bluuebunny/crossref_metadata_2025 huggingface dataset:
Selective isolation and distribution ofActinobisporastrains in soil
Authors: Suzuki Shin-ichi , Okuda Toru , Komatsubara Saburo
Published: April 2011

A simplified enrichment method for selective isolation of Actinobispora strains from soil is described. Actinobispora spores were tolerant to dry-heat treatment at 110°C for 15 min. Actinobispora was more resistant to 1 µg/mL leucomycin, 1 µg/mL novobiocin, and 0.5 µg/mL tunicamycin than Streptomyces dominant in soil, which prevents selective isolation of Actinobispora. Percentages of Actinobispora colonies on the isolation plate were increased by addition of antibiotics and dry-heat treatment of the soil samples. By combining the techniques described above, this genus was isolated from 105 out of 574 soil samples (18% of the samples tested). It was recovered from the soil samples with pH values ranging 5.0 to 8.9, and 78% of strains were isolated from neutral soil (pH 6.0-8.0). A number of Actinobispora strains were isolated from various soils around the world. Actinobispora strains are widely distributed in the world at relatively high frequency.Key words: Actinobispora, gellan gum, selective isolation, actinomycete, distribution.
The importance ofrecAmutant strains for the study of antifungal genes inPseudomonas aureofaciensPA147-2
Authors: Silby Mark W , Mahanty H Khris
Published: April 2011

Pseudomonas aureofaciens PA147-2 shows antifungal activity toward a variety of plant pathogenic fungi. We have been investigating the molecular mechanisms underlying the fungal inhibition, and during these studies it was observed that the use of pLAFR3-based cosmids for in trans complementation of mutants lacking antifungal activity is hindered by cosmid instability. It was hypothesised that the cosmid stability could be improved by inactivation of recA. The recA gene of PA147-2 was cloned and shown to complement recA mutants of E. coli, restoring RecA-dependent functions when expressed in trans. Two recA mutants of PA147-2 were constructed. Both of these mutants show sensitivity to DNA damage. Cosmid pPS2122 restores antifungal activity to a mutant by allele exchange, but is unstable in trans. The stability of pPS2122 is shown to be improved in a recA mutant of PA147-2 with respect to the wild type.Key words: antifungal, complementation, recA, Pseudomonas aureofaciens PA147-2.
Production of cellulases and xylanases by low-temperature basidiomycetes
Authors: Inglis G D , Popp A P , Selinger L B , Kawchuk L M , Gaudet D A , McAllister T A
Published: April 2011

Three of four isolates, representing phylogenetically distinct groupings of low-temperature basidiomycetes (LTB), were capable of utilizing wheat straw, and to a lesser extent conifer wood at 15°C. A cottony snow mould LTB (LRS 013) and a fruit rot LTB (LRS 241) grown on straw significantly degraded filter paper, carboxymethylcellulose (CMC), p-nitrophenyl β-glucopyranoside (i.e., β-glucosidases), and xylan. Enzymes produced by Coprinus psychromorbidus (LRS 067) were limited to xylanases from straw and wood and β-glucosidases from wood. A sclerotia-forming LTB (LRS 131) exhibited poor growth on both substrates, and did not produce detectable quantities of extracellular enzymes. None of the LTB isolates tested degraded avicel. The temperature optima of CMCases and xylanases in the filtrates from the straw medium ranged from 25°C to 55°C, and with the exception of LRS 067, significant activity was observed at 5°C. Two cellulases (25 and 31 kDa) and two xylanases (24 and 34 kDa) were observed on zymograms for LRS 013 and 241. Reduction of enzymes with 2-mercaptoethanol adversely affected their activity on zymograms, and an additional cellulase band was observed for non-reduced samples. This study indicates that LTB produce an array of cellulolytic and xylanolytic enzymes, and that some of these enzymes possess low-temperature optima which may facilitate degradation of plant fibre under low-temperature conditions.Key words: psychrophile, snow mould, cellulase, xylanase, enzymes.
Cholesterol is accumulated by mycobacteria but its degradation is limited to non-pathogenic fast-growing mycobacteria
Authors: Av-Gay Yossef , Sobouti Rafat
Published: April 2011

In this report we show that fast-growing non-pathogenic mycobacteria degrade cholesterol from liquid media, and are able to grow on cholesterol as a sole carbon source. In contrast, slow-growing mycobacteria, including pathogenic Mycobacterium tuberculosis and bacillus Calmette-Guérin (BCG), do not degrade and use cholesterol as a carbon source. Nevertheless, pathogenic mycobacteria are able to uptake, modify, and accumulate cholesterol from liquid growth media, and form a zone of clearance around a colony when plated on solid media containing cholesterol. These data suggest that cholesterol may have a role in mycobacterial infection other than its use as carbon source.Key words: mycobacteria, cholesterol, biodegradation.
Bile salt toxicity to some bifidobacteria strains: Role of conjugated bile salt hydrolase and pH
Authors: Grill J P , Perrin S , Schneider F
Published: April 2011

The purpose of this work was to study some aspects of bile salt toxicity towards bifidobacteria. A strain (Bifidobacterium coryneforme ATCC 25911) was selected for its lack of conjugated bile salt hydrolase activity (CBSH-), and was used with three deconjugating strains (CBSH+), for study of their growth and viability in the presence of two dihydroxylated conjugated bile salts (tauro- and glyco-deoxycholic acids). The presence of the glycoconjugate induced a more significant growth inhibition for the four strains than the tauroconjugate. The viability of the strains was measured at several pH levels. Glycodeoxycholic acid, but not taurodeoxycholic acid, exerted a lethal effect, which increased at low pH. This phenomenon was more pronounced for the CBSH-strain. We explain some of these results using an hypothesis based on the consequence of dissociation of conjugated and deconjugated bile salts, and the value of their pKa.Key words: Bifidobacterium, viability, bile salt, deconjugation.
Bartonella henselaeinfection in British Columbia: Evidence for an endemic disease among humans
Authors: Cimolai Nevio , Benoit Loralyn , Hill Alan , Lyons Christopher
Published: April 2011

Human bartonellosis in North America is mainly associated with Bartonella henselae, and the availability of laboratory diagnostic tools has significantly heightened awareness of the spectrum of human disease that is caused by this bacterium. We detail herein examples of illness in a pediatric population which serve to confirm that B. henselae-associated disease exists in British Columbia. Seroprevalence studies among asymptomatic adults and among children with symptomatic respiratory illness of other causation demonstrated that 36.8% and 18.5% of sera, respectively, had IFA-IgG titres [Formula: see text] 1:256. IFA-IgG titres did not vary significantly whether B. henselae ATCC 49793 or a local wild-type B. henselae isolate were used as substrate. An assessment of IgM response was consistent with the proposal that endemic seroprevalence is a function of past rather than recent exposure. Both clinical and serological studies are concordant in providing evidence that B. henselae is endemic in British Columbia.Key words: Bartonella henselae, cat scratch disease, serodiagnosis, seroprevalence.
A common molecular signature unifies the chitosanases belonging to families 46 and 80 of glycoside hydrolases
Authors: Tremblay Hugo , Blanchard Josée , Brzezinski Ryszard
Published: April 2011

The 3D structure-oriented alignment of the primary sequences of fourteen chitosanases, mainly of bacterial origin and belonging to families 46 and 80 of glycoside hydrolases, resulted in the identification of the following pattern common to all these enzymes: E-[DNQ]-x(8,17)-Y-x(7)-D-x-[RD]-[GP]-x-[TS]-x(3)-[AIVFLY]-G-x(5,11)-D. This pattern is proposed as the molecular signature of the chitosanases from families 46 and 80. It includes several amino acids essential for enzyme activity and (or) stability as shown by site-directed mutagenesis studies on the chitosanase from Streptomyces sp. N174. In particular, it includes two carboxylic residues directly involved in catalysis. We suggest that there is a continuum of sequence similarity between all the analyzed chitosanases, and that all these enzymes should probably be classified in one family.Key words: chitosanase, glycosyl hydrolase, protein motif.
Characterization of the anti-cancer-cell parasporal proteins of aBacillus thuringiensisisolate
Authors: Yamashita Satoko , Akao Tetsuyuki , Mizuki Eiichi , Saitoh Hiroyuki , Higuchi Kazuhiko , Park Yu Shin , Kim Ho-San , Ohba Michio
Published: April 2011

An unusual activity, associated with non-insecticidal and non-haemolytic parasporal inclusion proteins of a Bacillus thuringiensis soil isolate, designated 89-T-26-17, was characterized. The parasporal inclusion of this isolate was bipyramidal, rounded at both ends, containing proteins of 180, 150, 120, 100, and 88 kDa. No homologies with the Cry and Cyt proteins of B. thuringiensis were detected based on N-terminal sequences. Proteolytic processing of the inclusion proteins by proteinase K, trypsin, and chymotrypsin produced a major protein of 64 kDa exhibiting cytocidal activity against human leukaemic T cells and uterus cervix cancer (HeLa) cells. The protease-activated proteins showed no cytotoxicity to normal T cells.Key words: Bacillus thuringiensis parasporal inclusion, non-insecticidal, non-haemolytic, cytocidal activity, human cancer cell.
Subtilisins ofBacillusspp. hydrolyze keratin and allow growth on feathers
Authors: Evans Kristie L , Crowder Jamie , Miller Eric S
Published: April 2011

Keratinase is a serine protease produced by Bacillus licheniformis PWD-1 that effectively degrades keratin and confers the ability to grow on feathers to a protease-deficient B. subtilis strain. Studies presented herein demonstrate that B. licheniformis Carlsberg strain NCIMB 6816, which produces the well-characterized serine protease subtilisin Carlsberg, also degrades and grows on feathers. The PWD-1 and Carlsberg strains showed a similar time-course of enzyme production, and the purified serine proteases have similar enzymatic properties on insoluble azokeratin and soluble FITC-casein. Kinetic analysis of both enzymes demonstrated that they have high specificity for aromatic and hydrophobic amino acids in the P1 substrate position, although keratinase discriminates more than subtilisin Carlsberg against charged residues at this site. Nucleotide sequence analysis of the serine protease genes from B. licheniformis strains PWD-1, Carlsberg NCIMB 6816, ATCC 12759, and NCIMB 10689 showed that the kerA-encoded protease of PWD-1 differs from the others only by having V222, rather than A222, near the active site serine S220. Further, high-level expression of subE-encoded subtilisin from B. subtilis (78% similar to subtilisin Carlsberg) also confers growth on feathers on a protease-deficient B. subtilis strain. While strain PWD-1 and the kerA protease efficiently degrade keratin, keratin hydrolysis and growth on feathers is a property that can be conferred by appropriate expression of the major subtilisins, including the industrially produced enzymes.Key words: keratin hydrolysis, Bacillus, subtilisin, keratinase.
Use of lactose to induce expression of soluble NifA protein domains ofHerbaspirillum seropedicaeinEscherichia coli
Authors: Monteiro Rose A , Souza Emanuel M , Yates M Geoffrey , Pedrosa Fábio O , Chubatsu Leda S
Published: April 2011

Overexpression and purification are procedures used to allow functional and structural characterization of proteins. Many overexpressed proteins are partially or completely insoluble, and can not be easily purified. The NifA protein is an enhancer-binding protein involved in activating the expression of nif and some fix genes. The NifA protein from many organisms is usually insoluble when over-expressed, and therefore difficult to work with in vitro. In this work we have overexpressed the central+C-terminal and the central domains of the Herbaspirrilum seropedicae NifA protein in an Escherichia coli background. Expression was induced with either IPTG or lactose. The data showed that induction with lactose promoted a significantly higher percentage of these proteins in the soluble fraction than with IPTG. This probably reflects a slower kinetics of induction by lactose.Key words: Herbaspirillum seropedicae, NifA protein, transcriptional activator, nitrogen fixation, protein expression.
Genomic relatedness ofStaphylococcus aureusphages of the International Typing Set and detection of serogroup A, B, and F prophages in lysogenic strains
Authors: Dokar Jiri , Pallová Petra , Pantucek Roman , Rosypal Stanislav , Ruzicková Vladislava , Pantucková Pavla , Kailerová Jana , Klepárník Karel , Malá Zdena , Bocek Petr
Published: April 2011

On the basis of HindIII-restriction digest analysis of genomic DNAs, the S. aureus bacteriophages of the International Typing Set were divided into five clusters designated as A, F, Ba, Bb, and Bc. The clusters A and F include all the phages of serogroups A and F and correspond to species 3A and 77 proposed by Ackermann and DuBow (1987). On the other hand, the phages of serogroup B were divided into three clusters designated as Ba, Bb, and Bc that differ significantly each from the other in their restriction patterns. The clusters Ba and Bb may represent two separate species, while the cluster Bc may include more than one phage species. For each of the phage serogroups A, B, and F, common HindIII-restriction fragments of phage 3A (1700 bp), of 53 (4060 bp), and of 77 (8300 bp) were used for the preparation of probes specific to the phages of serogroups A, B, and F. These probes were very effective, making it possible to detect up to three different prophages in a given lysogenic strain at the same time. Restriction enzyme maps of phages 3A, 53, and 77, each representing a different serogroup, were constructed. The restriction maps of phage 3A and that of phage 77 are linear, whereas that of phage 53 is circular and exhibits a circular permutation. DNAs of the phages of serogroups A and F have cohesive ends. On each restriction map, the sites corresponding to specific probes are indicated. The size of intact genomic DNA of all phages estimated by PFGE varies within the range of 41.5-46.2 kb.Key words: Staphylococcus aureus, bacteriophages, prophage specific probes, restriction endonuclease maps.
Natural incidence of endophytic bacteria in pea cultivars under field conditions
Authors: Elvira-Recuenco M , van Vuurde J W L
Published: April 2011

Pea plants grown in the field were used to study the natural incidence of endophytic bacteria in the stem. Eleven pea cultivars at the flowering stage were screened for the presence of endophytic bacteria using a printing technique with surface disinfested stem cross-sections on 5% Trypticase Soy Agar (TSA). Five cultivars showed colonization. Cultivar Twiggy showed the highest and most consistent colonization and was further investigated. Stems of cv. Twiggy at the pod stage were analyzed for endophytic bacterial types and populations. Cross-sections of surface disinfested stems were printed on 5% TSA. Endophytic bacterial populations decreased from the lower to the upper part of the stem. One section from the third and the fourth internode was surface disinfested, homogenized, and spiral plated on the media 5% TSA, R2A, and SC (Davis et al. 1980). Over a series of 30 samples, 5% TSA gave significantly better recovery of bacterial endophytes compared with R2A and SC media. For most stems, populations ranged from 104 to 105 CFU/g except in one of the field blocks in which endophyte populations were uniformly higher. Comparison of colony counts by spiral plating and printing showed a positive correlation. The most frequently recovered bacterial types were Pantoea agglomerans and Pseudomonas fluorescens. Less frequently isolated were Pseudomonas viridiflava and Bacillus megaterium.Key words: endophytic bacteria, pea, stem colonization, cultivar screening, biodiversity.
Hydrogenase in Frankia KB5: Expression of and relation to nitrogenase
Authors: Mattsson Ulrika , Sellstedt Anita
Published: April 2011

The localization and expression of the hydrogenase in free-living Frankia KB5 was investigated immunologically and by monitoring activity, focusing on its relationships with nitrogenase and H2. Immunological studies revealed that the large subunit of the hydrogenase in Frankia KB5 was modified post-translationally, and transferred into the membrane after processing. The large subunit was constitutively expressed and no correlation was found between hydrogenase activity and synthesis. Although H2 was not needed for induction of hydrogenase synthesis, exogenously added H2 triggered hydrogen uptake in medium containing nitrogen, i.e., in the hyphae. A correlation between nitrogenase activity and hydrogen uptake was found in cultures grown in media without nitrogen, but interestingly the two enzymes showed no co-regulation.Key words: hydrogenase, expression, nitrogenase, Frankia KB5.
ThefliUandfliVgenes are expressed as a single ORF inSalmonellacholeraesuis
Authors: Ho Kuo-Chieh , Chang Gan-Nan
Published: April 2011

A DNA fragment carrying flagellar genes was cloned from Salmonella choleraesuis. Compared to the corresponding DNA fragment of Salmonella muenchen, this fragment contained three ORFs instead of four shown in S. muenchen. The DNA sequence data showed that there was an insertion of nucleotide C in the ORF of the S. choleraesuis fliU gene, which resulted in the disappearance of a termination codon downstream. The recombinant plasmid pFU11 containing the coding region of the fliU gene made by PCR on S. choleraesuis genomic DNA was constructed and expressed in Escherichia coli in the presence of IPTG. As expected, a 45 kDa protein band was observed on a SDS-PAGE gel, in contrast to two with each having about a half of the molecular weight. These results demonstrated that the DNA sequence encoding one protein (FliU) in S. choleraesuis corresponded to the DNA sequence encoding two proteins (FliU and FliV) in S. muenchen. The protein encoded by this single ORF might carry out the functions of two separated proteins by folding in such a way that its conformation could function like two interdependent protein subunits.Key words: Salmonella choleraesuis, Salmonella muenchen, fliU gene, gene expression.
Primary alcohols and di-alcohols as growth substrates for the purple nonsulfur bacteriumRhodobacter capsulatus
Authors: Pantazopoulous Panagiotis E , Madigan Michael T
Published: April 2011

Growth experiments were performed with the purple nonsulfur bacterium Rhodobacter capsulatus to test its ability to use aliphatic, methyl-substituted, and unsaturated alcohols, as well as di-alcohols, as carbon sources for growth. Both phototrophic and chemotrophic growth was observed on a wide variety of such alcohols. By contrast, secondary or tertiary alcohols, or primary alcohols containing an ethyl or propyl substituent, did not support growth. In addition, preculture history and serial subculturing were found to be important factors for obtaining reliable growth of R. capsulatus on alcohols. Collectively, these results suggest that the carbon nutritional diversity of Rhodobacter capsulatus is even greater than previously suspected and that besides metabolizing organic acids and fatty acids in nature, this species may also be a major consumer of alcohols.Key words: purple nonsulfur bacteria, Rhodobacter capsulatus, alcohol metabolism, primary alcohols, di-alcohols.
Ligninolytic characteristics of Pleurotus ostreatus strain F6 and its monokaryotic protoplast derivative P19
Authors: Eichlerová Ivana , Ruel Katia , Homolka Ladislav , Joseleau Jean-Paul , Nerud Frantiek
Published: April 2011

A stable isolate of Pleurotus ostreatus P19 differing in some morphological and physiological characteristics from its parental wild-type strain F6 was obtained via protoplast isolation during the preparation of strains with altered ligninolytic abilities. The isolate is monokaryotic, does not form clamp-connections, and produces much higher activities of enzymes involved in lignin modification (laccase, manganese peroxidase). Cellulase activity was comparable to that of wild-type strain F6, but the xylanase activity was slightly higher in isolate P19. However, this monokaryotic derivative degrades lignin at a slightly lower rate than its parental strain F6. Electron microscopy observations of wood degradation as a function of mycelium growth were performed on three zones of birch wafers delimited according to the distance from the point of inoculation. The different stages of fungal mycelium growth showed differences in the ultrastructural patterns of the decay not only between the strains P19 and F6, but also depending on the distance from the point of inoculation. This suggests a spatio-temporally controlled secretion of enzymes along the hyphae. The enhanced ability of P19 to degrade the condensed forms of lignin in middle lamellae is correlated to its higher laccase activity.Key words: electron microscopy, ligninolytic enzymes, Pleurotus ostreatus, wood degradation.
Assessing and analysing contamination of a dairy products processing plant byStaphylococcus aureususing antibiotic resistance and PFGE
Authors: Tondo E C , Guimarães MC M , Henriques J AP , Ayub M AZ
Published: April 2011

A dairy product processing plant was studied for 2.5 years to examine contamination with Staphylococcus aureus and try to correlate the source of contamination. Cultures were submitted to an antibiotic susceptibility test (AST) and characterised by Pulsed-field Gel Electrophoresis (PFGE) analysis. Results showed that 35.2% (19/51) of food handlers were asymptomatic carriers of S. aureus, and that 90.4% (19/21) of raw milk sampled was contaminated. Staphylococcus aureus was isolated from only 10 samples among more than 3200 investigated dairy products. No S. aureus contamination was found on machinery. The AST analysis demonstrated sensitivity of tested S. aureus to oxacillin, cephalothin, vancomycin, gentamicin, and sulfamethoxazole/trimethoprim. AST analysis generated eight different phenotypic profiles, but did not allow us to identify the source of contamination in seven of ten final products. PFGE analysis proved to be a sensitive method as it generated 42 different DNA banding profiles among the 48 S. aureus investigated, demonstrating a lack of predominance of endemic strains in the plant, contrary to suggestions raised by antibiotic resistance typing. Based on PFGE genotyping, S. aureus strains isolated from four contaminated final products were similar to four S. aureus isolated from raw milk. Five final products contained S. aureus different from all other strains collected, and one showed similarity to a strain isolated from a food handler. These results suggest contamination by raw milk as the main source of contamination of the final dairy products.Key words: Staphylococcus aureus, dairy products, antibiotic susceptibility, PFGE.
Effects of Cymbopogon nardus (L.) W. Watson essential oil on the growth and morphogenesis of Aspergillus niger
Authors: de Billerbeck Virginia G , Roques Christine G , Bessière Jean-Marie , Fonvieille Jean-Louis , Dargent Robert
Published: April 2011

The growth inhibitory effect of Cymbopogon nardus (L.) W. Watson var. nardus essential oil on Aspergillus niger (Van Tieghem) mycelium was determined on agar medium. The mycelium growth was completely inhibited at 800 mg/L. This concentration was found to be lethal under the test conditions. Essential oil at 400 mg/L caused growth inhibition of 80% after 4 days of incubation, and a delay in conidiation of 4 days compared with the control. Microscopic observations were carried out to determine the ultrastructural modifications of A. niger hyphae after treatment with C. nardus essential oil. The main change observed by transmission electron microscopy concerned the hyphal diameter and the hyphal wall, which appeared markedly thinner. These modifications in cytological structure might be caused by the interference of the essential oil with the enzymes responsible for wall synthesis which disturb normal growth. Moreover, the essential oil caused plasma membrane disruption and mitochondrial structure disorganization. The findings thus indicate the possibility of exploiting Cymbopogon nardus essential oil as an effective inhibitor of biodegrading and storage-contaminating fungi.Key words: essential oil, antifungal agent, hyphal ultrastructure, cell wall, scanning electron microscopy, transmission electron microscopy.
The effect of the addition of proteases and glucanases during yeast autolysis on the production and properties of yeast extracts
Authors: Conway John , Gaudreau Hélène , Champagne Claude P
Published: April 2011

Yeast extracts (YE) were produced with the addition of proteases or glucanases during bakers' yeast (Saccharomyces cerevisiae) autolysis. Chemical composition, physical properties, and biological value of the YE were examined. Proteases had the highest impact on the turbidity and filterability of YE. All 11 proteases and two glucanases increased YE yields (% yeast solids solubilized) obtained from heated (80°C/15 min) bakers' yeast creams (BYC). However, when proteases were added to native (unheated) BYC during autolysis, few increased YE yields, with papain being the most effective. The increased yields were generally related to increased levels of total nitrogen (TN) and α-amino nitrogen (α-AN) in the YE. Media were supplemented with the various yeast extracts, and the highest growth rates (µmax) and biomass values (ODmax) of Lactobacillus acidophilus were noted. The best growth was obtained with YE produced with native BYC treated with a fungal protease, and results of this study show that some enzymes could be used to produce improved YE for microbiological media.Key words: turbidity, filtration, amino acids, Lactobacillus.
Changes in enzymatic activities involved in glucose metabolism by acyl-CoAs inTrypanosoma cruzi
Authors: Lema M García de , Lucchesi G , Racagni G , Machado-Domenech E E
Published: April 2011

This study describes the effect of some saturated and unsaturated free fatty acids and acyl-CoA thioesters on Trypanosoma cruzi glucose 6-phosphate dehydrogenase and hexokinase activities. Glucose 6-phosphate dehydrogenase was sensitive to the destabilizing effect provoked by free fatty acids, while hexokinase remained unaltered. Glucose 6-phosphate dehydrogenase inhibition by free fatty acids was dependent on acid concentration and chain length. Both enzymes were inhibited when they were incubated with acyl-CoA thioesters. The acyl-CoA thioesters inhibited glucose 6-phosphate dehydrogenase at a lower concentration than the free fatty acids; the ligands glucose 6-phosphate and NADP+afforded protection. The inhibition of hexokinase by acyl-CoAs was not reverted when the enzyme was incubated with ATP. The type of inhibition found with acyl-CoAs in relation to glucose 6-phosphate dehydrogenase and hexokinase suggests that this type inhibition may produce an in vivo modulation of these enzymatic activities.Key words: Trypanosoma cruzi, fatty acids, acyl-CoAs, glucose 6-phosphate dehydrogenase, hexokinase.
Phospholipid analogue profiles of Peptostreptococcus, Micromonas, and Finegoldia species analysed by fast atom bombardment mass spectrometry
Authors: Radcliffe C E , Drucker D B , Boote V , Fletcher-Williams G , Claydon M A
Published: April 2011

Species of Peptostreptococcus cause a variety of infections, primarily abscesses of soft tissues, joints, and mucous membranes. The aim of this study was to compare the phospholipid analogue profiles of Peptostreptococcus species, represented by P. anaerobius, P. asaccharolyticus, P. indolicus, P. lacrimalis, and P. prevotii; Micromonas micros (P. micros) and Finegoldia magna (P. magnus). After anaerobic growth on blood-FAA, lipids extracted by chloroform-methanol (2:1 v/v) were purified, then analysed by fast atom bombardment mass spectrometry (FAB-MS) in negative ion mode. The major peaks with mass to charge (m/z) 719, 721, and 749, corresponded to phosphatidylglycerol analogues, namely PG (32:1), PG (32:0), and PG (34:0), which have been found previously in Lactobacillus spp., Clostridium difficile, and Staphylococcus spp. Other major peaks observed, with m/z 619, 647, 665, 675, 677, 687, 691, 693, 701, 703, 707, 733, and 746 have also been reported in one or more of these three species. However, other major peaks found here in Peptostreptococcus, Micromonas, and Finegoldia have not been described elsewhere; these are 501, 514, 515, 618, 659, 673, 676, 688, 690, 692, 694, 700, 706, 715, 718, 722, and 750. We conclude that Peptostreptococcus, Micromonas, and Finegoldia isolates are chemically unique.Key words: Peptostreptococcus, Micromonas, Finegoldia, phospholipids, FAB-MS.
Competitiveness in root colonization by Pseudomonas putida requires the rpoS gene
Authors: Miller Charles D , Kim Young-Cheol , Anderson Anne J
Published: April 2011

The rpoS gene in Pseudomonas putida was essential for plant root colonization under competitive conditions from other microbes. The RpoS- mutant survived less well than the wild-type strain in culture medium, and unlike the wild-type, failed to colonize the roots in a peat matrix containing an established diverse microflora. The RpoS-deficient P. putida isolate was generated by insertion of a glucuronidase-npt cassette into the rpoS gene. The RpoS- mutant had dose-dependent increased sensitivity to oxidative stress and produced Mn-superoxide dismutase activity earlier than the parent. While extracts from wild-type P. putida stationary-phase cells contained three isozymes of catalase (CatA, CatB, and CatC), the σ38-deficient P. putida lacked CatB. These results are consistent with previous findings that CatB is induced in stationary-phase.Key words: catalase, starvation, activated oxygen species.
Mapping of monoclonal antibodies specific to P64k: A common antigen of several isolates of Neisseria meningitidis
Authors: Nazábal C , Carmenate T , Cruz S , González S , Silva R , Musacchio A , Delgado M , Chinea G
Published: April 2011

P64k is a minor outer membrane protein from Neisseria meningitidis. This protein has been produced at high levels in Escherichia coli. We generated a group of monoclonal antibodies (mAbs) against recombinant P64k, which recognise four non-overlapping epitopes, as shown using competition assays with biotinylated mAbs. The P64k sequences involved in mAbs binding were mapped with synthetic overlapping peptides derived from the P64k protein, and located in the previously determined three-dimensional structure of the protein. These antibodies were also characterised by whole-cell ELISA and bactericidal tests against N. meningitidis. Only two of the recognised epitopes were exposed on the bacterial surface, and none of the mAbs showed bactericidal activity. The relationship between these results and the structural data on the epitopes bound by the mAbs is discussed.Key words: Neisseria meningitidis, P64k, monoclonal antibodies, epitope mapping.
Antibiotic production, accumulation of intracellular carbon reserves, and sporulation inMicromonospora echinospora(ATCC 15837)
Authors: Hoskisson Paul A , Hobbs Glyn , Sharples George P
Published: April 2011

The physiology of the actinomycete Micromonospora echinospora was examined during growth. Biphasic accumulation of glycogen occurred, initially during the early exponential growth phase, and again following the onset of sporulation at 120 h. Lipid levels increased during growth eventually representing 25% of the cell mass. A significant proportion of the lipid was found to be in the form of triacylglycerols, which were found to accumulate markedly during the sporulation phase. The disaccharide trehalose was also found to accumulate during growth with levels rising to 5% of the dry weight during the mycelial production phase, then remaining constant during sporulation. Antibiotic was produced transiently by the cultures over the period preceding sporulation.Key words: Micromonospora, glycogen, trehalose, lipids, triacylglycerol.
Catalase activity and the survival of Pseudomonas putida, a root colonizer, upon treatment with peracetic acid
Authors: Anderson Anne J , Miller Charles D
Published: April 2011

Peracetic acid is used as a sterilant in several industrial settings. Cells of a plant-colonizing bacterium, Pseudomonas putida in liquid suspension, were more sensitive to killing by peracetic acid when they lacked a major catalase activity, catalase A. Low doses of peracetic acid induced promoter activity of the gene encoding catalase A and increased total catalase specific activity in cell extracts. Microbes present in native agricultural soils rapidly degraded the active oxygen species present in peracetic acid. The simultaneous release of oxygen was consistent with a role for catalase in degrading the hydrogen peroxide that is part of the peracetic acid-equilibrium mixture. Amendment of sterilized soils with wild-type P. putida restored the rate of degradation of peracetic acid to a higher level than was observed in the soils amended with the catalase A-deficient mutant. The association of the bacteria with the plant roots resulted in protection of the wild-type as well as the catalase-deficient mutant from killing by peracetic acid. No differential recovery of the wild-type and catalase A mutant of P. putida was observed from roots after the growth matrix containing the plants was flushed with peracetic acid.Key words: Pseudomonas putida (Pp), activated oxygen species (AOS), hydrogen peroxide, luciferase, colonization.
Competition and antibiosis in the biological control of potato scab
Authors: Neeno-Eckwall Eric C , Kinkel Linda L , Schottel Janet L
Published: April 2011

Nonpathogenic, antibiotic-producing streptomycetes have been shown to reduce potato scab when added to disease-conducive soil. Spontaneous mutants of the pathogenic Streptomyces scabies RB4 that are resistant to at least one antibiotic activity produced by the nonpathogenic suppressive isolates Streptomyces diastatochromogenes strain PonSSII and S. scabies PonR have been isolated. To determine the importance of antibiosis in this biocontrol system, these mutants were investigated for their ability to cause disease in the presence of the two pathogen antagonists in a greenhouse assay. Disease caused by one of the mutant strains was reduced in the presence of both suppressive isolates, whereas disease caused by the other five mutants was not significantly reduced by either suppressive strain. In addition, a nonpathogenic mutant of S. scabies RB4 was isolated, which produced no detectable in vitro antibiotic activity and reduced disease caused by its pathogenic parent strain when the pathogen and mutant were coinoculated into soil. Population densities of the pathogen were consistently lower than those of the suppressive strains when individual strains were inoculated into soil. When a pathogen was coinoculated with a suppressive strain, the total streptomycete population density in the pot was always less than that observed when the suppressive isolate was inoculated alone. When the pathogens were inoculated individually into soil, a positive correlation was seen between population density and disease severity. In coinoculation experiments with pathogen and suppressive strains, higher total streptomycete population densities were correlated with lower amounts of disease.Key words: Streptomyces scabies, biological control, antibiotic resistance, potato scab disease.
A comparison of sole carbon source utilization patterns and phospholipid fatty acid profiles to detect changes in the root microflora of hydroponically grown crops
Authors: Khalil S , Bååth E , Alsanius B , Englund J -E , Sundin P , Gertsson U E , Jensén P
Published: April 2011

Sole carbon source utilization (SCSU) patterns and phospholipid fatty acid (PLFA) profiles were compared with respect to their potential to characterize root-inhabiting microbial communities of hydroponically grown crops. Sweet pepper (Capsicum annum cv. Evident), lettuce (Lactuca sativa cv. Grand Rapids), and four different cultivars of tomato (Lycopersicon esculentum cvs. Gitana, Armada, Aromata, and Elin) were grown in 1-L black plastic beakers placed in a cultivation chamber with artificial light. In addition to the harvest of the plants after 6 weeks, plants of one tomato cultivar, cv. Gitana, were also harvested after 4 and 8 weeks. The cultivation in this study was performed twice. Principal component analysis was used to analyze the data. Both characterization methods had the ability to discriminate between the root microflora of different plant species, cultivars, and one tomato cultivar at different ages. Differences in both SCSU patterns and PLFA profiles were larger between plant species than between cultivars, but for both methods the largest differences were between the two cultivations. Still, the differences between treatments were always due to differences in the same PLFAs in both cultivations. This was not the case for the SCSU patterns when different plant ages were studied. Furthermore, PLFA profiles showed less variation between replicates than did SCSU patterns. This larger variation observed among the SCSU data indicates that PLFA may be more useful to detect changes in the root microflora of hydroponically grown crops than the SCSU technique.Key words: sole carbon source utilization (SCSU) patterns, phospholipid fatty acid (PLFA) profiles, Lycopersicon esculentum, Lactuca sativa, Capsicum annum, indigenous microflora.
Production of transferrin receptors byHistophilus ovis: three of five strains require two signals
Authors: Ekins Andrew , Niven Donald F
Published: April 2011

Five strains of Histophilus ovis (9L, 642A, 714, 5688T, and 3384Y) were investigated with respect to iron acquisition. All strains used ovine, bovine, and goat transferrins (Tfs), but not porcine or human Tfs, as iron sources for growth. In solid phase binding assays, total membranes from only two (9L and 642A) of the five strains, grown under iron-restricted conditions, were able to bind Tfs (ovine, bovine, and goat, but not porcine or human). However, when the organisms were grown under iron-restricted conditions in the presence of bovine transferrin (Tf), total membranes from all strains exhibited Tf binding (as above); competition experiments demonstrated that all three Tfs (ovine, bovine, and goat) were bound by the same receptor(s). Membranes from organisms grown under iron-replete conditions in the presence or absence of bovine Tf failed to bind any of the test Tfs. An affinity-isolation procedure allowed the isolation of two putative Tf-binding polypeptides (78 and 66 kDa) from total membranes of strains 9L and 642A grown under iron-restricted conditions, and from membranes of all strains if the growth medium also contained Tf. It is concluded that all strains tested acquire Tf-bound iron by means of siderophore-independent mechanisms involving surface receptors analogous to the Tf-binding proteins (TbpA and TbpB) found in comparable organisms; although iron restriction alone is sufficient to promote the expression of these proteins by strains 9L and 642A, their production by strains 714, 5688T, and 3384Y appears to require two signals, iron restriction and the presence of Tf.Key words: Histophilus ovis, iron acquisition, transferrins, receptors, regulation.
Utilization of herpes simplex PCR assays for cerebrospinal fluid in a pediatric health care setting
Authors: Cimolai Nevio , Thomas Eva E , Tan Rusung , Hill Alan
Published: April 2011

An assessment was made of the utilization and impact of a diagnostic polymerase chain reaction (PCR) assay for the diagnosis of herpes simplex viruses (HSV) 1 and 2 in cerebrospinal fluid of children who attended a Canadian pediatric referral centre. One hundred and three assays were performed on specimens from 103 patients during the period August 1997 to September 1998. Patient ages ranged from newborn to 16 years. Indications for HSV PCR included seizures with or without fever (56.3%), aseptic meningitis (16.5%), and encephalopathy with or without fever (10.7%). Only 2 of 103 (1.9%) assays were positive, including one each for HSV1 and HSV2. Control specimens that were seeded with virus indicated inhibition for 24.3, 8.8, and 6.8% of assays for HSV1, HSV2, and both HSV1 and HSV2, respectively. The mean turn-around time for HSV PCR was 2.5 days, and 90.3% were completed in less than 5 days. Acyclovir was administered to 78.6% of the patients overall; the results of the HSV PCR impacted on the treatment courses for 36 individuals. Nevertheless, 16.5% of patients continued to receive extended courses of antiviral therapy despite negative HSV PCR assays. Although it is desirable to decrease the frequency of PCR inhibitions and to further decrease the interval to assay completion, HSV PCR does have a significant impact on antiviral use in this setting.Key words: herpes simplex, polymerase chain reaction, central nervous system.
Cytokinin production by plant growth promoting rhizobacteria and selected mutants
Authors: García de Salamone Ines E , Hynes Russell K , Nelson Louise M
Published: April 2011

One of the proposed mechanisms by which rhizobacteria enhance plant growth is through the production of plant growth regulators. Five plant growth promoting rhizobacterial (PGPR) strains produced the cytokinin dihydrozeatin riboside (DHZR) in pure culture. Cytokinin production by Pseudomonas fluorescens G2018, a rifampicin-resistant mutant (RIF), and two TnphoA-derived mutants (CNT1, CNT2), with reduced capacity to synthesize cytokinins, was further characterized in pure culture using immunoassay and thin layer chromatography. G2018 produced higher amounts of three cytokinins, isopentenyl adenosine (IPA), trans-zeatin ribose (ZR), and DHZR than the three mutants during stationary phase. IPA was the major metabolite produced, but the proportion of ZR and DHZR accumulated by CNT1 and CNT2 increased with time. No differences were observed between strain G2018 and the mutants in the amounts of indole acetic acid synthesized, nor were gibberellins detected in supernatants of any of the strains. Addition of 105 M adenine increased cytokinin production in 96- and 168-h cultures of strain G2018 by approximately 67%. G2018 and the mutants CNT1 and CNT2 may be useful for determination of the role of cytokinin production in plant growth promotion by PGPR.Key words: cytokinins, plant growth regulation, Pseudomonas fluorescens, rhizobacteria, plant growth promoting rhizobacteria (PGPR).
Regulation of Nod factor sulphation genes inRhizobiumtropiciCIAT899
Authors: Manyani Hamid , Sousa Carolina , Díaz María-Eugenia Soria , Gil-Serrano Antonio , Megías Manuel
Published: April 2011

Rhizobium tropici CIAT899 is a tropical symbiont able to nodulate various legumes such as Leucaena, Phaseolus, and Macroptilium. Broad host range of this species is related to its Nod factors wide spectrum. R. tropici contains Nod factors sulphation nod genes, nodHPQ genes, which control nodulation efficiency in Leucaena. To study nodHPQ regulation, we carried out different interposon insertions in its upstream region. One of these generated interruptions, nodI mutant produced nonsulphated Nod factors suggesting a possible dependence of these genes on nodI upstream region. Moreover, analysis results of lacZ transcriptional fusions with these genes in symbiotic plasmid showed dependence of these genes on NodD protein. In order to determine nodHPQ organization, we studied the effect of interposon insertion upstream of each lacZ transcriptional fusion, and the data obtained was used to indicate that nodHPQ belong to the nodABCSUIJ operon. However, comparison between nodP::lacZ β-galactosidase activity in the symbiotic plasmid and in the pHM500 plasmid (containing nodHPQ genes) suggested constitutive expression in free living, and flavonoid inducible expression in symbiotic conditions. Constitutive nodHPQ expression may play a role in bacterial house-keeping metabolism. On the other hand, the transference of R. tropici nodHPQ genes to other rhizobia that do not present sulphated substitutions demonstrated that NodH protein sulphotransference is specific to C6 at the reducing end.Key words: Nod factors, nodHPQ genes, Rhizobium tropici, nod-box.
Analysis of pFQ12, a 22.4-kbFrankiaplasmid
Authors: John Theodore R , Rice Jeffrey M , Johnson Jerry D
Published: April 2011

Frankia are gram-positive, filamentous bacteria capable of fixing atmospheric dinitrogen in symbiosis with a wide variety of woody plants and shrubs. Some isolates of Frankia harbor plasmids of 8.5 (pFQ11) and 22.4 kb (pFQ12) that have no known function but are transmitted through many generations in culture. We have sequenced the 22 437-bp pFQ12 plasmid that is present in isolates CpI1 and ArI3. This sequence, with 76% G+C, is almost totally unrelated to that of pFQ11 found in the same cells. However, four regions of identity, 40-90 bp each, are dispersed around the plasmids. The 22.4-kb plasmid has >50 open reading frames (ORFs) that encode putative proteins of more than 100 amino acids, with the largest being 2226 amino acids. Twenty of these ORFs are likely to encode proteins based on their codon bias as determined by two different algorithms. Transcripts from nine of these regions have been identified by reverse transcriptase-polymerase chain reaction (RT-PCR) or filter hybridization. The two Frankia plasmids each encode a protein similar to the korSA protein that regulates transmission of pSAM2 in Streptomyces. The origin of replication (ORI) region of pFQ12 was localized by intrastrand AT and GC equivalence switch. It includes a 40-bp, intergenic, A+T-rich region that has a strong identity in pFQ11.Key words: ORI analysis, RT-PCR, Glimmer, DNA sequence.
Extrusion of actin-positive strands from HEp-2 and Int 407 cells caused by outer membrane preparations of enteropathogenicEscherichia coliand specific attachment of wild type bacteria to the strands
Authors: Kumar Sukumaran Sunil , Malladi Vasantha , Sankaran Krishnan , Haigh Richard , Williams Peter , Balakrishnan Arun
Published: April 2011

Enteropathogenic Escherichia coli (EPEC) causes persistent infantile diarrhoea. This nontoxigenic E. coli exhibits a complicated pathogenic mechanism in which its outer membrane proteins and type III secretory proteins damage intestinal epithelium and cause diarrhoea. In accordance with this, our previous study using HEp-2 cells demonstrated cytopathic effects caused by cell-free outer membrane preparations of EPEC. In this study, we report the extrusion of actin-positive strands from HEp-2 and Int 407 cells when treated with outer membrane preparations. An interesting observation of this work, perhaps relevant to the characteristic localized three-dimensional colony formation of EPEC, is the attachment of a wild type EPEC strain to these actin-positive strands.Key words: enteropathogenic Escherichia coli, actin, outer membrane proteins, cytoskeletal elements.
The occurrence of cyanobacteria in pulp and paper waste-treatment systems
Authors: Kirkwood Andrea E , Nalewajko Czesia , Fulthorpe Roberta R
Published: April 2011

Pulp and paper secondary waste-treatment systems in Brazil, Canada, New Zealand, and the U.S.A. contained dynamic cyanobacterial communities, some of which exceeded heterotrophic bacterial biomass. No other viable photoautotrophic populations were detected in the ponds. Regardless of geographical location, Oscillatoriales including Phormidium, Geitlerinema, and Pseudanabaena were the dominant taxa. As well, Chroococcus (Chroococcales) was an important genus in Brazil and New Zealand. The possible impact of cyanobacteria on waste-treatment efficiency deserves further study given their large biomass and diverse metabolic characteristics.Key words: cyanobacteria, blue-green algae, heterotrophic bacteria, community structure, pulp and paper secondary waste treatment.