factors besides pH. The Swedish practice of storing urine for six months before its use thus appears satisfactory for the inactivation of Cryptosporidium oocysts.Key words: Cryptosporidium parvum, oocysts, human urine, survival, source separation.
Purification and characterization of 2-ethoxyphenol-induced cytochrome P450 fromCorynebacteriumsp. strain EP1
Authors: Kawahara Naoko , Ikatsu Hisayoshi , Kawata Hiroshige , Miyoshi Shin-ichi , Tomochika Ken-ichi , Sinoda Sumio
Published: April 2011

A soluble cytochrome P450 (P450EP1A) induced by 2-ethoxyphenol was purified to apparent homogeneity from Corynebacterium sp. strain EP1. The P450EP1Ashowed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular weight of about 45 kDa. The CO-reduced difference spectra of P450EP1Ahad a Soret maximum at 447.6 nm. The substrate difference spectra with 2-ethoxyphenol showed an absorption maximum at 394.0 nm. The purified P450EP1Adegraded 2-ethoxyphenol in an assay system composed of spinach ferredoxin-NADP+oxidoreductase and NADPH. The reaction activity decreased to 1.4% of its original activity by addition of CO. The existence of catechol in the reaction mixture was confirmed after the metabolic reaction, indicating that P450EP1Acatalyzes O-dealkylation of 2-ethoxyphenol. In addition to 2-ethoxyphenol, the P450EP1Ametabolized 2-methoxyphenol, 1,1,1-trichloroethane, carbon tetrachloride, benzene, and toluene.Key words: cytochrome P450, Corynebacterium sp., 2-ethoxyphenol, enzyme purification, biodegradation.
Transformation of aliphatic and aromatic nitriles by a nitrilase fromPseudomonassp.
Authors: Dhillon Jasvinder , Chhatre Suneel , Shanker Rishi , Shivaraman N
Published: April 2011

A Pseudomonas sp. (S1) isolated from a garden soil possessed a unique nitrilase, which is capable of catalyzing the direct hydrolysis of both potassium and organic cyanides to their corresponding carboxylic acids and ammonia, without the formation of amide as an intermediate. The nitrilase was purified with 4.8% recovery in three steps from a cell extract of the strain. The relative mobility of the homogenous enzyme preparation in SDS and native polyacrylamide gels indicated molecular weight of 41 kDa, approximately. Pseudomonas sp. (S1) utilized all the nitriles as carbon and nitrogen sources. The enzyme was induced by both aliphatic and aromatic nitriles, while the aliphatic olefinic nitrile - acrylonitrile was the most suitable substrate. The nitrilase also catalyzed the hydrolysis of acetonitrile, adiponitrile, benzonitrile, butyronitrile, glutaronitrile, phenylacetonitrile, succinodinitrile, and potassium cyanide, with the formation of ammonia and the corresponding carboxylic acids. The Michaelis-Menten constant, Km, of the partially purified nitrilase for acetonitrile, acrylonitrile, adiponitrile, benzonitrile, and potassium cyanide presented values of 11.26, 5.88, 10.28, 12.27, and 0.75 mM, respectively.Key words: nitriles, enzyme kinetics, nitrilase, partial purification, Pseudomonas sp.
Effect of carbon source, growth and temperature on the expression of thesecgenes ofStreptomyces lividans1326
Authors: Morosoli Rolf , Ostiguy Stéphane , Dupont Claude
Published: April 2011

The mRNA level in sec genes of Streptomyces lividans was studied as a function of growth temperature, glucose effect, and growth using two different carbon sources. Glucose and xylan, a complex hemicellulose, were used as carbon sources for the growth of S. lividans. For both substrates, the mRNA levels of secA, secD, secE, secF, and secY genes were almost constant during the early and log phases, but showed a marked decrease at the beginning of the stationary phase followed by a full recovery of mRNA level in the late stationary phase. This indicates that the sec genes are actively transcribed during the differentiation process. The mRNA level in xylan was generally from 1.5- to 2-fold that in glucose. At growth temperatures of 28°C, 34°C, or 40°C, there was no significant difference in the sec gene mRNA levels.Key words: Streptomyces lividans, sec genes, glucose repression, growth-phase dependent expression.
Conjugal transfer of a TOL-like plasmid and extension of the catabolic potential ofPseudomonas putidaF1
Authors: Hallier-Soulier S , Ducrocq V , Truffaut N
Published: April 2011

Strain mX was isolated from a petrol-contaminated soil, after enrichment on minimal medium with 0.5% (v/v) meta-xylene as a sole carbon source. The strain was tentatively characterized as Pseudomonas putida and harboured a large plasmid (pMX) containing xyl genes involved in toluene or meta-xylene degradation pathways via an alkyl monooxygenase and a catechol 2,3-dioxygenase. This new TOL-like plasmid was stable over two hundred generations and was self-transferable. After conjugal transfer to P. putida F1, which possesses the Tod chromosomal toluene biodegradative pathway, the transconjugant P. putida F1(pMX) was able to grow on benzene, toluene, meta-xylene, para-xylene, and ethylbenzene compounds as the sole carbon sources. Catechol 2,3-dioxygenases of the transconjugant cells presented a more relaxed substrate specificity than those of parental cells (strain mX and P. putida F1).Key words: biodegradation, conjugative transfer, toluene, xylene, Pseudomonas.
Relationships between fungal spore morphologies and surface properties for entomopathogenic members of the general Beauveria, Metarhizium, Paecilomyces,Tolypocladium, and Verticillium
Authors: Jeffs Lloyd B , Xavier Ilungo J , Matai Russell E , Khachatourians George G
Published: April 2011

The surface properties of aerial conidia (AC) from 24 strains of entomopathogenic fungi were studied and compared using the salt-mediated aggregation and sedimentation (SAS) assay, electron microscopy, FITC-labelled lectins, and spore dimensions. Spores with rugose surfaces were hydrophobic, whereas hydrophilic spores had smooth surfaces. Correlation analysis found no link between spore dimensions and either hydrophobicity or surface carbohydrates. However, there was a strong positive correlation between spore hydrophobicity and surface carbohydrates. The three spore types of Beauveria bassiana were all shown to possess discrete surface hydrophobicities, which were also strongly linked to surface carbohydrate profiles. Various chemical treatments had pronounced effects on spore surface properties, with sodium dodecyl sulfate (SDS) and formic acid (FA) reducing both lectin binding and surface hydrophobicity. When FA-protein extracts were separated and analysed using SDS-PAGE, only the hydrophobic spores had low molecular weight hydrophobin-like peptides that were unglycosylated and contained disulfide bonds. The strains with hydrophilic AC had much lower levels of FA-extractable protein per spore dry weight compared to their more hydrophobic counterparts. Moreover, extracts of the more hydrophobic spores tended to have greater protein:carbohydrate ratios.Key words: fungi, spores, hydrophobicity, lectins, morphology, microbial insecticides, protein.
Expression ofptxRand its effect ontoxAandregAexpression during the growth cycle ofPseudomonas aeruginosastrain PAO1
Authors: Colmer Jane A , Hamood Abdul N
Published: April 2011

The expression of the toxA and regA genes in Pseudomonas aeruginosa is negatively regulated by iron at the transcriptional level. We have previously described ptxR, an exotoxin A regulatory gene which appears to enhance toxA expression through regA. In this study, we have tried to determine if ptxR expression correlates with its effect on toxA and regA expression throughout the growth cycle of P. aeruginosa strain PAO1. This was done using Northern blot hybridization experiments (with toxA, regA, and ptxR probes), and ptxR transcriptional fusion studies. To avoid problems related to the presence of multiple copies of ptxR in PAO1, we have constructed a PAO1 strain (PAO1-XR) that carries only two ptxR genes in its chromosome. Our results showed that when PAO1-XR was grown in iron-limited conditions, the increase in exotoxin A activity and the accumulation of toxA mRNA appeared at about mid- to late-exponential phase. A similar increase in the accumulation of regA mRNA was detected. Both regA transcripts, T1 and T2, were enhanced in PAO1-XR. In iron-sufficient medium, neither toxA nor regA mRNA was detected at any time point in the growth cycle of PAO1-XR. In contrast, the accumulation of ptxR mRNA was detected throughout the growth cycle of PAO1-XR under both iron-deficient and iron-sufficient conditions. The presence of iron in the growth medium also had no effect on the level of β-galactosidase activity produced by a ptxR-lacZ fusion in PAO1. These results suggest that (i) the enhancement in toxA expression by ptxR correlates with the enhancement in regA expression; (ii) ptxR affects the expression of the regA P1 and P2 promoters; (iii) ptxR expression precedes its effect on toxA and regA expression; and (iv) unlike toxA and regA, the overall expression of ptxR throughout the growth cycle of PAO1 is not negatively regulated by iron.Key words: ptxR, differential expression, transcriptional regulation, regA, toxA.
Growth kinetics ofEscherichia coliwith galactose and several other sugars in carbon-limited chemostat culture
Authors: Lendenmann Urs , Snozzi Mario , Egli Thomas
Published: April 2011

Kinetic models for microbial growth describe the specific growth rate (μ) as a function of the concentration of the growth-limiting nutrient (s) and a set of parameters. A typical example is the model proposed by Monod, where μ is related to s using substrate affinity (Ks) and the maximum specific growth rate (μmax). The preferred method to determine such parameters is to grow microorganisms in continuous culture and to measure the concentration of the growth-limiting substrate as a function of the dilution rate. However, owing to the lack of analytical methods to quantify sugars in the microgram per litre range, it has not been possible to investigate the growth kinetics of Escherichia coli in chemostat culture. Using an HPLC method able to determine steady-state concentrations of reducing sugars, we previously have shown that the Monod model adequately describes glucose-limited growth of E. coli ML30. This has not been confirmed for any other sugar. Therefore, we carried out a similar study with galactose and found steady-state concentrations between 18 and 840 μg·L-1for dilution rates between 0.2 and 0.8·h-1, respectively. With these data the parameters of several models giving the specific growth rate as a function of the substrate concentration were estimated by nonlinear parameter estimation, and subsequently, the models were evaluated statistically. From all equations tested, the Monod model described the data best. The parameters for galactose utilisation were μmax= 0.75·h-1and Ks= 67 μg·L-1. The results indicated that accurate Ksvalues can be estimated from a limited set of steady-state data when employing μmaxmeasured during balanced growth in batch culture. This simplified procedure was applied for maltose, ribose, and fructose. For growth of E. coli with these sugars, μmaxand Kswere for maltose 0.87·h-1, 100 μg·L-1; for ribose 0.57·h-1, 132 μg·L-1, and for fructose 0.70·h-1, 125 μg·L-1. Key words: monod model, continuous culture, galactose, glucose, fructose, maltose, ribose.
PCR cloning, heterologous expression, and characterization of isopenicillin N synthase fromStreptomyces lipmaniiNRRL 3584
Authors: Loke Paxton , Ng Chee Pang , Sim Tiow-Suan
Published: April 2011

A key step which involves the cyclization of δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine to the bicyclic ring structure of isopenicillin N in the penicillin and cephalosporin biosynthetic pathway, is catalyzed by isopenicillin N synthase (IPNS). In this study, an IPNS gene from Streptomyces lipmanii NRRL 3584 (slIPNS) was cloned via PCR-based homology cloning, sequenced and expressed in Escherichia coli. Soluble slIPNS was overexpressed up to 21% of total soluble protein, and verified to be functionally active when in an IPNS enzymatic assay. Sequence comparison of the slIPNS gene obtained (excluding the consensus primer sequences) with another cloned IPNS from S. lipmanii 16884.3, revealed one three-nucleotide deletion and three closely-spaced single nucleotide deletions. Futhermore, this paper also reports the first instance of the usage of PCR as an alternative and rapid strategy for IPNS cloning using consensus primers. Key words: isopenicillin N synthase, β-lactam antibiotics, secondary metabolism, consensus primers.
Biodegradation of carbazole byRalstoniasp. RJGII.123 isolated from a hydrocarbon contaminated soil
Authors: Schneider Joanne , Grosser Robert J , Jayasimhulu Koka , Xue Weiling , Kinkle Brian , Warshawsky David
Published: April 2011

The use of microorganisms for bioremediation of contaminated soils may be enhanced with an understanding of the pathways involved in their degradation of hazardous compounds. Ralstonia sp. strain RJGII.123 was isolated from soil located at a former coal gasification plant, based on its ability to mineralize carbazole, a three-ring N-heterocyclic pollutant. Experiments were carried out with strain RJGII.123 and14C-carbazole (2 mg/L and 500 mg/L) as the sole organic carbon source. At 15 days, 80% of the 2 mg/L carbazole was recovered as CO2, and <1% remained as undegraded carbazole, while 24% of the 500 mg/L carbazole was recovered as CO2and ~70% remained as undegraded carbazole. Several stable intermediates were formed during this time. These intermediates were separated by high performance liquid chromatography (HPLC) and were characterized using high resolution mass spectroscopy (HR-MS) and gas chromatography - mass spectroscopy (GC-MS). At least 10 ring cleavage products of carbazole degradation were identified; four of these were confirmed as anthranilic acid, indole-2-carboxylic acid, indole-3-carboxylic acid, and (1H)-4-quinolinone by comparison with standards. These data indicate that strain RJGII.123 shares aspects of carbazole degradation with previously described Pseudomonas spp., and may be useful in facilitating the bioremediation of NHA from contaminated soils.Key words: mineralization, biodegradation, N-heterocyclic aromatic hydrocarbons, carbazole.
Aspects of the pathology and etiology of 'drippy gill' disease of the cultivated mushroom Agaricus bisporus
Authors: Gill Warwick , Cole Tony
Published: April 2011

Agaricus bisporus sporocarps exhibiting characteristic 'drippy gill' symptoms from a natural outbreak were examined. Discrete bacterial droplets on the hymenial lamellae often coalesced to form ribbons of bacterial ooze. Longitudinal splits on the stipe were lined with a similar bacterial ooze. Bacteria isolated from both the hymenium and stipe were identified as Pseudomonas agarici, and were confirmed to be the causal organism by satisfying Koch's postulates. By light and transmission electron microscopy, the causal bacteria were found to colonize the extrahyphal spaces and degrade the extracellular matrix within affected sporocarps. Degradation of the extracellular matrix was shown to reduce the integrity of the sporocarp, and result in stipe splitting and hymenium disruption. In artificial inoculations of the pileus, bacteria were shown to exist predominantly in sporocarp tissue below the point of inoculation and above affected areas of the hymenium, indicating an approximately vertical passage through the sporocarp via the extracellular spaces. The dissolution of the extracellular matrix, and the observed failure of the bacterium to produce a toxin active against A. bisporus, allow the bacteria to pass through protective membranes unnoticed, and infect the stipe and hymenium prior to veil break. These observations dispel previous assumptions of intrahyphal existence and transmission. In the few instances in which the bacteria were observed to be intrahyphal, the host fungal cell wall was often broken, suggesting intrahyphal existence was opportunistic rather than obligatory. The taxonomic position of a bacterium isolated previously from sporocarps exhibiting symptoms similar to those of drippy gill was determined by examining the biochemical and nutritional profiles of the bacterium, and comparing them with other Pseudomonas agarici isolates.Key words: Agaricus bisporus, drippy gill, extracellular, intracellular, Pseudomonas agarici.
Type I nitroreductases in soil enterobacteria reduce TNT (2,4,6-trinitrotoluene) and RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine)
Authors: Kitts Christopher L , Green Chad E , Otley Rebecca A , Alvarez Marc A , Unkefer Pat J
Published: April 2011

Many enteric bacteria express a type I oxygen-insensitive nitroreductase, which reduces nitro groups on many different nitroaromatic compounds under aerobic conditions. Enzymatic reduction of nitramines was also documented in enteric bacteria under anaerobic conditions. This study indicates that nitramine reduction in enteric bacteria is carried out by the type I, or oxygen-insensitive nitroreductase, rather than a type II enzyme. The enteric bacterium Morganella morganii strain B2 with documented hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) nitroreductase activity, and Enterobacter cloacae strain 96-3 with documented 2,4,6-trinitrotoluene (TNT) nitroreductase activity, were used here to show that the explosives TNT and RDX were both reduced by a type I nitroreductase. Morganella morganii and E. cloacae exhibited RDX and TNT nitroreductase activities in whole cell assays. Type I nitroreductase, purified from E. cloacae, oxidized NADPH with TNT or RDX as substrate. When expression of the E. cloacae type I nitroreductase gene was induced in an Escherichia coli strain carrying a plasmid, a simultaneous increase in TNT and RDX nitroreductase activities was observed. In addition, neither TNT nor RDX nitroreductase activity was detected in nitrofurazone-resistant mutants of M. morganii. We conclude that a type I nitroreductase present in these two enteric bacteria was responsible for the nitroreduction of both types of explosive.Key words: nitroreductase, TNT, RDX, Enterobacter cloacae, Morganella morganii.
Differential transport properties of D-leucine and L-leucine in the archaeon,Halobacterium salinarum
Authors: Tanaka Mikiei , Mukohata Yasuo , Yuasa Seiji
Published: April 2011

The transport of D-leucine was compared with that of L-leucine in Halobacterium salinarum. When a high-outside/low-inside Na+gradient was imposed, D-leucine as well as L-leucine accumulated in envelope vesicles, supporting the hypothesis that D-leucine is transported via a symport system along with Na+. Kinetic analyses, including inhibition experiments, indicated that both enantiomers are transported via a common carrier. However, a Hill plot indicated a single binding site for Na+during L-leucine transport, but dual binding sites for Na+during D-leucine transport. Furthermore, D-leucine transport was dependent on electrical membrane potential, suggesting that a transporter bound with D-leucine is positively charged. L-leucine transport was slightly, if at all, dependent on membrane potential, suggesting that a transporter bound with L-leucine is electrically neutral. These results indicate that the leucine carrier in Halobacterium salinarum translocates two moles of Na+per mole of D-leucine, and one mole of Na+per mole of L-leucine.Key words: D-leucine, sodium ion-dependent transport, stoichiometry, stereospecific recognition, halophilic archaea.
Introduction of theSerratia marcescens chiAgene into an endophyticPseudomonas fluorescensfor the biocontrol of phytopathogenic fungi
Authors: Downing Katrina J , Thomson Jennifer A
Published: April 2011

An endophytic strain of Pseudomonas fluorescens was isolated from micropropagated apple plantlets and introduced into beans (Phaseolus vulgaris) via their root tips. It was shown to be present as an endophyte in the roots at a level of 1.2 × 105CFU/g fresh weight. The gene coding for the major chitinase of Serratia marcescens, chiA, was cloned under the control of the tac promoter into the broad-host-range plasmid pKT240 and the integration vector pJFF350. Pseudomonas fluorescens carrying tacchiA either on the plasmid or integrated into the chromosome is an effective biocontrol agent of the phytopathogenic fungus Rhizoctonia solani on bean seedlings under plant growth chamber conditions.Key words: endophyte, biological control, chitinase.
Early outplanting performance of nutrient-loaded containerized black spruce seedlings inoculated withLaccaria bicolor: a bioassay study
Authors: Quoreshi A M , Timmer V R
Published: April 2011

Early growth potential of nutrient-loaded and (or) Laccaria bicolor (R. Mre.) Orton inoculated (Picea mariana (Mill.) BSP) seedlings was investigated using pot bioassays retrieved from a low-competition feathermoss site and a high competition hardwood-Alnus site in the boreal forest. Mycorrhizal seedlings were similar in biomass and shoot/root ratio to nonmycorrhizal seedlings at planting but significantly higher in nutrient content depending on fertilization regime and loading rate. After transplanting, both nutrient-loaded and inoculated seedlings outperformed conventional seedlings, increasing dry matter production by 20-49% with loading and by 45-92% with combined treatments. Nutrient uptake followed similar trends, increasing N, P, and K uptake by 80-124, 89-129, and 72-106%, respectively, for combined treatments compared with conventional seedlings, demonstrating the advantage of both nutrient loading and inoculation in early plantation establishment. Seedling response was greater on the feathermoss site, presumably because of less competition for nutrients and light. Vector diagnosis indicated the response was associated with a primary limitation of N and P that was alleviated by nutrient loading and mycorrhizal inoculation, particularly when treatments were combined. A strong correlation between preplant N content and outplant biomass suggests that initial nutritional status is a better criterion for predicting stock quality than traditional morphological parameters of seedlings.
Growth response of spruce saplings in relation to climatic conditions along a gradient of gap size
Authors: Drobyshev Igor , Nihlgård Bengt
Published: April 2011

To evaluate the relative importance of climatic factors and the level of natural canopy disturbance on sapling growth rates, terminal shoot increment of Norway spruce (Picea abies (L.) Karst.) saplings was analyzed in old-growth Sphagnum-Myrtillus forests of the European southern boreal zone (Tver Region, Russia). For a 5-year period, terminal increments were retrospectively measured in 225 saplings in a range of naturally created canopy gaps. Climatic variability was estimated by Seljaninov hydrothermal coefficient. Variation in the growth rate was partitioned as (i) annual variation observed within a particular sapling over a 5-year period ("within-stem variation," WSV) and (ii) variation of 5-year cumulative height increments within a particular location ("within-location variation," WLV). Sapling growth was positively related to gap size and, except when under canopy location, with the height of the saplings. For the growth, differences in sapling location along a gradient of gap sizes were more important than annual dynamics of water availability. Impact of the annual climatic variability was less pronounced in large gap, compared with other locations. Absolute values of WSV and WLV were similar under an intact canopy. WSV had a tendency to decrease in greater gaps, whereas WLV variation increased.
Spatiotemporal variation in black spruce cone and seed crops along a boreal forest - tree line transect
Authors: Sirois Luc
Published: April 2011

To assess the relationship between the regenerative potential of black spruce (Picea mariana (Mill.) BSP) and the latitudinal and thermal gradients, the cone crop was monitored in the same selection of trees during the 1989-1995 period in the northern boreal forest (sites A, n = 49, and B, n = 48), in the southern forest-tundra transition zone (site C, n = 35), and at the tree line (site D, n = 21). The size of the cone crop, the amount of seeds extracted per cone, along with the percentages of filled seed and germination were measured on each tree. There was no south to north trend associated with the cone crop. The cone crop at tree line was not significantly lower than in either of the southerly sites in six of the seven observed years. The number of seeds extracted per cone, the percentage of filled seeds, and the germination of filled seeds showed significant decrease northward according to year. Although there was no significant relationship between temperature and the cone production over the study area, the percentages of filled seeds and germination were significantly (0.51 [Formula: see text] r2 [Formula: see text] 0.44; p < 0.001) associated with the regional variation in heat sum.
Molybdenum at German Norway spruce sites: contents and mobility
Authors: Lang Friederike , Kaupenjohann Martin
Published: April 2011

Molybdenum plays an important role in the nitrogen turnover of ecosystems. However, very little is known about Mo availability in forest soils. We measured the oxalate-extractable Mo concentrations of acid forest soils, the Mo, nitrate, phosphate, and sulfate fluxes from the organic forest floor into the mineral soil using resin tubes and the Mo concentrations of the tree needles at 28 different Norway spruce (Picea abies (L.) Karst.) sites in southern Germany. The supply of oxalate-extractable Mo varied from 51 to 3400 g·ha-1, with the lowest values occurring in sandstone-derived soils (370 ± 212 g·ha-1; mean ± SD). Molybdenum concentrations of current-year needles were in the range of 5 to 48 ng·g-1. The Mo needle concentrations and oxalate-extractable Mo of soils did not correlate. However, Mo fluxes (6-60 g·ha-1·a-1) from the organic forest floor into the mineral soils were correlated to needle concentrations and to the NO3fluxes. We conclude that Mo turnover within forest ecosystems is governed by Mo plant availability of mineral soils as well as by plant Mo uptake. In addition, Mo cycling strongly affects Mo distribution within soil profiles and Mo fluxes out of the organic layer.
Size- and age-class distributions of Fraser fir following balsam woolly adelgid infestation
Authors: Smith George F , Nicholas N S
Published: April 2011

Fraser fir (Abies fraseri (Pursh) Poir.) has suffered catastrophic mortality throughout most of its native range from an exotic insect, the balsam woolly adelgid (Adelges piceae Ratz.). To assess the regeneration potential and viability of fir populations, overstory and understory Fraser fir size and age structure were analyzed. The data were collected from thirty-six 400-m2 permanent plots, stratified into four stand canopy composition types, established near the summits of five mountains in the Great Smoky Mountains. We found that, where canopy mortality was severe, fir advance regeneration was re-entering the overstory. In seriously impacted stands, mortality of large fir and increased recruitment have produced distributions characterized by few large fir and relatively high densities of small fir. Densities of 0- to 10-year-old fir seedlings and fir seedlings [Formula: see text]0.25 m tall were much lower in stands dominated by dead fir than in mostly intact fir stands. While the lack of reproducing adults appears to be the main cause, competition with invasive species and higher seedling mortality from environmental factors probably contribute. These results lead to a hypothesis that Fraser fir will undergo a regeneration-mortality cycle with a decrease in the numbers of each successive generation.