bluuebunny/crossref_metadata_2025 huggingface dataset:
Optimal staining and sample storage time for direct microscopic enumeration of total and active bacteria in soil with two fluorescent dyes
Authors: Yu W , Dodds W K , Banks M K , Skalsky J , Strauss E A
Published: January 2020

Direct counting techniques, first developed for aquatic samples, can be used to enumerate bacteria in soil and groundwater sediments. Two fluorescent dyes, 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) for actively respiring bacteria and 4(prm1),6-diamidino-2-phenylindole (DAPI) for total bacteria, were tested for their usefulness in epifluorescent direct bacterial enumeration in soil. Both dyes can be used for the same soil sample without affecting enumeration results. Staining for 8 h with CTC and for 40 min with DAPI resulted in maximum numbers of stained cells. The optimal DAPI staining concentration is 10 mg liter(sup-1). After preparation, slides should be stored at 4(deg)C and counted within 2 days for CTC and within 24 h for DAPI. Sodium PP(infi) or sodium chloride solutions were used to desorb bacteria from soil prior to counting. Counts were significantly higher when sodium chloride was used.
Survival and Activity of lux-Marked Aeromonas salmonicida in Seawater
Authors: Ferguson Y , Glover L A , McGillivray D M , Prosser J I
Published: January 2020

The fish pathogen Aeromonas salmonicida was chromosomally marked with genes encoding bacterial luciferase, luxAB, isolated from Vibrio fischeri, resulting in constitutive luciferase production. During exponential growth in liquid batch culture, luminescence was directly proportional to biomass concentration, and luminometry provided a lower detection limit of approximately 10(sup3) cells ml(sup-1), 1 order of magnitude more sensitive than enzyme-linked immunosorbent assay detection. In sterile seawater at 4(deg)C, lux-marked A. salmonicida entered a dormant, nonculturable state and population activity decreased rapidly. The activity per viable cell, however, increased by day 4, indicating that a proportion of the population remained active and culturable. Putative dormant cells were not resuscitated after the addition of a range of substrates.
Purification and Properties of Component B of 2,4,5-Trichlorophenoxyacetate Oxygenase from Pseudomonas cepacia AC1100
Authors: Xun L , Wagnon K B
Published: January 2020

Pseudomonas cepacia AC1100 degrades 2,4,5-trichlorophenoxyacetate (2,4,5-T), an herbicide and chlorinated aromatic compound. Although some progress has been made in understanding 2,4,5-T degradation by AC1100 by molecular analysis, little is known about the biochemistry involved. Enzymatic activity converting 2,4,5-T to 2,4,5-trichlorophenol in the presence of NADH and O(inf2) was detected in cell extracts of AC1100. Phenyl agarose chromatography of the ammonium sulfate-fractionated cell extracts yielded no active single fractions, but the mixing of two fractions, named component A and component B, resulted in the recovery of enzyme activity. Component B was further purified to homogeneity by hydroxyapatite and DEAE chromatographies. Component B had a native molecular weight of 140,000, and it was composed of two 49-kDa (alpha)-subunits and two 24-kDa (beta)-subunits. Component B was red, and its spectrum in the visible region had maxima at 430 and 560 nm (shoulder), whereas upon reduction it had maxima at 420 (shoulder) and 530 nm. Each mole of (alpha)(beta) heterodimer contained 2.9 mol of iron and 2.1 mol of labile sulfide. These properties suggest strong similarities between component B and the terminal oxygenase components of the aromatic ring-hydroxylating dioxygenases. Component A was highly purified but not to homogeneity. The reconstituted 2,4,5-T oxygenase, consisting of components A and B, converted 2,4,5-T quantitatively into 2,4,5-trichlorophenol and glyoxylate with the coconsumption of NADH and O(inf2).
In vivo production, stabilization, and infectivity of baculovirus preoccluded virions
Authors: Hughes P R , Wood H A
Published: January 2020

Wild-type and polyhedrin-negative isolates of Autographa californica nuclear polyhedrosis virus were replicated in fifth-instar Trichoplusia ni larvae. Insect tissues infected with wild-type virus contained two types of virions that are highly infectious when ingested, those occluded in polyhedra and preoccluded virions. Tissue infected with the polyhedrin-negative virus contained only preoccluded virions. The relative potencies of the two types of infected tissue were determined by dose-mortality bioassays by using the neonate droplet feeding procedure. On a fresh weight basis, preparations of tissues infected with the polyhedrin-negative virus were approximately four times more potent than equivalent preparations of tissue infected with wild-type virus. Approximately half of the observed potency of the wild-type-virus preparations was due to polyhedra, and the remaining activity was due to preoccluded virions present in the tissue. The potency of the polyhedrin-negative preparations was not reduced significantly by lyophilization. The polyhedrin-negative isolate produced about 60% more infectious virus per unit of larval weight than did the wild-type isolate. The ability to produce large amounts of high-potency viral preparations in larvae and the convenience of being able to lyophilize the preparations for long-term storage shows promise for the use of preoccluded virus preparations as biopesticides.
Effect of signal peptide alterations and replacement on export of xylanase A in Streptomyces lividans
Authors: Pagé N , Kluepfel D , Shareck F , Morosoli R
Published: January 2020

Starting from its translation initiation site, the Streptomyces lividans xylanase A signal peptide consists of 41 amino acids. This signal peptide was deleted and successively replaced with one of six signal peptides from other enzymes secreted by S. lividans and by a signal peptide from the outer membrane protein (LamB) of Escherichia coli. Deletion of the xylanase A signal peptide or modification of its cleavage site abolished secretion of the enzyme. Replacement with the signal peptides of either xylanase B, cellulase A, mannanase, or acetylxylan esterase produced equivalent amounts of xylanase A, while the signal peptides of cellulase B, xylanase C, and LamB secreted less enzyme than did the wild type. All the clones exhibited the same transcription levels, which indicated that the variations in xylanase production were due to the natures of the signal sequences.
Potential virulence of viable but nonculturable Shigella dysenteriae type 1
Authors: Rahman I , Shahamat M , Chowdhury M A , Colwell R R
Published: January 2020

We examined a virulent strain of Shigella dysenteriae type 1 after induction into the viable but nonculturable (VBNC) state for its ability to (i) maintain the Shiga toxin (stx) gene; (ii) maintain biologically active Shiga toxin (ShT); and (iii) adhere to intestinal epithelial cells (Henle 407 cell line). PCR was used to amplify the stx gene from VBNC cells of S. dysenteriae type 1, thereby establishing its presence even when cells are in the VBNC state. VBNC S. dysenteriae type 1 ShT was monitored by the enzyme-linked immunosorbent assay with mouse monoclonal antibodies against the B subunit of ShT and affinity-purified rabbit polyclonal antibodies against ShT. We used the Henle 407 cell line to study the adhesive property of VBNC S. dysenteriae type 1 cells in a series of tissue culture experiments. Results showed that VBNC S. dysenteriae type 1 not only maintained the stx gene and biologically active ShT but also remained capable of adhering to Henle 407 cells. However, S. dysenteriae type 1 cells lost the ability to invade Henle 407 cells after entering the VBNC state. From results of the study, we conclude that VBNC cells of S. dysenteriae type 1 retain several virulence factors and remain potentially virulent, posing a public health problem.
Degradation of pyrene, benz[a]anthracene, and benzo[a]pyrene by Mycobacterium sp. strain RJGII-135, isolated from a former coal gasification site
Authors: Schneider J , Grosser R , Jayasimhulu K , Xue W , Warshawsky D
Published: January 2020

The degradation of three polycyclic aromatic hydrocarbons (PAH), pyrene (PYR), benz[a]anthracene (BAA), and benzo[a]pyrene (BaP), by Mycobacterium sp. strain RJGII-135 was studied. The bacterium was isolated from an abandoned coal gasification site soil by analog enrichment techniques and found to mineralize [14C]PYR. Further degradation studies with PYR showed three metabolites formed by Mycobacterium sp. strain RJGII-135, including 4,5-phenanthrene-dicarboxylic acid not previously isolated, 4-phenanthrene-carboxylic acid, and 4,5-pyrene-dihydrodiol. At least two dihydrodiols, 5,6-BAA-dihydrodiol and 10,11-BAA-dihydrodiol, were confirmed by high-resolution mass spectral and fluorescence analyses as products of the biodegradation of BAA by Mycobacterium sp. strain RJGII-135. Additionally, a cleavage product of BAA was also isolated. Mass spectra and fluorescence data support two different routes for the degradation of BaP by Mycobacterium sp. strain RJGII-135. The 7,8-BaP-dihydrodiol and three cleavage products of BaP, including 4,5-chrysene-dicarboxylic acid and a dihydro-pyrene-carboxylic acid metabolite, have been isolated and identified as degradation products formed by Mycobacterium sp. strain RJGII-135. These latter results represent the first example of the isolation of BaP ring fission products formed by a bacterial isolate. We propose that while this bacterium appears to attack only one site of the PYR molecule, it is capable of degrading different sites of the BAA and BaP molecules, and although the sites of attack may be different, the ability of this bacterium to degrade these PAH is well supported. The proposed pathways for biodegradation of these compounds by this Mycobacterium sp. strain RJGII-135 support the dioxygenase enzymatic processes reported previously for other bacteria. Microorganisms like Mycobacterium sp. strain RJGII-135 will be invaluable in attaining the goal of remediation of sites containing mixtures of these PAH.
Dynamics of Substrate Consumption and Enzyme Synthesis in Chelatobacter heintzii during Growth in Carbon-Limited Continuous Culture with Different Mixtures of Glucose and Nitrilotriacetate
Authors: Bally M , Egli T
Published: January 2020

Regulation of nitrilotriacetate (NTA) degradation and expression of NTA monooxygenase (NTA-MO) in the NTA-degrading strain Chelatobacter heintzii ATCC 29600 in continuous culture at a dilution rate of 0.06 h(sup-1) under transient growth conditions when the feed was switched between media containing NTA, glucose, or different mixtures thereof as the sole carbon and energy sources was investigated. A transition from NTA to glucose was accompanied by a rapid loss of NTA-MO. A transition from glucose to NTA resulted in a lag phase of some 25 h until NTA-MO expression started, and approximately 100 h was needed before a steady state for NTA-MO specific activity was reached. This transient lag phase was markedly shortened when mixtures of NTA plus glucose were supplied instead of NTA only; for example, when a mixture of 90% glucose and 10% NTA was used, induction of NTA-MO was detected after 30 min. This suggests a strong positive influence of alternative carbon substrates on the expression of other enzymes under natural environmental conditions. Regulation of NTA-MO expression and the fate of NTA-MO were also studied during starvation of both glucose-grown and NTA-grown cultures. Starvation of NTA-grown cells led to a loss of NTA-MO protein. No synthesis of NTA-MO (derepression) was observed when glucose-grown cells were starved.
Production of R-(-)-Ketoprofen from an Amide Compound by Comamonas acidovorans KPO-2771-4
Authors: Yamamoto K , Otsubo K , Matsuo A , Hayashi T , Fujimatsu I , Komatsu K
Published: January 2020

R-(-)-2-(3(prm1)-Benzoylphenyl)propionic acid [R-(-)-ketoprofen] was produced from racemic 2-(3(prm1)-benzoylphenyl)propionamide (keto-amide) by the isolated bacterial strain Comamonas acidovorans KPO-2771-4. Sodium fumarate as the carbon source and 2-azacyclononanone or isobutyronitrile as the enhancer in the culture medium were effective for bacterial growth and the enhancement of R-(-)-ketoprofen-producing activity. R-(-)-Ketoprofen produced from the keto-amide by resting cells was present in 99% enantiomeric exess. C. acidovorans KPO-2771-4 has an R-enantioselective amidase for keto-amide because the purified amidase from the bacterium hydrolyzed keto-amide, producing optically pure R-ketoprofen and ammonia.
A new in vivo fluorimetric technique to measure growth of adhering phototrophic microorganisms
Authors: Karsten U , Klimant I , Holst G
Published: January 2020

We developed a noninvasive rapid fluorimetric method for the investigation of growth of adhering (benthic) phototrophic microorganisms. The technique is based on the sensitive detection of the in vivo fluorescence of chlorophylls chlorophyll a and bacteriochlorophyll a and monitors increases in signal over time as an indicator for growth. The growth fluorimeter uses modulated excitation light of blue-light-emitting diodes and a photodiode as the detector. The light-emitting diodes are mounted geometrically in an aluminum housing for efficient and uniform illumination of the bottoms of the growth containers. The fluorimeter was characterized with respect to detection limit and dynamic range. This system is capable of resolving in vivo chlorophyll a concentrations of 0.5 (mu)g liter(sup-1) in cyanobacteria and 0.03 (mu)g liter(sup-1) in diatoms as well as in vivo bacteriochlorophyll a concentrations in phototrophic bacteria of 0.3 (mu)g liter(sup-1), which points to an extremely high sensitivity compared with that of similar available techniques. Thus, the new fluorimeter allows the determination of growth at extremely low cell densities. The instrument was used successfully to measure the growth of several adhering isolates of the filamentous cyanobacterium Microcoleus chthonoplastes from benthic microbial mats in seawater of different salinities. The data obtained demonstrate broad growth responses for all strains, which thus can be characterized as euryhaline organisms.
Diversity of aquatic actinomycetes in lakes of the middle plateau, yunnan, china
Authors: Jiang C , Xu L
Published: January 2020

A total of 749 sediment and water samples were collected from 12 lakes of the Middle Plateau of Yunnan from 1983 to 1993. The diversity and biological characteristics of the aquatic actinomycetes in these lakes were studied. Sixteen genera of actinomycetes were isolated from these samples. Micromonospores assumed a notable dominance (from 39 to 89%) in the actinomycete populations of these lake sediments. Streptomycetes were the second most abundant organisms. The diversity and counts of actinomycetes varied with the season. Thermophilic actinomycetes have a wide distribution in these lakes, but their counts were smaller. The cell wall compositions of certain Micromonospora and Streptomyces strains from an alkaline lake revealed an unusual combination of glycine and isomers of diaminopimelic acid. It seems that aquatic actinomycetes play a significant role in the decomposition of organic substances, including some toxic compounds such as phenol, in these lakes. It also appears that aquatic actinomycetes are one of the important resources for screening useful enzymes and metabolites.
Effect of compatible solutes on survival of lactic Acid bacteria subjected to drying
Authors: Kets E , Teunissen P , de Bont J
Published: January 2020

Four strains of lactic acid bacteria were investigated to determine if a relationship exists between accumulation of compatible solutes and the ability of cells to survive drying. Betaine was the major solute found in these lactic acid bacteria subjected to salt stress. Survival of cultures subjected to drying was considerably enhanced when this solute was accumulated by cells.
Effects of Calcium and Calmodulin on Spore Germination and Appressorium Development in Colletotrichum trifolii
Authors: Warwar V , Dickman M B
Published: January 2020

Spore germination and appressorium formation are important steps in the process of fungal development and pathogenesis. These prepenetration events, which begin with spore attachment and culminate with appressorium maturation, a common scheme for many pathogenic fungi, are prerequisites for penetration of host external barriers and subsequent colonization. Conditions for in vitro spore germination and appressorium development in Colletotrichum trifolii are described. In addition, effects of Ca(sup2+) and calmodulin on these processes have been examined. Results indicate that, as for other pathogenic fungi, appressorium development is induced on a hard surface. The data suggest that disturbance of calcium homeostasis, by ethylene-bis(oxy-ethylenenitrolo)tetraacetic acid (EGTA) or calcium channel blockers, impairs appressorium development. Moreover, calmodulin inhibitors affect both germination and differentiation, implying that the Ca(sup2+)/calmodulin signal transduction pathway is important in the early development of C. trifolii on the plant host surface.
Synergistic activity of a Bacillus thuringiensis delta-endotoxin and a bacterial endochitinase against Spodoptera littoralis larvae
Authors: Regev A , Keller M , Strizhov N , Sneh B , Prudovsky E , Chet I , Ginzberg I , Koncz-Kalman Z , Koncz C , Schell J , Zilberstein A
Published: January 2020

In an attempt to increase the insecticidal effect of the delta-endotoxin crystal protein CryIC on the relatively Cry-insensitive larvae of Spodoptera littoralis, a combination of CryIC and endochitinase was used. CryIC comprising the first 756 amino acids from Bacillus thuringiensis K26-21 and endochitinase ChiAII encoded by Serratia marcescens were separately produced in Escherichia coli carrying the genes in overexpression vectors. The endochitinase on its own, even at very low concentrations (0.1 microgram/ml), perforated the larval midgut peritrophic membrane. When applied together with low concentrations of CryIC, a synergistic toxic effect was obtained. In the absence of chitinase, about 20 micrograms of CryIC per ml was required to obtain maximal reduction in larval weight, while only 3.0 micrograms of CryIC per ml caused a similar toxic effect in the presence of endochitinase. Thus, a combination of the Cry protein and an endochitinase could result in effective insect control in transgenic systems in which the Cry protein is not expressed in a crystalline form.
Kinetics of adhesion of selected fish-pathogenic Vibrio strains of skin mucus of gilt-head sea bream (Sparus aurata L.)
Authors: Bordas M A , Balebona M C , Zorrilla I , Borrego J J , Moriñigo M A
Published: January 2020

The kinetics of adhesion of Vibrio strains isolated from diseased fish to skin mucus of gilt-head sea bream was studied. A modified Langmuir adsorption isotherm was calculated, and the results obtained indicate that the strains tested (Vibrio alginolyticus DP1HE4 and Vibrio anguillarum-like DC12R8 and DC12R9) showed a saturation kinetics except for V. alginolyticus (CAN), which showed a proportional adsorption kinetics. The adhesive capability for skin mucus does not seem to be an essential virulence factor of pathogenic strains of Vibrio, since this specific interaction depended on several environmental factors, temperature and salinity being the most important. However, the absence of an inhibitory effect of mucus on the pathogenic microorganisms, and the capability of the Vibrio strains to utilize mucus as a carbon source, could favor their settlement on the skin with a potential for infection of cultured, stressed fish.
Virulence determinants invA and spvC in salmonellae isolated from poultry products, wastewater, and human sources
Authors: Swamy S C , Barnhart H M , Lee M D , Dreesen D W
Published: January 2020

The presence of two virulence foci, invA and spvC, in Salmonella isolates obtained from poultry, wastewater, and human sources was determined. All isolates (n = 245) were positive for the invA gene sequence. Differences in degree of invasiveness were apparent with the Madin Darby canine kidney cell line, as only 79 of 159 randomly selected isolates (49.7%) tested were invasive at > 0.1% of the inoculum. 25% were invasive between 0.1 and 1.0% of the inoculum, and 24.5% were invasive at > 1.0% of the inoculum. There was a significant correlation between degree of invasion and source from which the isolate was recovered but no correlation between geographic origin of poultry isolates and degree of invasion. Only 37 of 245 isolates (15.1%) hybridized with the spvC DNA probe. All isolates that were recovered from a commercial egg production environment and chicken eggs and whose sequences exhibited homology with the spvC gene sequence were determined to be either Salmonella enteritidis PT 23 or PT 13. The sequences of few isolates from ceca and none from wastewater or humans demonstrated homology with the spvC gene.
Stereospecific production of the herbicide phosphinothricin (glufosinate): purification of aspartate transaminase from Bacillus stearothermophilus, cloning of the corresponding gene, aspC, and application in a coupled transaminase process
Authors: Bartsch K , Schneider R , Schulz A
Published: January 2020

We have isolated and characterized an aspartate transaminase (glutamate:oxalacetate transaminase, EC 2.6.1.1) from the thermophilic microorganism Bacillus stearothermophilus. The purified enzyme has a molecular mass of 40.5 kDa by sodium dodecyl sulfate gel analysis, a temperature optimum of 95 degrees C, and a pH optimum of 8.0. The corresponding gene, aspC, was cloned and overexpressed in Escherichia coli. The recombinant glutamate:oxalacetate transaminase protein was used in immobilized form together with 4-aminobutyrate:2-ketoglutarate transaminase (EC 2.6.1.19) from E. coli for the production of L-phosphinothricin [L-homoalanin-4-yl-(methyl)phosphinic acid], the active ingredient of the herbicide Basta (AgrEvo GmbH), from its nonchiral 2-keto acid precursor 2-oxo-4-[(hydroxy)(methyl)phosphinoyl]butyric acid (PPO). In this new coupled process conversion rates of ca. 85% were obtained with substrate solutions containing 10% PPO by using only slight excesses of the amino donors glutamate and aspartate. The contamination of the reaction broth with amino acid by-products was < 3%.
Formation of 30- to 40-micrometer-thick laminations by high-speed marine bacteria in microbial mats
Authors: Barbara G M , Mitchell J G
Published: January 2020

The precision with which motile heterotrophic bacteria could position themselves in microbial mats was determined. This required the development of a technique to view motile bacteria in situ. This was successfully achieved by replacing a 1-cm-diameter minicore from the mat sediment with 210- to 300-(mu)m-diameter glass beads or sieved agar. After allowing 3 days for regrowth of the mat into the transparent medium, a cross section showed that bacteria formed a layer as thin as 30 to 40 (mu)m at a depth of 500 (mu)m below the surface. Bacterial concentrations in this microlamination were 20 times above background. Mean speeds were 200 (mu)m s(sup-1) inside and 60 (mu)m s(sup-1) outside the microlamination. The percentages of bacteria turning per 30 s were 93% inside and 10% outside the microlamination. Artificial chemical gradients were unsuccessful in stimulating microlamination formation or in eliciting the same extent of speed and turning responses. The significance of the results is that it is now possible to microscopically examine sedimentary bacteria in situ. Our first examination indicates that some bacteria form chemotactic microlaminations by increasing their turning frequency. This behavior is opposite that described in the enteric-based model of chemotactic movement, in which positive chemotaxis is achieved by decreasing the turning frequency.
Identification of root rot fungi in nursery seedlings by nested multiplex PCR
Authors: Hamelin R C , Bérubé P , Gignac M , Bourassa M
Published: January 2020

The internal transcribed spacer (ITS) of the ribosomal DNA (rDNA) subunit repeat was sequenced in 12 isolates of Cylindrocladium floridanum and 11 isolates of Cylindrocarpon destructans. Sequences were aligned and compared with ITS sequences of other fungi in GenBank. Some intraspecific variability was present within our collections of C. destructans but not in C. floridanum. Three ITS variants were identified within C. destructans, but there was no apparent association between ITS variants and host or geographic origin. Two internal primers were synthesized for the specific amplification of portions of the ITS for C. floridanum, and two primers were designed to amplify all three variants of C. destructans. The species-specific primers amplified PCR products of the expected length when tested with cultures of C, destructans and C. floridanum from white spruce, black spruce, Norway spruce, red spruce, jack pine, red pine, and black walnut from eight nurseries and three plantations in Quebec. No amplification resulted from PCR reactions on fungal DNA from 26 common contaminants of conifer roots. For amplifications directly from infected tissues, a nested primer PCR using two rounds of amplification was combined with multiplex PCR approach resulting in the amplification of two different species-specific PCR fragments in the same reaction. First, the entire ITS was amplified with one universal primer and a second primer specific to fungi; a second round of amplification was carried out with species-specific primers that amplified a 400-bp PCR product from C. destructans and a 328-bp product from C. floridanum. The species-specific fragments were amplified directly from infected roots from which one or the two fungi had been isolated.
Phylogenetic analysis of the bacterial communities in marine sediments
Authors: Gray J P , Herwig R P
Published: January 2020

For the phylogenetic analysis of microbial communities present in environmental samples microbial DNA can be extracted from the sample, 16S rDNA can be amplified with suitable primers and the PCR, and clonal libraries can be constructed. We report a protocol that can be used for efficient cell lysis and recovery of DNA from marine sediments. Key steps in this procedure include the use of a bead mill homogenizer for matrix disruption and uniform cell lysis and then purification of the released DNA by agarose gel electrophoresis. For sediments collected from two sites in Puget Sound, over 96% of the cells present were lysed. Our method yields high-molecular-weight DNA that is suitable for molecular studies, including amplification of 16S rRNA genes. The DNA yield was 47 micrograms per g (dry weight) for sediments collected from creosote-contaminated Eagle Harbor, Wash. Primers were selected for the PCR amplification of (eu)bacterial 16S rDNA that contained linkers with unique 8-base restriction sites for directional cloning. Examination of 22 16S rDNA clones showed that the surficial sediments in Eagle Harbor contained a phylogenetically diverse population of organisms from the Bacteria domain (G. J. Olsen, C. R. Woese, and R. Overbeek, J. Bacteriol. 176:1-6, 1994) with members of six major lineages represented: alpha, delta, and gamma Proteobacteria; the gram-positive high G+C content subdivision; clostridia and related organisms; and planctomyces and related organisms. None of the clones were identical to any representatives in the Ribosomal Database Project small subunit RNA database. The analysis of clonal representives in the first report using molecular techniques to determine the phylogenetic composition of the (eu)bacterial community present in coastal marine sediments.
Cloning and characterization of a locus encoding an indolepyruvate decarboxylase involved in indole-3-acetic acid synthesis in Erwinia herbicola
Authors: Brandl M T , Lindow S E
Published: January 2020

Erwinia herbicola 299R synthesizes indole-3-acetic acid (IAA) primarily by the indole-3-pyruvic acid pathway. A gene involved in the biosynthesis of IAA was cloned from strain 299R. This gene (ipdC) conferred the synthesis of indole-3-acetaldehyde and tryptophol upon Escherichia coli DH5 alpha in cultures supplemented with L-tryptophan. The deduced amino acid sequence of the gene product has high similarity to that of the indolepyruvate decarboxylase of Enterobacter cloacae. Regions within pyruvate decarboxylases of various fungal and plant species also exhibited considerable homology to portions of this gene. This gene therefore presumably encodes an indolepyruvate decarboxylase (IpdC) which catalyzes the conversion of indole-3-pyruvic acid to indole-3-acetaldehyde. Insertions of Tn3-spice within ipdC abolished the ability of strain 299R to synthesize indole-3-acetaldehyde and tryptophol and reduced its IAA production in tryptophan-supplemented minimal medium by approximately 10-fold, thus providing genetic evidence for the role of the indolepyruvate pathway in IAA synthesis in this strain. An ipdC probe hybridized strongly with the genomic DNA of all E. herbicola strains tested in Southern hybridization studies, suggesting that the indolepyruvate pathway is common in this species. Maximum parsimony analysis revealed that the ipdC gene is highly conserved within this group and that strains of diverse geographic origin were very similar with respect to ipdC.
Molecular diversity of soil and marine 16S rRNA gene sequences related to beta-subgroup ammonia-oxidizing bacteria
Authors: Stephen J R , McCaig A E , Smith Z , Prosser J I , Embley T M
Published: January 2020

We have conducted a preliminary phylogenetic survey of ammonia-oxidizing beta-proteobacteria, using 16S rRNA gene libraries prepared by selective PCR and DNA from acid and neutral soils and polluted and nonpolluted marine sediments. Enrichment cultures were established from samples and analyzed by PCR. Analysis of 111 partial sequences of c. 300 bases revealed that the environmental sequences formed seven clusters, four of which are novel, within the phylogenetic radiation defined by cultured autotrophic ammonia oxidizers. Longer sequences from 13 cluster representatives support their phylogenetic positions relative to cultured taxa. These data suggest that known taxa may not be representative of the ammonia-oxidizing beta-proteobacteria in our samples. Our data provide further evidence that molecular and culture-based enrichment methods can select for different community members. Most enrichments contained novel Nitrosomonas-like sequences whereas novel Nitrosospira-like sequences were more common from gene libraries of soils and marine sediments. This is the first evidence for the occurrence of Nitrosospira-like strains in marine samples. Clear differences between the sequences of soil and marine sediment libraries were detected. Comparison of 16S rRNA sequences from polluted and nonpolluted sediments provided no strong evidence that the community composition was determined by the degree of pollution. Soil clone sequences fell into four clusters, each containing sequences from acid and neutral soils in varying proportions. Our data suggest that some related strains may be present in both samples, but further work is needed to resolve whether there is selection due to pH for particular sequence types.
Hydrolysis of feather keratin by immobilized keratinase
Authors: Lin X , Shih J , Swaisgood H E
Published: January 2020

Keratinase isolated from Bacillus licheniformis PWD-1 was immobilized on controlled-pore glass beads. The immobilized keratinase demonstrated proteolytic activities against both insoluble feather keratin and soluble casein. It also displayed a higher level of heat stability and an increased tolerance toward acidic pHs compared with the free keratinase. During a continuous reaction at 50(deg)C, the immobilized keratinase retained 40% of the original enzyme activity after 7 days. The immobilized keratinase exhibits improved stability, thereby increasing its potential for use in numerous applications.
Thermal inactivation of bovine immunodeficiency virus
Authors: Moore E C , Keil D , Coats K S
Published: January 2020

Cell-associated bovine immunodeficiency virus (BIV) and cell-free BIV were subjected to increasing temperatures, including pasteurization conditions. To determine the effect of heat treatment on BIV viability, reverse transcriptase activity and infectivity of the heat-treated virus were assessed. BIV was inactivated by heating to 47 degrees C for 30 min and by low- and high-temperature pasteurization conditions.
Polyunsaturated fatty acid biosynthesis in Saccharomyces cerevisiae: expression of ethanol tolerance and the FAD2 gene from Arabidopsis thaliana
Authors: Kajiwara S , Shirai A , Fujii T , Toguri T , Nakamura K , Ohtaguchi K
Published: January 2020

The Arabidopsis thaliana delta-12 fatty acid desaturase gene (FAD2) was overexpressed in Saccharomyces cerevisiae by using the GAL1 promoter. S. cerevisiae harboring the FAD2 gene was capable of forming hexadecadienoyl (16:2) and linoleoyl (18:2) residues in the membrane lipid when cultured in medium containing galactose. Gas-liquid chromatography analysis of total lipids indicated that the transformed S. cerevisiae accumulated these dienoic fatty acyl residues and that they accounted for approximately 50% of the total fatty acyl residues. Phospholipid analysis of this strain indicated that the oleoyl (18:1) residue binding phosphatidylcholine (PC) was mostly converted to the 18:2 residue binding PC, whereas 50% of the palmitoleoyl (16:1) residue binding PC was converted to the 16:2 residue binding PC. A marked effect on the unsaturation of 16:1 and 18:1 was observed when S. cerevisiae harboring the FAD2 gene was cultured at 8 degrees C. To assess the ethanol tolerance of S. cerevisiae producing polyunsaturated fatty acids, the cell viability of this strain in the presence of ethanol was examined. The results indicated that S. cerevisiae cells overexpressing the FAD2 gene had greater resistance to 15% (vol/vol) ethanol than did the control cells.
l-Isoleucine Production with Corynebacterium glutamicum: Further Flux Increase and Limitation of Export
Authors: Morbach S , Sahm H , Eggeling L
Published: January 2020

The synthesis of l-isoleucine with Corynebacterium glutamicum involves 11 reaction steps, in at least five of which activity or expression is regulated. We used four genes and alleles encoding feedback-resistant enzymes (Fbr) in various combinations to assay flux increase through the sequence. During strain construction, the order of genes overexpressed was important. Only when ilvA(Fbr) was first overexpressed could hom(Fbr) be introduced. This succession apparently prevents the toxic accumulation of biosynthesis intermediates. The best strain constructed (SM13) was characterized by high-level expression of hom(Fbr), thrB, and ilvA(Fbr). With this strain a yield of 0.22 g of l-isoleucine per g of glucose was obtained, with a maximal specific productivity of 0.10 g of l-isoleucine per g (dry weight) per h. In strain SM13, with the high metabolite flux through the reaction sequence, effects on (i) other enzyme levels, (ii) time-dependent variations with process time, and (iii) concentrations of cytosolic intermediates were quantified. Most importantly, the intracellular l-isoleucine concentration is always higher at all process times than the extracellular concentration. The intracellular concentration rises to 110 mM, whereas extracellularly only 60 mM is accumulated. Also the immediate l-isoleucine precursor 2-ketomethyl valerate accumulates in the cell. Therefore, in the high-level l-isoleucine producer SM13, the export of this amino acid is the major limiting reaction step and therefore is a new target of strain design for biotechnological purposes.
Accelerated Mineralization of Pentachlorophenol in Soil upon Inoculation with Mycobacterium chlorophenolicum PCP1 and Sphingomonas chlorophenolica RA2
Authors: Miethling R , Karlson U
Published: January 2020

Mineralization of pentachlorophenol (PCP) was studied in nonsterile soil from a PCP-contaminated site upon inoculation with two PCP-degrading bacterial strains. At spiked [(sup14)C]PCP concentrations of 30 and 100 mg/kg, the effects of organism type, different inoculation techniques, including structural amendment with sawdust and cell attachment to polyurethane (PU), as well as the effect of different inoculum sizes of 10(sup4) to 10(sup8) cells per g (dry weight) of soil were compared with PCP mineralization by indigenous bacteria. Gas chromatographic analysis was used to monitor PCP disappearance and to check mass balances. The survival and activity of the released bacteria were examined by immunofluorescence microscopy and respiking experiments. Noninoculated soil completely mineralized 30 mg of PCP per kg within 7 months but showed no or only low degradation activity at 100 mg/kg in the same period. Structural amendment with PU or sawdust initiated slow mineralization after half a year. Soil inoculation with Sphingomonas chlorophenolica RA2 shortened the mineralization time drastically to 1 month at 30 mg of PCP per kg using 10(sup8) cells per g, with approximately 80% of the added radioactivity being converted to CO(inf2). The inoculated cells disappeared rapidly, with a count of 2 x 10(sup6) cells per g after 2.3 months and nondetectability after 7 months. At 100 mg/kg, mineralization was slower because of PCP toxicity but approached completion within 7.5 months. The inhibition could be overcome by addition of sawdust (1 g/kg of soil), resulting in a mineralization rate of 3 to 4 mg/kg(middot)d. PU had the opposite effect. Lower inoculum densities resulted in prolonged lag phases and lower rates, although mineralization was still enhanced over the background level. At 30 mg of PCP per kg, inoculation with Mycobacterium chlorophenolicum PCP1 increased mineralization slightly over the indigenous bacterial activity, regardless of inoculum size, but only when the organisms were attached to PU. At 100 mg of PCP per kg, only 27% were mineralized within 7.5 months. After 7 months, the original strain PCP1 inoculum of 10(sup8) cells per g was recovered at 5 x 10(sup6) to 3 x 10(sup7) cells per g, depending on the PCP concentration, but independent of PU amendment. Amendment with sawdust had no effect on the performance of this organism. Possible reasons for the poor performance of this strain include its sensitivity to PCP and its preference for slightly acidic soil conditions.
Production of pediocin AcH by Lactobacillus plantarum WHE 92 isolated from cheese
Authors: Ennahar S , Aoude-Werner D , Sorokine O , Van Dorsselaer A , Bringel F , Hubert J C , Hasselmann C
Published: January 2020

Among 1,962 bacterial isolates from a smear-surface soft cheese (Munster cheese) screened for activity against Listeria monocytogenes, six produced antilisterial compounds other than organic acids. The bacterial strain WHE 92, which displayed the strongest antilisterial effect, was identified at the DNA level as Lactobacillus plantarum. The proteinaceous nature, narrow inhibitory spectrum, and bactericidal mode of action of the antilisterial compound produced by this bacterium suggested that it was a bacteriocin. Purification to homogeneity and sequencing of this bacteriocin showed that it was a 4.6-kDa, 44-amino-acid peptide, the primary structure of which was identical to that of pediocin AcH produced by different Pediococcus acidilactici strains. We report the first case of the same bacteriocin appearing naturally with bacteria of different genera. Whereas the production of pediocin AcH from P. acidilactici H was considerably reduced when the final pH of the medium exceeded 5.0, no reduction in the production of pediocin AcH from L. plantarum WHE 92 was observed when the pH of the medium was up to 6.0. This fact is important from an industrial angle. As the pH of dairy products is often higher than 5.0, L. plantarum WHE 92, which develops particularly well in cheeses, could constitute an effective means of biological combat against L. monocytogenes in this type of foodstuff.
Initial reactions in the oxidation of 1,2-dihydronaphthalene by Sphingomonas yanoikuyae strains
Authors: Eaton S L , Resnick S M , Gibson D T
Published: January 2020

The substrate oxidation profiles of Sphingomonas yanoikuyae B1 biphenyl-2,3-dioxygenase and cis-biphenyl dihydrodiol dehydrogenase activities were examined with 1,2-dihydronaphthalene and various cis-diols as substrates. m-Xylene-induced cells of strain B1 oxidized 1,2-dihydronaphthalene to (-)-(1R,2S)-cis-1,2-dihydroxy-1,2-3,4-tetrahydronaphthalene as the major product (73% relative yield). Small amounts of (+)-(R)-2-hydroxy-1,2-dihydronaphthalene (15%), naphthalene (6%), and alpha-tetralone (6%) were also formed. Strain B8/36, which lacks an active cis-biphenyl dihydrodiol dehydrogenase, formed (+)-(1R,2S)-cis-1,2-dihydroxy-1,2-dihydronaphthalene (51%), in addition to (-)-(1R,2S)-cis-1,2-dihydroxy-1,2,3,4-tetrahydronaphthalene (44%) and (+)-(R)-2-hydroxy-1,2-dihydronaphthalene (5%). The cis-biphenyl dihydrodiol dehydrogenase of strain B1 oxidized both enantiomers of cis-1,2-dihydroxy-1,2-dihydronaphthalene, but only the (+)-(1S,2R)-enantiomers of cis-1,2-dihydroxy-1,2,3,4-tetrahydronaphthalene and cis-1,2-dihydroxy-3-phenylcyclohexa-3,5-diene. The results show that biphenyl dioxygenase expressed by S. yanoikuyae catalyzes dioxygenation, monooxygenation, and desaturation reactions with 1,2-dihydronaphthalene as the substrate, and cis-biphenyl dihydrodiol dehydrogenase catalyzes the enantioselective dehydrogenation of (+)-(1S,2R)-cis-1,2-dihydroxy-1,2,3,4-tetrahydronaphthalene and (+)-(1S,2R)-cis-1,2-dihydroxy-3-phenylcyclohexa-3,5-diene.
Physiologic Mechanisms Involved in Accumulation of 3-Hydroxypropionaldehyde during Fermentation of Glycerol by Enterobacter agglomerans
Authors: Barbirato F , Soucaille P , Bories A
Published: January 2020

When grown in 700 mM glycerol within the pH range 6.0 to 7.5, anaerobic pH-regulated cultures of Enterobacter agglomerans exhibited an extracellular accumulation of 3-hydroxypropionaldehyde (3-HPA). This phenomenon, which causes fermentation cessation, occurred earlier when pH was low. In contrast, substrate consumption was complete at pH 8. Levels of glycerol-catabolizing enzymes, i.e., glycerol dehydrogenase and dihydroxyacetone kinase for the oxidative route and glycerol dehydratase and 1,3-propanediol dehydrogenase for the reductive route, as well as the nucleotide pools were determined periodically in the pH 7- and pH 8-regulated cultures. A NAD/NADH ratio of 1.7 was correlated with the beginning of the production of the inhibitory metabolite. Further accumulation was dependent on the ratio of glycerol dehydratase activity to 1,3-propanediol dehydrogenase activity. For a ratio higher than 1, 3-HPA was produced until fermentation ceased, which occurred for the pH 7-regulated culture. At pH 8, a value below 1 was noticed and 3-HPA accumulation was transient, while the NAD/NADH ratio decreased. The low rate of glycerol dissimilation following the appearance of 3-HPA in the culture medium was attributed to the strong inhibitory effect exerted by 3-HPA on glycerol dehydrogenase activity.
Functional analysis of the gene encoding immunity to lactacin F, lafI, and its use as a Lactobacillus-specific, food-grade genetic marker
Authors: Allison G E , Klaenhammer T R
Published: January 2020

Lactacin F is a two-component class II bacteriocin produced by Lactobacillus johnsonii VPI 11088. The laf operon is composed of the bacteriocin structural genes, lafA and lafX, and a third open reading frame, ORFZ. Two strategies were employed to study the function of ORFZ. This gene was disrupted in the chromosome of NCK64, a lafA729 lafX ORFZ derivative of VPI 11088. A disruption cassette consisting of ORFZ interrupted with a cat gene was cloned into pSA3 and introduced into NCK64. Manipulation of growth temperatures and antibiotic selection resulted in homologous recombination which disrupted the chromosomal copy of ORFZ with the cat gene. This ORFZ mutation resulted in loss of immunity to lactacin F but had little effect on production of LafX, which is not bactericidal without LafA. Expression of ORFZ in this ORFZ- background rescued the immune phenotype. Expression of ORFZ in a bacteriocin-sensitive derivative of VPI 11088 also reestablished immunity. These data indicate that ORFZ, renamed lafI, encodes the immunity factor for the lactacin F system. The sensitivity of various Lactobacillus strains to lactacin F was further evaluated. Lactacin F inhibited 11 strains including several members of the A1, A2, A3, A4, B1, and B2 L. acidophilus homology groups. Expression of lafI in bacteriocin-sensitive strains L. acidophilus ATCC 4356, L. acidophilus NCFM/N2, L. fermentum NCDO1750, L. gasseri ATCC 33323, and L. johnsonii ATCC 33200 provided immunity to lactacin F. Furthermore, it was shown that lactacin F production by VPI 11088 could be used to select for L. fermentum NCDO1750 transformants containing the recombinant plasmid encoding LafI. The data demonstrate that lafI is functional in heterologous hosts, suggesting that it may be a suitable food-grade genetic marker for use in lactobacillus species.
Characterization of universal small-subunit rRNA hybridization probes for quantitative molecular microbial ecology studies
Authors: Zheng D , Alm E W , Stahl D A , Raskin L
Published: January 2020

Universal oligonucleotide hybridization probes targeting the small-subunit rRNA are commonly used to quantify total microbial representation in environmental samples. Universal probes also serve to normalize results obtained with probes targeting specific phylogenetic groups of microorganisms. In this study, six universal probes were evaluated for stability of probe-target duplexes by using rRNA from nine organisms representing the three domains of Bacteria, Archaea, and Eucarya. Domain-specific variations in dissociation temperatures were observed for all probes. This could lead to a significant bias when these probes are used to quantify microbial populations in environmental samples. We suggest lowering the posthybridization wash stringency for two of the universal probes (S-*-Univ-1390-a-A-18 and S-*-Univ-1392-a-A-15) examined. These two probes were evaluated with traditional and modified hybridization conditions to characterize defined mixtures of rRNAs extracted from pure cultures and rRNA samples obtained from anaerobic digester samples. Probe S-*-Univ-1390-a-A-18 provided excellent estimations of domain-level community composition of these samples and is recommended for future use in microbial ecology studies.
Relationship between the Intracellular Integrity and the Morphology of the Capsular Envelope in Attached and Free-Living Marine Bacteria
Authors: Heissenberger A , Leppard G G , Herndl G J
Published: January 2020

The integrity of the intracellular structures and the presence and dimension of the capsular envelope were investigated in marine snow-associated and marine free-living bacteria by transmission electron microscopy and special fixation techniques. Three categories depending on the presence of internal structures were differentiated. In marine snow, 51% of the marine snow-associated bacterial community was considered intact, 26% had a partly degraded internal structure, and 23% were empty with only the cell wall remaining. For the free-living bacterial community, 34% were intact cells, 42% exhibited damage, and 24% of the cells were lacking any internal structure. We also investigated the morphology and the extent of the bacterial capsular envelope. More than 95% of all intact marine snow-associated bacteria were surrounded by a capsule while (apprx=)55% of empty marine snow-associated bacteria had no capsule. For free-living bacteria, (apprx=)65% of the intact cells had a capsule while (apprx=)80% of the empty free-living bacteria lacked a capsule. Thus there is a clear trend from intact cells which are commonly surrounded by a capsular envelope to empty bacteria for which only the cell wall is remaining. Since bacterioplankton represent the largest living surface in the ocean, it is concluded that the release of intracellular material from bacteria into the environment as well as the release of extracellular capsular material might fuel the dissolved organic matter pool of the ocean.