bluuebunny/crossref_metadata_2025 huggingface dataset:
Population Sizes, Immigration, and Growth of Epiphytic Bacteria on Leaves of Different Ages and Positions of Field-Grown Endive (Cichorium endivia var. latifolia)
Authors: Jacques M , Kinkel L L , Morris C E
Published: January 2020

Total, fluorescent, and pectolytic epiphytic bacterial population sizes were quantified on leaves of different age groups of broad-leaved endive during field cultivation from leaf emergence until harvest. Greater bacterial population densities (log(inf10) CFU per square centimeter) were observed on outer leaves than on inner leaves of the plants throughout the growing season. These differences were statistically significant for total bacterial populations at all sampling times and were often significant for fluorescent and pectolytic bacterial populations. At harvest, a linear gradient of decreasing densities of epiphytic bacteria from outer (older) to inner (younger) leaves of the head was significant. Leaf age influenced the frequency distribution and variability of bacterial population sizes associated with leaves of broad-leaved endive. Total bacterial population sizes were greater at leaf emergence for leaves emerging during the second half of the cultivation period than for leaves emerging earlier. The size of fluorescent and pectolytic bacterial populations on newly emerged leaves increased throughout the season as plants aged. To assess the importance of plant age on bacterial immigration at leaf emergence, bacterial densities were quantified on leaves emerging simultaneously on plants of different ages. In two of the three experiments, greater bacterial population sizes were observed on leaves emerging on younger plants. This indicates that factors other than an increase in concentration of airborne bacteria can lead to increases in population sizes at leaf emergence as plants age in the field. Results of leaf pruning experiments suggested that adjacent leaves may act as a barrier for immigration of fluorescent bacteria on newly emerged leaves. Survival of an inoculated strain of Pseudomonas fluorescens on newly emerged leaves generally did not vary with the age of plants. However, these effects were not consistent among experiments, suggesting that interactions among micro- and macroenvironmental conditions, physiological condition of leaves, and accessibility of leaves to airborne bacteria are important in controlling epiphytic bacterial population sizes.
A High-Molecular-Weight, Cell-Associated Xylanase Isolated from Exponentially Growing Thermoanaerobacterium sp. Strain JW/SL-YS485
Authors: Shao W , Deblois S , Wiegel J
Published: January 2020

An unusual cell-associated (beta)-1,4-xylanase was purified to gel electrophoretic homogeneity from a cell extract of the bacterium Thermoanaerobacterium sp. strain JW/SL-YS485 harvested at the late exponential growth phase. The molecular mass of the xylanase was 350 kDa as determined by gel filtration and 234 kDa as determined by native gradient gel electrophoresis. The enzyme contained 6% carbohydrates. Heterosubunits of 180 and 24 kDa were observed for the xylanase on sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis gels. The xylanase had a pI of 4.37 and a half-life of 1 h at 70(deg)C. Using a 5-min assay, we observed the highest level of activity at pH 6.2 and 80(deg)C. The K(infm) and k(infcat) values when oat spelt xylan was used were 3 mg/ml and 26,680 U/(mu)mol, respectively. The Arrhenius energy was 41.8 kJ/mol. The purified enzyme differed in size, subunit structure, and location from other xylanases that have been described. The cell-associated enzyme activity appeared in the S-layer fraction.
Development and testing of improved suicide functions for biological containment of bacteria
Authors: Knudsen S , Saadbye P , Hansen L H , Collier A , Jacobsen B L , Schlundt J , Karlström O H
Published: January 2020

We have developed very efficient suicide functions for biological containment based on the lethal Escherichia coli relF gene. The suicide functions are placed in duplicate within a plasmid and arranged to prevent inactivation by deletion, recombination, and insertional inactivation. The efficiency of this concept was tested in a plasmid containment system that prevents transfer of plasmids to wild-type bacteria. Protection against plasmid transfer was assayed in test tubes and in rat intestine. Protection was efficient and refractory to inactivation by mutation and transposons. The efficiency of the suicide system was also tested in soil and seawater. We show that unprecedented suicide efficiency can be achieved in soil and seawater after suicide induction by IPTG (isopropyl-beta-D-thiogalactopyranoside). More than 7 orders of magnitude reduction in suicide bacteria was achieved.
Diversity among Field Populations of Bradyrhizobium japonicum in Poland
Authors: Madrzak C J , Golinska B , Kroliczak J , Pudelko K , Lazewska D , Lampka B , Sadowsky M J
Published: January 2020

Genetic structure in field populations of Bradyrhizobium japonicum isolated in Poland was determined by using several complementary techniques. Of the 10 field sites examined, only 4 contained populations of indigenous B. japonicum strains. The Polish bradyrhizobia were divided into at least two major groups on the basis of protein profiles on polyacrylamide gels, serological reaction with polyclonal antisera, repetitive extragenic palindromic PCR fingerprints of genomic DNA, and Southern hybridization analyses with nif and nod gene probes. Serological analyses indicated that 87.5% of the Polish B. japonicum isolates tested were in serogroups 123 and 129, while seven (12.5%) of the isolates tested belonged to their own unique serogroup. These seven strains also could be grouped together on the basis of repetitive extragenic palindromic PCR fingerprints, protein profiles, and Southern hybridization analyses. Cluster analyses indicated that the seven serologically undefined isolates were genetically dissimilar from the majority of the Polish B. japonicum strains. Moreover, immuno-cross-adsorption studies indicated that although the Polish B. japonicum strains reacted with polyclonal antisera prepared against strain USDA123, the majority failed to react with serogroup 123- and 129-specific antisera, suggesting that Polish bradyrhizobia comprise a unique group of root nodule bacteria which have only a few antigens in common with strains USDA123 and USDA129. Nodulation studies indicated that members of the serologically distinct group were very competitive for nodulation of Glycine max cv. Nawiko. None of the Polish serogroup 123 or 129 isolates were restricted for nodulation by USDA123- and USDA129-restricting soybean plant introduction genotypes. Taken together, our results indicate that while genetically diverse B. japonicum strains were isolated from some Polish soils, the majority of field sites contained no soybean-nodulating bacteria. In addition, despite the lack of long-term soybean production in Poland, field populations of unique B. japonicum strains are present in some Polish soils and these strains are very competitive for nodulation of currently used Polish soybean varieties.
Detection of Campylobacter jejuni added to foods by using a combined selective enrichment and nucleic acid sequence-based amplification (NASBA)
Authors: Uyttendaele M , Schukkink R , van Gemen B , Debevere J
Published: January 2020

An assay to detect Campylobacter jejuni in foods that uses a short selective enrichment culture, a simple and rapid isolation procedure, NASBA amplification of RNA, and a nonradioactive in solution hybridization was studied. The presence of high numbers of indigenous flora affected the sensitivity of the assay. However, detection of C. jejuni was possible up to a ratio of indigenous flora to C. jejuni of 10,000:1. Interference by food components was eliminated by centrifugation following the enrichment step. Fourteen food samples artificially inoculated with C. jejuni (1 to 1,000 CFU/10 g) were analyzed with the NASBA assay and the conventional culture method with Campylobacter charcoal differential agar (CCDA). A few false-negative results were obtained by both NASBA (1.42%) and CCDA (2.86%) isolation. Yet the use of enrichment culture and NASBA shortened the analysis time from 6 days to 26 h. The relative simplicity and rapidity of the NASBA assay make it an attractive alternative for detection of C. jejuni in food samples.
Analysis of factors affecting the accuracy, reproducibility, and interpretation of microbial community carbon source utilization patterns
Authors: Haack S K , Garchow H , Klug M J , Forney L J
Published: January 2020

We determined factors that affect responses of bacterial isolates and model bacterial communities to the 95 carbon substrates in Biolog microtiter plates. For isolates and communities of three to six bacterial strains, substrate oxidation rates were typically nonlinear and were delayed by dilution of the inoculum. When inoculum density was controlled, patterns of positive and negative responses exhibited by microbial communities to each of the carbon sources were reproducible. Rates and extents of substrate oxidation by the communities were also reproducible but were not simply the sum of those exhibited by community members when tested separately. Replicates of the same model community clustered when analyzed by principal-components analysis (PCA), and model communities with different compositions were clearly separated on the first PCA axis, which accounted for >60% of the dataset variation. PCA discrimination among different model communities depended on the extent to which specific substrates were oxidized. However, the substrates interpreted by PCA to be most significant in distinguishing the communities changed with reading time, reflecting the nonlinearity of substrate oxidation rates. Although whole-community substrate utilization profiles were reproducible signatures for a given community, the extent of oxidation of specific substrates and the numbers or activities of microorganisms using those substrates in a given community were not correlated. Replicate soil samples varied significantly in the rate and extent of oxidation of seven tested substrates, suggesting microscale heterogeneity in composition of the soil microbial community.
Inhibition, Inactivation, and Recovery of Ammonia-Oxidizing Activity in Cometabolism of Trichloroethylene by Nitrosomonas europaea
Authors: Hyman M R , Russell S A , Ely R L , Williamson K J , Arp D J
Published: January 2020

The kinetics of the cometabolism of trichloroethylene (TCE) by the ammonia-oxidizing soil bacterium Nitrosomonas europaea in short-term (<10-min) incubations were investigated. Three individual effects of TCE cometabolism on this bacterium were characterized. First, we observed that TCE is a potent competitive inhibitor of ammonia oxidation by N. europaea. The K(infi) value for TCE (30 (mu)M) is similar to the K(infm) for ammonia (40 (mu)M). Second, we examined the toxicity associated with TCE cometabolism by N. europaea. Stationary-phase cells of N. europaea oxidized approximately 60 nmol of TCE per mg of protein before ammonia-oxidizing activity was completely inactivated by reactive intermediates generated during TCE oxidation. At the TCE concentrations used in these experiments, ammonia did not provide significant protection against inactivation. Third, we have determined the ability of cells to recover ammonia-oxidizing activity after exposure to TCE. Cells recovering from TCE inactivation were compared with cells recovering from the specific inactivation of ammonia-oxidizing activity by light. The recovery kinetics were indistinguishable when 40% or less of the activity was inactivated. However, at increased levels of inactivation, TCE-inactivated cells did not recover as rapidly as light-inactivated cells. The kinetics of recovery appear to be dependent on both the extent of inactivation of ammonia-oxidizing activity and the degree of specificity of the inactivating treatment.
Microorganism gram-type differentiation based on pyrolysis-mass spectrometry of bacterial Fatty Acid methyl ester extracts
Authors: Basile F , Voorhees K J , Hadfield T L
Published: January 2020

Curie-point pyrolysis (Py)-mass spectrometry has been used to differentiate 19 microorganisms by Gram type on the basis of the methyl esters of their fatty acid distribution. The mass spectra of gram-negative microorganisms were characterized by the presence of palmitoleic acid (C(inf16:1)) and oleic acid (C(inf18:1)), as well as a higher abundance of palmitic acid (C(inf16:0)) than pentadecanoic acid (C(inf15:0)). For gram-positive microorganisms, a signal of branched C(inf15:0) (isoC(inf15:0) and/or anteisoC(inf15:0)) more intense than that of palmitic acid was observed in the mass spectra. Principal components analysis of these mass spectral data segregated the microorganisms investigated in this study into three discrete clusters that correlated to their gram reactions and pathogenicities. Further tandem mass spectrometric analysis demonstrated that the nature of the C(inf15:0) fatty acid isomer (branched or normal) present in the mass spectrum of each microorganism was important for achieving the classification into three clusters.
Anaerobic electron acceptor chemotaxis in Shewanella putrefaciens
Authors: Nealson K H , Moser D P , Saffarini D A
Published: January 2020

Shewanella putrefaciens MR-1 can grow either aerobically or anaerobically at the expense of many different electron acceptors and is often found in abundance at redox interfaces in nature. Such redox interfaces are often characterized by very strong gradients of electron acceptors resulting from rapid microbial metabolism. The coincidence of S. putrefaciens abundance with environmental gradients prompted an examination of the ability of MR-1 to sense and respond to electron acceptor gradients in the laboratory. In these experiments, taxis to the majority of the electron acceptors that S. putrefaciens utilizes for anaerobic growth was seen. All anaerobic electron acceptor taxis was eliminated by the presence of oxygen, nitrate, nitrite, elemental sulfur, or dimethyl sulfoxide, even though taxis to the latter was very weak and nitrate and nitrite respiration was normal in the presence of dimethyl sulfoxide. Studies with respiratory mutants of MR-1 revealed that several electron acceptors that could not be used for anaerobic growth nevertheless elicited normal anaerobic taxis. Mutant M56, which was unable to respire nitrite, showed normal taxis to nitrite, as well as the inhibition of taxis to other electron acceptors by nitrite. These results indicate that electron acceptor taxis in S. putrefaciens does not conform to the paradigm established for Escherichia coli and several other bacteria. Carbon chemo-taxis was also unusual in this organism: of all carbon compounds tested, the only positive response observed was to formate under anaerobic conditions.
Aerobic and anaerobic starvation metabolism in methanotrophic bacteria
Authors: Roslev P , King G M
Published: January 2020

The capacity for anaerobic metabolism of endogenous and selected exogenous substrates in carbon- and energy-starved methanotrophic bacteria was examined. The methanotrophic isolate strain WP 12 survived extended starvation under anoxic conditions while metabolizing 10-fold less endogenous substrate than did parallel cultures starved under oxic conditions. During aerobic starvation, the cell biomass decreased by 25% and protein and lipids were the preferred endogenous substrates. Aerobic protein degradation (24% of total protein) took place almost exclusively during the initial 24 h of starvation. Metabolized carbon was recovered mainly as CO(inf2) during aerobic starvation. In contrast, cell biomass decreased by only 2.4% during anaerobic starvation, and metabolized carbon was recovered mainly as organic solutes in the starvation medium. During anaerobic starvation, only the concentration of intracellular low-molecular-weight compounds decreased, whereas no significant changes were measured for cellular protein, lipids, polysaccharides, and nucleic acids. Strain WP 12 was also capable of a limited anaerobic glucose metabolism in the absence of added electron acceptors. Small amounts of CO(inf2) and organic acids, including acetate, were produced from exogenous glucose under anoxic conditions. Addition of potential anaerobic electron acceptors (fumarate, nitrate, nitrite, or sulfate) to starved cultures of the methanotrophs Methylobacter albus BG8, Methylosinus trichosporium OB3b, and strain WP 12 did not stimulate anaerobic survival. However, anaerobic starvation of these bacteria generally resulted in better survival than did aerobic starvation. The results suggest that methanotrophic bacteria can enter a state of anaerobic dormancy accompanied by a severe attenuation of endogenous metabolism. In this state, maintenance requirements are presumably provided for by fermentation of certain endogenous substrates. In addition, low-level catabolism of exogenous substrates may support long-term anaerobic survival of some methanotrophic bacteria.
Alterations in structure, chemistry, and biodegradability of grass lignocellulose treated with the white rot fungi Ceriporiopsis subvermispora and Cyathus stercoreus
Authors: Akin D E , Rigsby L L , Sethuraman A , Morrison W H , Gamble G R , Eriksson K E
Published: January 2020

The white rot fungi Ceriporiopsis subvermispora FP-90031-sp and Cyathus stercoreus ATCC 36910 were evaluated for their ability to delignify Bermuda grass (Cynodon dactylon) stems and improve biodegradability. Compositional and structural alterations in plant cell walls effected by the fungi were determined by nuclear magnetic resonance spectroscopy, gas chromatography of alkali-treated residues, microspectrophotometry, and electron microscopy. Contaminating bacteria and fungi, which grew from unsterilized Bermuda grass stems, did not alter the improvement in grass biodegradability by either of the fungi from that of gas-sterilized stems. The biodegradation of stems by ruminal microorganisms, after treatment for 6 weeks with C. subvermispora or C. stercoreus, was improved by 29 to 32% and by 63 to 77%, respectively; dry weight losses caused by pretreatment with the fungi were about 20% over that in untreated, control stems. Both fungi preferentially removed aromatics to carbohydrates, and C. subvermispora removed proportionately more guaiacyl units than did C. stercoreus. Substantial amounts of ester-linked p-coumaric and ferulic acids were removed by both fungi, and about 23 and 41% of total aromatics (determined after 4 M NaOH direct treatment) were removed from the plant biomass after incubation with C. subvermispora and C. stercoreus, respectively. UV absorption microspectrophotometry indicated that ester-linked phenolic acids were totally removed from the parenchyma cell walls, and these cells were readily and completely degraded by both fungi. However, aromatic constituents were only partially removed from the more recalcitrant sclerenchyma cell walls, resulting in variation in electron density and random digestion pits after incubation with fiber-degrading bacteria.(ABSTRACT TRUNCATED AT 250 WORDS)
Genetic and phenetic analyses of Bradyrhizobium strains nodulating peanut (Arachis hypogaea L.) roots
Authors: Van Rossum D , Schuurmans F P , Gillis M , Muyotcha A , Van Verseveld H W , Stouthamer A H , Boogerd F C
Published: January 2020

Seventeen Bradyrhizobium sp. strains and one Azorhizobium strain were compared on the basis of five genetic and phenetic features: (i) partial sequence analyses of the 16S rRNA gene (rDNA), (ii) randomly amplified DNA polymorphisms (RAPD) using three oligonucleotide primers, (iii) total cellular protein profiles, (iv) utilization of 21 aliphatic and 22 aromatic substrates, and (v) intrinsic resistances to seven antibiotics. Partial 16S rDNA analysis revealed the presence of only two rDNA homology (i.e., identity) groups among the 17 Bradyrhizobium strains. The partial 16S rDNA sequences of Bradyrhizobium sp. strains form a tight similarity (> 95%) cluster with Rhodopseudomonas palustris, Nitrobacter species, Afipia species, and Blastobacter denitrificans but were less similar to other members of the alpha-Proteobacteria, including other members of the Rhizobiaceae family. Clustering the Bradyrhizobium sp. strains for their RAPD profiles, protein profiles, and substrate utilization data revealed more diversity than rDNA analysis. Intrinsic antibiotic resistance yielded strain-specific patterns that could not be clustered. High rDNA similarity appeared to be a prerequisite, but it did not necessarily lead to high similarity values between RAPD profiles, protein profiles, and substrate utilization. The various relationship structures, coming forth from each of the studied features, had low compatibilities, casting doubt on the usefulness of a polyphasic approach in rhizobial taxonomy.
Frequency of generalized transducing phages in natural isolates of the Salmonella typhimurium complex
Authors: Schicklmaier P , Schmieger H
Published: January 2020

From 85 natural isolates of the Salmonella typhimurium complex, including the Salmonella reference collection A (P. Beltran, S. A. Plock, N. H. Smith, T. S. Whittam, D. C. Old, and R. K. Selander, J. Gen. Microbiol. 137:601-606, 1991), 65 strains (76.5%) released 71 different temperate phages. Forty-three (93.5%) of 46 tested phages were able to transduce the chromosomal markers his+ and trp+ and the cloning vector pBR325.
Detection of Lyme disease spirochetes in the skin of naturally infected wild sika deer (Cervus nippon yesoensis) by PCR
Authors: Kimura K , Isogai E , Isogai H , Kamewaka Y , Nishikawa T , Ishii N , Fujii N
Published: January 2020

We demonstrated the presence of Borrelia burgdorferi sensu lato DNA in the skin tissues of naturally infected wild sika deer, using PCR. The risk of transmission of B. burgdorferi sensu lato is recognized in sika deer.
Recovery and phylogenetic analysis of novel archaeal rRNA sequences from a deep-sea deposit feeder
Authors: McInerney J O , Wilkinson M , Patching J W , Embley T M , Powell R
Published: January 2020

In 1992, two independent reports based on small-subunit rRNA gene (SSU rDNA) cloning revealed the presence of novel Archaea among marine bacterioplankton. Here, we report the presence of further novel Archaea SSU rDNA sequences recovered from the midgut contents of a deep-sea marine holothurian. Phylogenetic analyses show that these abyssal Archaea are a paraphyletic component of a highly divergent clade that also includes some planktonic sequences. Our data confirm that this clade is a deep-branching lineage in the tree of life.
An apparent anomaly in the calculation of ash-free dry weights for the determination of cellular yields
Authors: Battley E H
Published: January 2020

The use of ash-free dry weights results in a determination of CHON-containing cell mass that is 5 to 6% less than that determined on the basis of total cellular composition. This discrepancy derives from the fact that phosphorus and sulfur contribute greater weight to ash as oxides and salts of P and S than the contribution they make as part of the cellular fabric. In addition, in the absence of P and S, the unit carbon formula for this mass becomes changed, on the basis of an equivalent number of available electrons per unit of carbon formula weight.
Identification of possible artifacts in the association of official analytical chemists sporicidal test
Authors: Miner N A , Mulberry G K , Starks A N , Powers-Prather A , Entrup M , Armstrong M , Maida B
Published: January 2020

Two laboratories tested four different brands of alkaline 2% glutaraldehyde sterilants by the Association of Official Analytical Chemists sporicidal test. Each laboratory found survival of Clostridium sporogenes spores on spore-labeled unglazed porcelain penicylinders (cylinders) to vary from test to test, and survival did not always correlate with increasing sterilant exposure time. These results were consistent with a theory that there may be random conditions within the test that prevent the sterilant from contacting all spores. Further studies indicated that the prior history of the unglazed porcelain cylinders and whether the C. sporogenes culture grown in egg-meat media had been processed (homogenized) to eliminate visible pieces of egg-meat media were important factors affecting the results and repeatability of this test.
Xylanase IV, an Exoxylanase of Aeromonas caviae ME-1 Which Produces Xylotetraose as the Only Low-Molecular-Weight Oligosaccharide from Xylan
Authors: Kubata B K , Takamizawa K , Kawai K , Suzuki T , Horitsu H
Published: January 2020

A novel xylanase (xylanase IV) which produces xylotetraose as the only low-molecular-weight oligosaccharide from oat spelt xylan was isolated from the culture medium of Aeromonas caviae ME-1. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the xylanase IV molecular weight was 41,000. Xylanase IV catalyzed the hydrolysis of oat spelt xylan, producing exclusively xylotetraose. The acid hydrolysate of the product gave d-xylose. The enzyme did not hydrolyze either p-nitrophenyl-(beta)-d-xyloside, small oligosaccharides (xylobiose and xylotetraose), or polysaccharides, such as starch, cellulose, carboxymethyl cellulose, laminarin, and (beta)-1,3-xylan.
Methanogenic and other strictly anaerobic bacteria in desert soil and other oxic soils
Authors: Peters V , Conrad R
Published: January 2020

Strictly anaerobic bacteria such as methanogenic, sulfate-reducing, and homoacetogenic bacteria could be enriched from all five oxic soils tested. The number of cells was lower than that in typical anoxic habitats. Spores did not always dominate the population of sulfate-reducing bacteria. In all soils, the methanogenic population displayed a long lag phase after anoxic conditions were imposed before methane production began.
Phylogenetic characterization of the epibiotic bacteria associated with the hydrothermal vent polychaete Alvinella pompejana
Authors: Haddad A , Camacho F , Durand P , Cary S C
Published: January 2020

Alvinella pompejana is a polychaetous annelid that inhabits active deep-sea hydrothermal vent sites along the East Pacific Rise, where it colonizes the walls of actively venting high-temperature chimneys. An abundant, morphologically diverse epibiotic microflora is associated with the worm's dorsal integument, with a highly integrated filamentous morphotype clearly dominating the microbial biomass. It has been suggested that this bacterial population participates in either the nutrition of the worm or in detoxification of the worm's immediate environment. The primary goal of this study was to phylogenetically characterize selected epibionts through the analysis of 16S rRNA gene sequences. Nucleic acids were extracted from bacteria collected from the dorsal surface of A. pompejana. 16S rRNA genes were amplified with universal bacterial primers by the PCR. These genes were subsequently cloned, and the resulting clone library was screened by restriction fragment length polymorphism analysis to identify distinct clone types. The restriction fragment length polymorphism analysis identified 32 different clone families in the library. Four of these families were clearly dominant, representing more than 65% of the library. Representatives from the four most abundant clone families were chosen for complete 16S rRNA gene sequencing and phylogenetic analysis. These gene sequences were analyzed by a variety of phylogenetic inference methods and found to be related to the newly established epsilon subdivision of the division Proteobacteria. Secondary structural model comparisons and comparisons of established signature base positions in the 16S rRNA confirmed the placement of the Alvinella clones in the epsilon subdivision of the Proteobacteria.
Chitinolytic Enterobacter agglomerans Antagonistic to Fungal Plant Pathogens
Authors: Chernin L , Ismailov Z , Haran S , Chet I
Published: January 2020

Three Enterobacter agglomerans strains which produce and excrete proteins with chitinolytic activity were found while screening soil-borne bacteria antagonistic to fungal plant pathogens. The chitinolytic activity was induced when the strains were grown in the presence of colloidal chitin as the sole carbon source. It was quantitated by using assays with chromogenic p-nitrophenyl analogs of disaccharide, trisaccharide, and tetrasaccharide derivatives of N-acetylglucosamine. A set of three fluorescent substrates with a 4-methylumbelliferyl group linked by (beta)-1,4 linkage to N-acetylglucosamine mono- or oligosaccharides were used to identify the chitinolytic activities of proteins which had been renatured following their separation by electrophoresis. This study provides the most complete evidence for the presence of a complex of chitinolytic enzymes in Enterobacter strains. Four enzymes were detected: two N-acetyl-(beta)-d-glucosaminidases of 89 and 67 kDa, an endochitinase with an apparent molecular mass of 59 kDa, and a chitobiosidase of 50 kDa. The biocontrol ability of the chitinolytic strains was demonstrated under greenhouse conditions. The bacteria decreased the incidence of disease caused by Rhizoctonia solani in cotton by 64 to 86%. Two Tn5 mutants of one of the isolates, which were deficient in chitinolytic activity, were unable to protect plants against the disease.
Influence of various dust sampling and extraction methods on the measurement of airborne endotoxin
Authors: Douwes J , Versloot P , Hollander A , Heederik D , Doekes G
Published: January 2020

The influence of various filter types and extraction conditions on the quantitation of airborne endotoxin with the Limulus amebocyte lysate test was studied by using airborne dusts sampled in a potato processing plant. Samples were collected with an apparatus designed to provide parallel samples. Data from the parallel-sampling experiment were statistically evaluated by using analysis of variance. In addition, the influence of storage conditions on the detectable endotoxin concentration was investigated by using commercially available lipopolysaccharides (LPS) and endotoxin-containing house dust extracts. The endotoxin extraction efficiency of 0.05% Tween 20 in pyrogen-free water was seven times higher than that of pyrogen-free water only. Two-times-greater amounts of endotoxin were extracted from glass fiber, Teflon, and polycarbonate filters than from cellulose ester filters. The temperature and shaking intensity during extraction were not related to the extraction efficiency. Repeated freeze (-20 degrees C)-and-thaw cycles with commercial LPS reconstituted in pyrogen-free water had a dramatic effect on the detectable endotoxin level. A 25% loss in endotoxin activity per freeze-thaw cycle was observed. Storage of LPS samples for a period of 1 year at 7 degrees C had no effect on the endotoxin level. House dust extracts showed a decrease of about 20% in the endotoxin level after they had been frozen and thawed for a second time. The use of different container materials (borosilicate glass, "soft" glass, and polypropylene) did not result in different endotoxin levels. This study indicates that the assessment of endotoxin exposure may differ considerably between groups when different sampling, extraction, and storage procedures are employed.
Insertional Mutations in the Hydrogenase vhc and frc Operons Encoding Selenium-Free Hydrogenases in Methanococcus voltae
Authors: Berghofer Y , Klein A
Published: January 2020

Methanococcus voltae, which contains four different gene groups that encode [NiFe]-hydrogenases, was transformed with integration vectors to achieve polar inactivation of two of the four hydrogenase operons that encode the selenium-free enzymes Vhc and Frc. Transformants which were selected by their acquired puromycin resistance showed site-specific insertions in either the vhc or frc operon by single crossover events. Southern hybridization revealed tandem integrations of whole vectors in the vhc operon, whereas only one vector copy was found in the frc operon. Northern (RNA) hybridizations showed a pac transcript of defined size, indicating strong termination in front of the hydrogenase genes downstream. In spite of the apparent abolition of expression of selenium-free hydrogenases through these polar insertions, they were not lethal to cells upon growth in selenium-deprived minimal medium, which we had previously shown to strongly induce transcription of the respective operons in M. voltae. Instead, like wild-type control cultures, transformants responded to selenium deprivation only with a reduction in growth rate. We conclude that loss of the potential to express a selenium-free hydrogenase can nevertheless be balanced by very small amounts of selenium hydrogenases under laboratory conditions in which the hydrogen supply is not likely to be a limiting growth factor.
Flow cytometric analysis of activated sludge with rRNA-targeted probes
Authors: Wallner G , Erhart R , Amann R
Published: January 2020

Samples from a wastewater treatment plant were hybridized with fluorescein-labeled oligonucleotide probes specific for members of the domains Bacteria and Eucarya; the alpha, beta, and gamma subclasses of the class Proteobacteria; or the genus Acinetobacter. Subsequently, they were counterstained with the DNA-specific dye Hoechst 33342 and analyzed by flow cytometry. By quantifying forward angle light scatter and Hoechst- and probe-conferred fluorescence as measures for cell size, DNA content, and rRNA content, respectively, not only relative abundances but also assessments of general metabolic activity for each of these groups were obtained. Hybridizations with a positive control probe binding to all bacteria showed that in the activated-sludge samples examined, 70 to 80% of the Hoechst-stained cells could unambiguously be identified by this method. The majority of the detected cells (approximately 40%) were beta-subclass Proteobacteria. Flow cytometric and microscopic counts were in general agreement. Discrepancies were found in particular for those populations that occurred predominantly in flocs (alpha subclass of the Proteobacteria) or chains (Acinetobacter spp.). Although the dispersal of aggregates needs to be improved, flow cytometry combined with rRNA-based in situ probing appears to be a powerful tool for the rapid and highly automated analysis of the microbial communities in activated sludge.
xylA cloning and sequencing and biochemical characterization of xylose isomerase from Thermotoga neapolitana
Authors: Vieille C , Hess J M , Kelly R M , Zeikus J G
Published: January 2020

The xylA gene coding for xylose isomerase from the hyperthermophile Thermotoga neapolitana 5068 was cloned, sequenced, and expressed in Escherichia coli. The gene encoded a polypeptide of 444 residues with a calculated molecular weight of 50,892. The native enzyme was a homotetramer with a molecular weight of 200,000. This xylose isomerase was a member of the family II enzymes (these differ from family I isomerases by the presence of approximately 50 additional residues at the amino terminus). The enzyme was extremely thermostable, with optimal activity above 95 degrees C. The xylose isomerase showed maximum activity at pH 7.1, but it had high relative activity over a broad pH range. The catalytic efficiency (kcat/Km) of the enzyme was essentially constant between 60 and 90 degrees C, and the catalytic efficiency decreased between 90 and 98 degrees C primarily because of a large increase in Km. The T. neapolitana xylose isomerase had a higher turnover number and a lower Km for glucose than other family II xylose isomerases. Comparisons with other xylose isomerases showed that the catalytic and cation binding regions were well conserved. Comparison of different xylose isomerase sequences showed that numbers of asparagine and glutamine residues decreased with increasing enzyme thermostability, presumably as a thermophilic strategy for diminishing the potential for chemical denaturation through deamidation at elevated temperatures.
Lignin oxidation by laccase isozymes from Trametes versicolor and role of the mediator 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate) in kraft lignin depolymerization
Authors: Bourbonnais R , Paice M G , Reid I D , Lanthier P , Yaguchi M
Published: January 2020

Two laccase isozymes (I and II) produced by the white-rot fungus Trametes versicolor were purified, and their reactivities towards various substrates and lignins were studied. The N-terminal amino acid sequences of these enzymes were determined and compared to other known laccase sequences. Laccase II showed a very high sequence similarity to a laccase which was previously reported to depolymerize lignin. The reactivities of the two isozymes on most of the substrates tested were similar, but there were some differences in the oxidation rate of polymeric substrates. We found that the two laccases produced similar qualitative effects on kraft lignin and residual lignin in kraft pulp, with no evidence of a marked preference for depolymerization by either enzyme. However, the presence of the mediator 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate) prevented and reversed the polymerization of kraft lignin by either laccase. The delignification of hardwood and softwood kraft pulps with the two isozymes and the mediator was compared; either laccase was able to reduce the kappa number of pulp, but only in the presence of 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate).
Evidence for the Adhesive Function of the Exopolysaccharide of Hyphomonas Strain MHS-3 in Its Attachment to Surfaces
Authors: Quintero E J , Weiner R M
Published: January 2020

Hyphomonas strain MHS-3 (MHS-3) is a marine procaryote with a biphasic life cycle and which has prosthecate stages that adhere to submerged substrata. We found that adherent forms produced an exopolysaccharide (EPS) capsule that bound Glycine max lectin, Arachis hypogaea lectin, and Bauhinia purpurea lectin (BPA), each having affinity for N-acetyl-d-galactosamine. It also bound the dye Calcofluor. BPA and Calcofluor were tested for the ability to hinder MHS-3 adhesion to glass surfaces; they reduced attachment by >50 and >85%, respectively. Periodate treatment also reduced attachment (by >80%), but pronase treatment did not. Furthermore, an EPS(sup-) variant, Hyphomonas strain MHS-3 rad, did not attach well to surfaces. These results suggest that the MHS-3 EPS capsule is an adhesin.
Tri6 encodes an unusual zinc finger protein involved in regulation of trichothecene biosynthesis in Fusarium sporotrichioides
Authors: Proctor R H , Hohn T M , McCormick S P , Desjardins A E
Published: January 2020

In Fusarium sporotrichioides, several genes required for biosynthesis of the trichothecene mycotoxin T-2 toxin are closely linked. Further characterization of this gene cluster has revealed a gene, Tri6, that specifies a 217-amino-acid protein with regions similar to Cys2His2 zinc finger proteins. Temporal expression of Tri6 is similar to that of trichothecene biosynthetic pathway genes. Analysis of Tri6 transcripts indicated that transcription is initiated in two regions and that within each region there may be at least four initiation sites. Disruption of Tri6 resulted in a mutant that did not produce trichothecenes but that did accumulate low levels of the trichothecene precursor trichodiene. The Tri6 mutant was unable to convert six trichothecene biosynthetic intermediates to T-2 toxin, and transcription of two biosynthetic genes, Tri4 and Tri5, was greatly reduced in the mutant relative to the wild type. In addition, the product of Tri6 functioned as a transcriptional activator in Saccharomyces cerevisiae when fused to the DNA binding region of GAL4. These results indicate that Tri6 encodes a protein involved in the transcriptional regulation of trichothecene biosynthetic genes in F. sporotrichioides.
DNA polymorphisms in strains of Legionella pneumophila serogroups 3 and 4 detected by macrorestriction analysis and their use for epidemiological investigation of nosocomial legionellosis
Authors: Lück P C , Köhler J , Maiwald M , Helbig J H
Published: January 2020

Genomic DNAs of clinical and environmental isolates of Legionella pneumophila belonging to serogroups 3 and 4 were analyzed by macrorestriction analysis by pulsed-field gel electrophoresis. The restriction enzymes SfiI and NotI allowed easy visual separation of epidemiologically unrelated serogroup 3 strains. Three unrelated serogroup 3 strains that were isolated from different locations were identical by this genome mapping technique. Five unrelated serogroup 4 strains were separable by this technique. The electrophoretic patterns obtained after SfiI or NotI cleavage of the DNA of strains isolated from four patients with hospital-acquired legionellosis were identical to the patterns of strains isolated from the hot water supply systems of the buildings in which the patients were hospitalized. In conclusion, macrorestriction analysis is a valuable tool for epidemiological studies of infections caused by L. pneumophila serogroups 3 and 4.
A unique endoglucanase-encoding gene cloned from the phytopathogenic fungus Macrophomina phaseolina
Authors: Wang H , Jones R W
Published: January 2020

The deduced amino acid sequence derived from a Macrophomina phaseolina beta-1,4-endoglucanase-encoding gene revealed 48% identity (over 119 amino acids) with egl1 from the phytopathogen Pseudomonas solanacearum. Its similarity to saprophyte endoglucanases was not significant. Its minimum substrate size, unlike that of any known saprophyte endoglucanase, was cellopentaose. The unique characteristics of M. phaseolina egl1-encoded endoglucanase suggest that it is phytopathogen specific.
An indirect enzyme immunoassay for the mycotoxin citrinin
Authors: Abramson D , Usleber E , Märtlbauer E
Published: January 2020

An indirect competitive enzyme immunoassay using rabbit antisera could detect citrinin in buffer solutions at 1 to 13 ng/ml (0.05 to 0.65 ng per assay). Cross-reactivity with austdiol, alternariol, ochratoxin A, and deoxynivalenol was < 0.1% relative to citrinin. Recovery of citrinin added to wheat flour at 200 to 2,000 ng/g was 89 to 104%, with a coefficient of variation of 6.9 to 13%.
Cloning and characterization of the abortive infection genetic determinant abiD isolated from pBF61 of Lactococcus lactis subsp. lactis KR5
Authors: McLandsborough L A , Kolaetis K M , Requena T , McKay L L
Published: January 2020

A 6.3-kb fragment from pBF61 in Lactococcus lactis subsp. lactis KR5 was cloned and found to confer an abortive phage infection (Abi+) phenotype exhibiting a reduction in efficiency of plating and plaque size for small isometric- and prolate-headed bacteriophages sk1 and c2, respectively, and to produce a 10-fold decrease in c2 phage burst size. Phage adsorption was not significantly reduced. An open reading frame of 1,098 bp was sequenced and designated abiD. Tn5 mutagenesis confirmed that abiD was required for the Abi+ phenotype.
Starvation- and stationary-phase-induced acid tolerance in Escherichia coli O157:H7
Authors: Arnold K W , Kaspar C W
Published: January 2020

Stationary phase and the starvation of log-phase cells increased the acid tolerance of Escherichia coli O157:H7 strains. Although the degree of acid tolerance varied, the survival of most O157:H7 strains exceeded that of other, related, pathogens in a synthetic gastric fluid.
Development of Bacillus thuringiensis CryIC Resistance by Spodoptera exigua (Hubner) (Lepidoptera: Noctuidae)
Authors: Moar W J , Pusztai-Carey M , Van Faassen H , Bosch D , Frutos R , Rang C , Luo K , Adang M J
Published: January 2020

Selection of resistance in Spodoptera exigua (Hubner) to an HD-1 spore-crystal mixture, CryIC (HD-133) inclusion bodies, and trypsinized toxin from Bacillus thuringiensis subsp. aizawai and B. thuringiensis subsp. entomocidus was attempted by using laboratory bioassays. No resistance to the HD-1 spore-crystal mixture could be achieved after 20 generations of selection. Significant levels of resistance (11-fold) to CryIC inclusion bodies expressed in Escherichia coli were observed after seven generations. Subsequent selection of the CryIC-resistant population with trypsinized CryIC toxin resulted, after 21 generations of CryIC selection, in a population of S. exigua that exhibited only 8% mortality at the highest toxin concentration tested (320 (mu)g/g), whereas the 50% lethal concentration was 4.30 (mu)g/g for the susceptible colony. Insects resistant to CryIC toxin from HD-133 also were resistant to trypsinized CryIA(b), CryIC from B. thuringiensis subsp. entomocidus, CryIE-CryIC fusion protein (G27), CryIH, and CryIIA. In vitro binding experiments with brush border membrane vesicles showed a twofold decrease in maximum CryIC binding, a fivefold difference in K(infd), and no difference in the concentration of binding sites for the CryIC-resistant insects compared with those for the susceptible insects. Resistance to CryIC was significantly reduced by the addition of HD-1 spores. Resistance to the CryIC toxin was still observed 12 generations after CryIC selection was removed. These results suggest that, in S. exigua, resistance to a single protein is more likely to occur than resistance to spore-crystal mixtures and that once resistance occurs, insects will be resistant to many other Cry proteins. These results have important implications for devising S. exigua resistance management strategies in the field.
Detection and counting of Nitrobacter populations in soil by PCR
Authors: Degrange V , Bardin R
Published: January 2020

Although the biological conversion of nitrite to nitrate is a well-known process, studies of Nitrobacter populations are hindered by their physiological characteristics. This report describes a new method for detecting and counting Nitrobacter populations in situ with the PCR. Two primers from the 16S rRNA gene were used to generate a 397-bp fragment by amplification of Nitrobacter species DNA. No signal was detected from their phylogenetic neighbors or the common soil bacteria tested. Extraction and purification steps were optimized for minimal loss and maximal purity of soil DNA. The detection threshold and accuracy of the molecular method were determined from soil inoculated with 10, 10(2), or 10(3) Nitrobacter hamburgensis cells per g of soil. Counts were also done by the most-probable-number (MPN)-Griess and fluorescent antibody methods. PCR had a lower detection threshold (10(2) Nitrobacter cells per g of soil) than did the MPN-Griess or fluorescent antibody method. When PCR amplification was coupled with the MPN method, the counting rate reached 65 to 72% of inoculated Nitrobacter cells. Tested on nonsterile soil, this rapid procedure was proved efficient.
Quantitation of fungal mRNAs in complex substrates by reverse transcription PCR and its application to Phanerochaete chrysosporium-colonized soil
Authors: Lamar R T , Schoenike B , Vanden Wymelenberg A , Stewart P , Dietrich D M , Cullen D
Published: January 2020

Thorough analysis of fungi in complex substrates has been hampered by inadequate experimental tools for assessing physiological activity and estimating biomass. We report a method for the quantitative assessment of specific fungal mRNAs in soil. The method was applied to complex gene families of Phanerochaete chrysosporium, a white-rot fungus widely used in studies of organopollutant degradation. Among the genes implicated in pollutant degradation, two closely related lignin peroxidase transcripts were detected in soil. The pattern of lignin peroxidase gene expression was unexpected; certain transcripts abundant in defined cultures were not detected in soil cultures. Transcripts encoding cellobiohydrolases and beta-tubulin were also detected. The method will aid in defining the roles of specific genes in complex biological processes such as organopollutant degradation, developing strategies for strain improvement, and identifying specific fungi in environmental samples.