bluuebunny/crossref_metadata_2025 huggingface dataset:
A Dual-Vector Method to Derive the Equivalent Screw of Two Successive Finite Screw Displacements for Line Segments
Authors: Yu Zetao , Ting Kwun-Lon
Published: January 2020

For finite rigid body motion, every two successive screw displacements can be represented by one equivalent screw displacement. However, such phenomenon should not be considered naturally to be valid for incompletely specified displacements (ISDs). There is neither a precise statement for such phenomenon nor an understanding of its range of validity within ISD, such as line segment displacements. As one of the main contributions in this paper, based on dual vector algebra and screw theory, an algorithm is provided to prove the existence of the subset within the scope of the line segment motion, which expresses the similar relation as shown in finite rigid body motion. A numerical example is presented for illustrative purpose.
Suboptimal Bacillus licheniformis and Bacillus weihenstephanensis Spore Incubation Conditions Increase Heterogeneity of Spore Outgrowth Time
Authors: Trunet C. , Mtimet N. , Mathot A.-G. , Postollec F. , Leguerinel I. , Couvert O. , Broussolle V. , Carlin F. , Coroller L.
Published: January 2020

Sporulation and incubation conditions have an impact on the numbers of spores able to recover after exposure to sublethal heat treatment. Using flow cytometry, we were able to follow at a single-cell level the changes in the physiological states of heat-stressed spores of
Bacillus
spp. and to discriminate between dormant spores, germinated spores, and outgrowing vegetative cells. We developed original mathematical models that describe (i) the changes with time of the proportion of cells in their different states during germination and outgrowth and (ii) the influence of temperature and pH on the kinetics of spore recovery using the growth limits of the tested strains as model parameters. We think that these models better predict spore recovery after a sublethal heat treatment, a common situation in food processing and a concern for food preservation and safety.
Effect of compost on rhizosphere microflora of the tomato and on the incidence of plant growth-promoting rhizobacteria
Authors: de Brito A M , Gagne S , Antoun H
Published: January 2020

Four commercial composts were added to soil to study their effect on plant growth, total rhizosphere microflora, and incidence of plant growth-promoting rhizobacteria (PGPR) in the rhizosphere of tomato plants. Three of the compost treatments significantly improved plant growth, while one compost treatment significantly depressed it. Compost amendments caused only small variations in the total numbers of bacteria, actinomycetes, and fungi in the rhizosphere of tomato plants. A total of 709 bacteria were isolated from the four compost treatments and the soil control to determine the percentage of PGPR in each treatment. The PGPR tests measured antagonism to soilborne root pathogens, production of indoleacetic acid, cyanide, and siderophores, phosphate solubilization, and intrinsic resistance to antibiotics. Our results show that the addition of some composts to soil increased the incidence in the tomato rhizosphere of bacteria exhibiting antagonism towards Fusarium oxysporum f. sp. radicis-lycopersici, Pyrenochaeta lycopersici, Pythium ultimum, and Rhizoctonia solani. The antagonistic effects observed were associated with marked increases in the percentage of siderophore producers. No significant differences were observed in the percentage of cyanogens, whereas the percentages of phosphate solubilizers and indoleacetic acid producers were affected, respectively, by one and two compost treatments. Intrinsic resistance to antibiotics was only marginally different among the rhizobacterial populations. Our results suggest that compost may stimulate the proliferation of antagonists in the rhizosphere and confirm previous reports indicating that the use of composts in container media has the potential to protect plants from soilborne root pathogens.
A di- and tripeptide transport system can supply Listeria monocytogenes Scott A with amino acids essential for growth
Authors: Verheul A , Hagting A , Amezaga M R , Booth I R , Rombouts F M , Abee T
Published: January 2020

Listeria monocytogenes takes up di- and tripeptides via a proton motive force-dependent carrier protein. This peptide transport system resembles the recently cloned and sequenced secondary di- and tripeptide transport system of Lactococcus lactis (A. Hagting, E. R. S. Kunji, K. J. Leenhouts, B. Poolman, and W. N. Konings, J. Biol. Chem. 269:11391-11399, 1994). The peptide permease of L. monocytogenes has a broad substrate specificity and allows transport of the nonpeptide substrate 5-aminolevulinic acid, the toxic di- and tripeptide analogs, alanyl-beta-chloroalanine and alanyl-alanyl-beta-chloroalanine, and various di- and tripeptides. No extracellular peptide hydrolysis was detected, indicating that peptides are hydrolyzed after being transported into the cell. Indeed, peptidase activities in response to various synthetic substrates were detected in cell extracts obtained from L. monocytogenes cells grown in brain heart infusion broth or defined medium. The di- and tripeptide permease can supply L. monocytogenes with essential amino acids for growth and might contribute to growth of this pathogen in various foods where peptides are supplied by proteolytic activity of other microorganisms present in these foods. Possible roles of this di- and tripeptide transport system in the osmoregulation and virulence of L. monocytogenes are discussed.
Proposals for optimization of the international phage typing system for Listeria monocytogenes: combined analysis of phage lytic spectrum and variability of typing results
Authors: Marquet-Van der Mee N , Audurier A
Published: January 2020

Combined analysis of 5,179 serial phage reactions of 20 Listeria monocytogenes propagating strains over 14 years and phage typing results from 2,659 further L. monocytogenes strains allowed us to estimate lytic spectrum specificity and the variability of the lytic reactions of 35 phages. These included the 26 phages recommended for the international method for phage typing defined in 1985 by Rocourt et al. (J. Rocourt, A. Audurier, A. L. Courtieu, J. Durst, S. Ortel, A. Schrettenbrunner, and A. G. Taylor, Zentralbl. Bakteriol. Abt. 1 Orig. A 259:489-497, 1985). The results are discussed individually for each phage. Proposals for modifying the present system are made with the aim of producing an optimal bacteriophage set for routine use.
Analysis of viral RNA persistence in seawater by reverse transcriptase-PCR
Authors: Tsai Y L , Tran B , Palmer C J
Published: January 2020

It is important to determine the stability of naked viral RNA in seawater, since false-positive results can occur when reverse transcriptase-PCR (RT-PCR) is used to detect viruses if the RT-PCR amplifies free RNA instead of RNA from intact viruses. An acid guanidinium thiocyanate-phenol-chloroform method was used to extract total RNA from a filtered poliovirus cell culture suspension. The sensitivity of detection in this viral RNA study was 600 fg when RT-PCR was used. The extracted total RNA was seeded into filtered and unfiltered seawater, and the resulting preparations were incubated at 4 degrees C and at room temperature (23 +/- 1 degrees C). Our results showed that the seeded RNA was more stable in filtered seawater than in unfiltered seawater at both temperatures. The viral RNA could not be detected by the RT-PCR after 2 days of incubation in unfiltered seawater and after 28 days of incubation in filter-sterilized seawater. Therefore, because of the relatively short life of viral RNA in natural water, the detection of virus in environmental samples by the RT-PCR was mainly due to the presence of well-protected viral particles and not due to the presence of naked viral RNA.
Purification and characterization of an extracellular pectate lyase from an Amycolata sp
Authors: Br�hlmann F
Published: January 2020

The extracellular pectate lyase (EC 4.2.2.2) of a nonsporulating Amycolata sp. was purified to homogeneity by anion- and cation-exchange chromatographies followed by hydrophobic interaction chromatography. The enzyme cleaved polygalacturonate but not highly esterified pectin in a random endolytic transeliminative mechanism that led to the formation of a wide range of 4,5-unsaturated oligogalacturonates. As shown by high-performance anion-exchange chromatography and pulsed amperometric detection, these unsaturated oligogalacturonates were further depolymerized by the enzyme to the unsaturated dimer and trimer as final products. The pectate lyase had a molecular weight of 31,000 determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a molecular mass of 30,000 Da determined by matrix-assisted laser desorption ionization mass spectrometry. The isoelectric point of the protein was 10. Maximum activity occurred at pH 10.25. Calcium was essential for activity, and EDTA inactivated the enzyme under standard assay conditions. Interestingly, EDTA did not inhibit the ability of the enzyme to cleave the native pectin (protopectin) of ramie (Boehmeria nivea) fibers. The Km value with sodium polygalacturonate as the substrate was 0.019 g liter-1. The purified enzyme lost its activity after a 1-h incubation at 50 degrees C but was stabilized by calcium or polygalacturonate. The N-terminal sequence showed high similarity within a stretch of 13 amino acids to the N-terminal sequences of pectate lyases PLa and PLe from Erwinia chrysanthemi. The Amycolata sp. did not produce additional isozymes of pectate lyase but produced further activities of pectinesterase, xylanase, and carboxymethyl cellulase when grown in a medium with decorticated bast fibers from ramie as the sole carbon source.
Specific PCR primers directed to identify cryI and cryIII genes within a Bacillus thuringiensis strain collection
Authors: Cerón J , Ortíz A , Quintero R , Güereca L , Bravo A
Published: January 2020

In this paper we describe a PCR strategy that can be used to rapidly identify Bacillus thuringiensis strains that harbor any of the known cryI or cryIII genes. Four general PCR primers which amplify DNA fragments from the known cryI or cryIII genes were selected from conserved regions. Once a strain was identified as an organism that contains a particular type of cry gene, it could be easily characterized by performing additional PCR with specific cryI and cryIII primers selected from variable regions. The method described in this paper can be used to identify the 10 different cryI genes and the five different cryIII genes. One feature of this screening method is that each cry gene is expected to produce a PCR product having a precise molecular weight. The genes which produce PCR products having different sizes probably represent strains that harbor a potentially novel cry gene. Finally, we present evidence that novel crystal genes can be identified by the method described in this paper.
Development of a PCR protocol for sensitive detection of Cryptosporidium oocysts in water samples
Authors: Johnson D W , Pieniazek N J , Griffin D W , Misener L , Rose J B
Published: January 2020

The development of a reliable method of using PCR for detection of Cryptosporidium oocysts in environmental samples with oligonucleotide primers which amplify a portion of the sequence encoding the small (18S) subunit of rRNA producing a 435-bp product was demonstrated. The PCR assay was found to provide highly genus-specific detection of Cryptosporidium spp. after release of nucleic acids from oocysts by a simple freeze-thaw procedure. The assay routinely detected 1 to 10 oocysts in purified oocyst preparations, as shown by direct microscopic counts and by an immunofluorescence assay. The sensitivity of the PCR assay in some seeded environmental water samples was up to 1,000-fold lower. However, this interference was eliminated by either flow cytometry or magnetic-antibody capture. Sensitivity was also improved 10- to 1,000-fold by probing of the PCR product on dot blots with an oligonucleotide probe detected by chemiluminescence. Confirmation of the presence of Cryptosporidium oocysts in water samples from the outbreak in Milwaukee, Wis., was obtained with this technique, and PCR was found to be as sensitive as immunofluorescence for detection of oocysts in wastewater concentrates.
Toxicity of N-substituted aromatics to acetoclastic methanogenic activity in granular sludge
Authors: Donlon B A , Razo-Flores E , Field J A , Lettinga G
Published: January 2020

N-substituted aromatics are important priority pollutants entering the environment primarily through anthropogenic activities associated with the industrial production of dyes, explosives, pesticides, and pharmaceuticals. Anaerobic treatment of wastewaters discharged by these industries could potentially be problematical as a result of the high toxicity of N-substituted aromatics. The objective of this study was to examine the structure-toxicity relationships of N-substituted aromatic compounds to acetoclastic methanogenic bacteria. The toxicity was assayed in serum flasks by measuring methane production in granular sludge. Unacclimated cultures were used to minimize the biotransformation of the toxic organic chemicals during the test. The nature and the degree of the aromatic substitution were observed to have a profound effect on the toxicity of the test compound. Nitroaromatic compounds were, on the average, over 500-fold more toxic than their corresponding aromatic amines. Considering the facile reduction of nitro groups by anaerobic microorganisms, a dramatic detoxification of nitroaromatics towards methanogens can be expected to occur during anaerobic wastewater treatment. While the toxicity exerted by the N-substituted aromatic compounds was closely correlated with compound apolarity (log P), it was observed that at any given log P, N-substituted phenols had a toxicity that was 2 orders of magnitude higher than that of chlorophenols and alkylphenols. This indicates that toxicity due to the chemical reactivity of nitroaromatics is much more important than partitioning effects in bacterial membranes.
Resistance of Spiroplasma citri Lines to the Virus SVTS2 Is Associated with Integration of Viral DNA Sequences into Host Chromosomal and Extrachromosomal DNA
Authors: Sha Y , Melcher U , Davis R E , Fletcher J
Published: January 2020

Spiroplasmavirus SVTS2, isolated from Spiroplasma melliferum TS2, produces plaques when inoculated onto lawns of Spiroplasma citri M200H, a derivative of the type strain Maroc R8A2. S. citri strains MR2 and MR3, originally selected as colonies growing within plaques on a lawn of M200H inoculated with SVTS2, were resistant to SVTS2. Genomic DNA fingerprints and electrophoretic protein profiles of M200H, MR2, and MR3 were similar, but three proteins present in M200H were missing or significantly reduced in both resistant lines. None of these three polypeptides reacted with antiserum against S. citri membrane proteins, indicating that they probably are not surface-located virus receptors. Electroporation with SVTS2 DNA produced 1.5 x 10(sup5) transfectants per (mu)g of DNA in M200H but none in MR2 or MR3, suggesting that resistance may result from inhibition of viral replication. The digestion patterns of the extrachromosomal double-stranded (ds) DNA of these lines were similar. Three TaqI fragments of MR2 extrachromosomal DNA that were not present in M200H extrachromosomal DNA hybridized strongly to an SVTS2 probe, and two of these fragments plus an additional one hybridized with the MR3 extrachromosomal DNA, indicating that a fragment of SVTS2 DNA was present in the extrachromosomal ds DNA of MR2 and MR3 but not of M200H. When the restricted genomes of all three lines were probed with SVTS2 DNA, strong hybridization to two EcoRI fragments of chromosomal MR2 and MR3 DNA but not M200H DNA indicated that SVTS2 DNA had integrated into the genomes of MR2 and MR3 but not of M200H. When MR3 extrachromosomal ds DNA containing a 2.1-kb SVTS2 DNA fragment was transfected into M200H, the transformed spiroplasmas were resistant to SVTS2. These results suggest that SVTS2 DNA fragments, possibly integrated into the chromosomal or extrachromosomal DNA of a previously susceptible spiroplasma, may function as viral incompatibility elements, providing resistance to superinfection by SVTS2.
Application of reverse transcriptase PCR for monitoring expression of the catabolic dmpN gene in a phenol-degrading sequencing batch reactor
Authors: Selvaratnam S , Schoedel B A , McFarland B L , Kulpa C F
Published: January 2020

A modified freeze-thaw method in combination with reverse transcriptase PCR was developed for monitoring gene expression in activated sludge. The sensitivity of the methodology was determined by inoculating non-sterile activated sludge samples with 3-chlorobenzoate-degrading Pseudomonas putida PPO301(pRO103), which contains the catabolic tfdB gene. tfdB mRNA was detected in 10 mg of activated sludge inoculated with 10(4) CFU of the target organism. This technique was subsequently utilized to analyze the in situ expression of the catabolic dmpN gene in a sequencing batch reactor (SBR) bioaugmented with phenol-degrading P. putida ATCC 11172. Greatest dmpN expression was observed 15 min after maximum phenol concentration was reached in the reactor and 15 min after the start of aeration. Decreased phenol concentrations in the reactor corresponded to reduced levels of dmpN expression, although low levels of dmpN mRNA were observed throughout the SBR cycle. These results indicate that concentration of phenol in the reactor and the onset of aeration stimulated transcriptional activity of the dmpN gene. The information obtained from this study can be used to alter SBR operational strategies so as to lead to more effective bioaugmentation practices.
Comparative Physiology of Dimethyl Sulfide Production by Dimethylsulfoniopropionate Lyase in Pseudomonas doudoroffii and Alcaligenes sp. Strain M3A
Authors: de Souza M P , Yoch D C
Published: January 2020

Dimethylsulfoniopropionate (DMSP) lyase enzymatically cleaves DMSP, an algal metabolite, to produce acrylate, a proton, and dimethyl sulfide (DMS), the most abundant volatile sulfur compound emitted from oceans. The physiology of DMS production by DMSP lyase was studied in vivo in an Alcaligenes-like organism, strain M3A, a salt marsh bacterial isolate, and in a marine strain, Pseudomonas doudoroffii. Enzymes from both strains were induced at optimum rates by 1 mM DMSP and vigorous aeration. P. doudoroffii was very sensitive to continued aeration and lost activity rapidly; the enzyme was more stable when aeration ceased. In addition to DMSP, acrylate and several of its analogs acted as inducers of DMSP lyase in Alcaligenes sp. strain M3A but not in P. doudoroffii. Turnover of DMSP by P. doudoroffii was enhanced by 3.5% NaCl or seawater, whereas the Alcaligenes sp. strain M3A enzyme was not salt dependent and salt did not greatly affect its activity. The pH profile showed two peaks of DMSP lyase activity (6.5 and 8.8) for Alcaligenes sp. strain M3A and a single peak at pH 8 for P. doudoroffii. Enzyme activity in both organisms was inhibited by methyl-3-mercaptopropionate and homocysteine. Cyanide, azide and p-chloromercuribenzoate inhibited only the P. doudoroffii DMSP lyase. The apparent K(infm) values for DMSP for cell cultures of Alcaligenes sp. strain M3A and P. doudoroffii were ca. 2 mM and <20 (mu)M, respectively. The differences in the physiology of DMSP metabolism in these two bacterial isolates may enable them to exist in diverse ecological niches.
Carbohydrate Transport by the Anaerobic Thermophile Clostridium thermocellum LQRI
Authors: Strobel H J , Caldwell F C , Dawson K A
Published: January 2020

Clostridium thermocellum is an anaerobic thermophilic bacterium which degrades cellulose and ferments the resulting glucose, cellobiose, and cellodextrins predominantly to ethanol. However, relatively little information was available on carbohydrate uptake by this bacterium. Washed cells internalized intact oligomers as large as cellopentaose. Since cellobiose and cellodextrin phosphorylase activities were detected in the cytosol and were not associated with cell membranes, phosphorylation of carbohydrates occurred intracellularly. Kinetic studies indicated that cellobiose and larger cellodextrins were taken up by a common uptake system while glucose entered via a separate mechanism. When cells were treated with metabolic inhibitors including iodoacetate and arsenate, the uptake of radiolabeled glucose or cellobiose was reduced by as much as 90%, and this reduction was associated with a 95% decline in intracellular ATP content. A combination of the ionophores nigericin and valinomycin abolished the proton-motive force but only slightly decreased transport and ATP. These results suggested that the two modes of carbohydrate transport in C. thermocellum were ATP dependent. This work is the first demonstration of cellodextrin transport by a cellulolytic bacterium.
Maintenance and induction of naphthalene degradation activity in Pseudomonas putida and an Alcaligenes sp. under different culture conditions
Authors: Guerin W F , Boyd S A
Published: January 2020

The expression of xenobiotic-degradative genes in indigenous bacteria or in bacteria introduced into an ecosystem is essential for the successful bioremediation of contaminated environments. The maintenance of naphthalene utilization activity is studied in Pseudomonas putida (ATCC 17484) and an Alcaligenes sp. (strain NP-Alk) under different batch culture conditions. Levels of activity decreased exponentially in stationary phase with half-lives of 43 and 13 h for strains ATCC 17484 and NP-Alk, respectively. Activity half-lives were 2.7 and 5.3 times longer, respectively, in starved cultures than in stationary-phase cultures following growth on naphthalene. The treatment of starved cultures with chloramphenicol caused a loss of activity more rapid than that measured in untreated starved cultures, suggesting a continued enzyme synthesis in starved cultures in the absence of a substrate. Following growth in nutrient medium, activity decreased to undetectable levels in the Alcaligenes sp. but remained at measurable levels in the pseudomonad even after 9 months. The induction of naphthalene degradation activities in these cultures, when followed by radiorespirometry with 14C-labeled naphthalene as the substrate, was consistent with activity maintenance data. In the pseudomonad, naphthalene degradation activity was present constitutively at low levels under all growth conditions and was rapidly (in approximately 15 min) induced to high levels upon exposure to naphthalene. Adaptation in the uninduced Alcaligenes sp. occurred after many hours of exposure to naphthalene. In vivo labeling with 35S, to monitor the extent of de novo enzyme synthesis by naphthalene-challenged cells, provided an independent confirmation of the results.
Characterization of Serratia entomophila Bacteriophages and the Phage-Resistant Mutant Strain BC4B
Authors: Grkovic S , O'callaghan M , Mahanty H K
Published: January 2020

Successful large-scale fermentations of the bacterium Serratia entomophila for use in biological control of the soil-dwelling insect Costelytra zealandica has required the development of a phage-resistant mutant, BC4B. We report our investigations into S. entomophila phages and the nature of the phage resistance mechanism of strain BC4B. The parental strain of BC4B, A1MO2, was found to contain two previously unidentified prophages, (phi)9A and (phi)9B, which were UV inducible and also released spontaneously in large numbers. BC4B was shown to be completely cured of (phi)9A. Single lysogens of (phi)9A and (phi)9B were not homoimmune to any other S. entomophila phages. However, on the basis of DNA-DNA homology, all S. entomophila phages except (phi)CW3 were shown to have significant regions of homology and also packaged their DNA via pac-like mechanisms. The failure of phage particles to adsorb was identified as the basis of phage resistance in BC4B. In addition, it was demonstrated that all known S. entomophila phages are naturally temperature sensitive.
Structure and function of Tn5467, a Tn21-like transposon located on the Thiobacillus ferrooxidans broad-host-range plasmid pTF-FC2
Authors: Clennel A M , Johnston B , Rawlings D E
Published: January 2020

A 3.5-kb region of plasmid pTF-FC2, which contains a transposon-like element designated Tn5467, has been sequenced, and its biological activity has been investigated. The transposon is bordered by two 38-bp inverted repeat sequences which have sequence identity in 37 of 38 and in 38 of 39 bp to the tnpA distal and tnpA proximal inverted repeats of Tn21, respectively. Within these borders, open reading frames with amino acid similarity to a glutaredoxin-like protein, a MerR regulatory protein, and a multidrug-resistant-membrane transport-like protein were found. The gene for the glutaredoxin-like protein was expressed in Escherichia coli and enabled growth of a glutathione-requiring E. coli trxA gshA mutant on minimal medium and the reduction of methionine sulfoxide to methionine. In addition, there were two regions which, when translated, had homology to 85% of the N-terminal region of the Tn21 resolvase (tnpR) and to 15% of the C terminus of the Tn21 transposase (tnpA). A region containing res-like sites was located immediately upstream of the partial tnpR gene. Neither the partial transposase nor the resolvase genes of Tn5467 were biologically active, but Tn5467 was transposed and resolved when the Tn21 transposase and resolvase were provided in trans. Tn5467 appears to be a defective transposon which belongs to the Tn21 subgroup of the Tn3 family.
Cloning and expression of a novel toxin gene from Bacillus thuringiensis subsp. jegathesan encoding a highly mosquitocidal protein
Authors: Del�cluse A , Rosso M L , Ragni A
Published: January 2020

A gene, designated cry11B, encoding a 81,293-Da crystal protein of Bacillus thuringiensis subsp. jegathesan was cloned by using a gene-specific oligonucleotide probe. The sequence of the Cry11B protein, as deduced from the sequence of the cry11B gene, contains large regions of similarity with the Cry11A toxin (previously CryIVD) from B. thuringiensis subsp. israelensis. The Cry11B protein was immunologically related to both Cry11A and Cry4A proteins. The cry11B gene was expressed in a nontoxic strain of B. thuringiensis, in which Cry11B was produced in large amounts during sporulation and accumulated as inclusions. Purified Cry11B inclusions were highly toxic for mosquito larvae of the species Aedes aegypti, Culex pipiens, and Anopheles stephensi. The activity of Cry11B toxin was higher than that of Cry11A and similar to that of the native crystals from B. thuringiensis subsp. jegathesan, which contain at least seven polypeptides.
Effects of a Bacterial Hay Preservative (Pediococcus pentosaceus) on Hay under Experimental Storage Conditions
Authors: Duchaine C , Lavoie M C , Cormier Y
Published: January 2020

The abundant growth of molds and thermophilic actinomycetes in stored hay decreases its quality and can be hazardous for the producer who inhales these contaminants when the moldy hay is fed in closed barns. These microbes are responsible for a respiratory disease called farmer's lung. Products, including bacterial cultures that can be inoculated in hay, are available to prevent hay deterioration by molds and bacteria. The aim of this study was to verify the effectiveness of Pediococcus pentosaceus (a bacterial inoculant) in preventing hay deterioration at different humidity levels in a laboratory experiment. Mixtures of grasses (mostly alfalfa, timothy, and clover) placed in plastic bags were treated with the commercially available product (live culture of P. pentosaceus) at 500,000 and 5,000,000 CFU/g of hay and humidified at different levels (20, 25, 30, and 35%). Control batches of hay (untreated) were prepared at the same humidity levels. The growth of inoculated bacteria in hay, pH level, and hay deterioration were evaluated. Under these experimental conditions, the growth of P. pentosaceus was abundant only when it was inoculated in very moist hay (35% moisture), resulting in bacterium levels of 6.3 x 10(sup8) CFU/g after 30 days. This abundant growth did not prevent the pH from increasing (final pH of about 9.0), nor did it prevent molding. At lower humidity levels (20, 25, and 30%), the bacterial inoculant used did not grow and did not prevent hay deterioration.
Cloning and characterization of the genes for p-nitrobenzoate degradation from Pseudomonas pickettii YH105
Authors: Yabannavar A V , Zylstra G J
Published: January 2020

Pseudomonas pickettii YH105 was isolated for its ability to utilize p-nitrobenzoate as the sole source of carbon, nitrogen, and energy. Degradation of p-nitrobenzoate by this strain proceeds through a reductive route as evidenced by the accumulation of ammonia in the culture medium during growth on p-nitrobenzoate. Enzyme assays and high-performance liquid chromatography (HPLC) analysis of culture supernatants indicate that p-nitrobenzoate is degraded through p-hydroxylaminobenzoate and protocatechuate. In order to clone the genes responsible for the initial steps in the catabolic pathway, a cosmid library was constructed with P. pickettii YH105 genomic DNA. The library was screened for clones capable of transforming p-nitrobenzoate to protocatechuate, using a plate assay specific for diphenolic compounds. HPLC analysis of culture supernatants confirmed that the cosmid clones did indeed produce protocatechuate from p-nitrobenzoate. Five positive cosmid clones that possessed this activity were identified. Restriction digests of the cosmid clones indicated that all of the clones had two EcoRI fragments in common (3.9 and 1.0 kb). One of these cosmid clones, designated pGJZ1601, was chosen for further analysis. Subcloning and activity assay experiments localized the genes responsible for the conversion of p-nitrobenzoate to protocatechuate to a 1.4-kb SalI-SphI DNA fragment. Further subcloning experiments localized the gene coding for p-nitrobenzoate reductase, responsible for the first enzymatic step in the catabolic pathway, to a 0.8-kb SalI-ApaI DNA fragment. The gene for the second step in the catabolic pathway, coding for hydroxylaminolyase, was located adjacent to the gene for the p-nitrobenzoate reductase.
Statistical approach for comparison of the growth rates of five strains of Staphylococcus aureus
Authors: Dengremont E , Membr� J M
Published: January 2020

The interaction of temperature (10, 14, 25, 31, and 37 degrees C), pH (pH 5, 5.6, 6.5, 7.4, and 8), and NaCl (0, 2, 5, 8, and 10%) in a laboratory medium affects the specific growth of Staphylococcus aureus. From growth curves obtained by the turbidimetric technique, a nonlinear model in which the specific growth rate (mu) is fitted directly, without data transformation and with the residual error variations taken into account, is proposed. This model correctly fits experimental data and gives more biological information than the quadratic polynomial model. Moreover, the comparison of five strains of S. aureus was performed by a principal-component analysis in which the specific growth rate was the identifying characteristic for S. aureus strains. The results obtained from model coefficient comparison among the five strains and from multivariate data analysis allow the same classification of strains to be performed. Two of them have similar behaviors during food spoilage, two others could be distinguished by their capacity to grow at a low temperature, whereas the last one was markedly different from the others.
Effect of Chloramphenicol on Denitrification in Flexibacter canadensis and "Pseudomonas denitrificans"
Authors: Wu Q , Knowles R
Published: January 2020

It was recently reported that chloramphenicol inhibits existing denitrification enzyme activity in sediments and carbon-starved cultures of "Pseudomonas denitrificans." Therefore, we studied the effect of chloramphenicol on denitrification by Flexibacter canadensis and "P. denitrificans." Production of N(inf2)O from nitrate by F. canadensis cells decreased as the concentration of chloramphenicol was increased, and 10.0 mM chloramphenicol completely inhibited N(inf2)O production. "P. denitrificans" was less sensitive to chloramphenicol, and production of N(inf2)O from nitrate was inhibited by only about 50% even in the presence of 10.0 mM chloramphenicol. These results suggested that inhibition of denitrification enzyme activity depended on the concentration of chloramphenicol. Increasing the concentration of chloramphenicol decreased the rate of production of nitrite from nitrate by F. canadensis cells, and the concentration of chloramphenicol which resulted in 50% inhibition of production of nitrite from nitrate was 2.5 mM. In contrast, the rates of production of nitrite from nitrate by intact cells and cell extracts of "P. denitrificans" were inhibited by only 58 and 54%, respectively, at a chloramphenicol concentration of 10.0 mM. Chloramphenicol caused accumulation of NO from nitrite but not from nitrate and inhibited NO consumption in F. canadensis; however, it had neither effect in "P. denitrificans." Chloramphenicol did not affect N(inf2)O consumption by these organisms. We concluded that chloramphenicol inhibits denitrification at the level of nitrate reduction and, in F. canadensis, also at the level of NO reduction.
Sandwich enzyme-linked immunosorbent assay for Vibrio vulnificus hemolysin to detect V. vulnificus in environmental specimens
Authors: Parker R W , Lewis D H
Published: January 2020

Vibrio vulnificus hemolysin, purified by quantitative isoelectric focusing, was used to prepare rabbit and goat anti-hemolysin. The resulting antibodies were used as capture and detector antibody reagents in a sandwich enzyme-linked immunosorbent assay (ELISA) to detect V. vulnificus in environmental samples. By this technique, 4 laboratory-maintained V. vulnificus strains and 33 environmental V. vulnificus isolates were detected. Also, the technique distinguished five other Vibrio species from V. vulnificus, and when it was used in combination with colistin-polymyxin-cellobiose agar, 31 non-V. vulnificus isolated were excluded. This sandwich ELISA compared favorably with the current Food and Drug Administration standard immunoassay in confirming presumptive V. vulnificus colonies from environmental specimens: oysters, sediment, and seawater. Among 340 presumptive V. vulnificus colonies, the sandwich ELISA detected 95% of the confirmed V. vulnificus colonies. Equally important, the technique correctly distinguished 99% of the non-V. vulnificus colonies. The sandwich ELISA offers time-saving and labor-saving advantages over the currently accepted immunoassay.
Identification of methanotrophic lipid biomarkers in cold-seep mussel gills: chemical and isotopic analysis
Authors: Jahnke L L , Summons R E , Dowling L M , Zahiralis K D
Published: January 2020

A lipid analysis of the tissues of a cold-seep mytilid mussel collected from the Louisiana slope of the Gulf of Mexico was used in conjunction with a compound-specific isotope analysis to demonstrate the presence of methanotrophic symbionts in the mussel gill tissue and to demonstrate the host's dependence on bacterially synthesized metabolic intermediates. The gill tissue contained large amounts of group-specific methanotrophic biomarkers, bacteriohopanoids, 4-methylsterols, lipopolysaccharide-associated hydroxy fatty acids, and type I-specific 16:1 fatty acid isomers with bond positions at delta 8, delta 10, and delta 11. Only small amounts of these compounds were detected in the mantle or other tissues of the host animal. A variety of cholesterol and 4-methylsterol isomers were identified as both free and steryl esters, and the sterol double bond positions suggested that the major bacterially derived gill sterol [11.0% 4 alpha-methyl-cholesta-8(14),24-dien-3 beta-ol] was converted to host cholesterol (64.2% of the gill sterol was cholest-5-en-3 beta-ol). The stable carbon isotope values for gill and mantle preparations were, respectively, -59.0 and -60.4% for total tissue, -60.6 and -62.4% for total lipids, -60.2 and-63.9% for phospholipid fatty acids, and -71.8 and 73.8% for sterols. These stable carbon isotope values revealed that the relative fractionation pattern was similar to the patterns obtained in pure culture experiments with methanotrophic bacteria (R.E. Summons, L.L. Jahnke, and Z. Roksandic, Geochim. Cosmochim. Acta 58: 2853-2863, 1994) further supporting the conversion of the bacteria methylsterol pool.
Biopreservation of Brined Shrimp (Pandalus borealis) by Bacteriocins from Lactic Acid Bacteria
Authors: Einarsson H , Lauzon H L
Published: January 2020

In brined shrimp (ca. 3% NaCl), the effects of three different lactic acid bacteria bacteriocins (crude [6.54 x 10(sup10) U of bacteriocin activity {BU}/g] and purified [8.13 x 10(sup23) BU/g] nisin Z, carnocin UI49 [2.32 x 10(sup4) BU/g], and crude bavaricin A [2.78 BU/g]) on bacterial growth and shelf life were compared with those of a benzoate-sorbate solution (0.1% each [wt/wt]) and a control with no preservatives. The shelf life of shrimp subjected to the control treatment was found to be 10 days. Carnocin UI49 did not extend the shelf life, while crude bavaricin A (a cell-free supernatant of Lactobacillus bavaricus MI 401) resulted in a shelf life of 16 days, as opposed to 31 days with nisin Z for both its crude and purified forms. The benzoate-sorbate solution preserved the brined shrimp for the whole storage period (59 days). In the control, carnocin UI49, and crude bavaricin A treatments, a gram-positive flora dominated towards the end of the storage period while in the nisin Z treatment a gram-negative flora was more pronounced.
Cell Cycle Regulation in Marine Synechococcus sp. Strains
Authors: Binder B J , Chisholm S W
Published: January 2020

The cell cycle behavior of four marine strains of the unicellular cyanobacterium Synechococcus sp. was analyzed by examining the DNA frequency distributions of exponentially growing and dark-blocked populations and by considering the patterns of change in these distributions during growth under a diel light-dark cycle. The two modes of cell cycle regulation previously identified in a freshwater and coastal marine Synechococcus isolate, respectively, were represented among the three open-ocean strains we examined. The first of these modes of regulation is consistent with the slow-growth case of the widely accepted prokaryotic cell cycle paradigm. The second appears to involve asynchronous initiation of chromosome replication, the presence of multiple chromosome copies at low growth rates, and variability in chromosome copy number among cells in the population. These characteristics suggest the involvement of a large probabilistic component in cell cycle regulation which could make the application of cell cycle-based estimators of in situ growth rate to Synechococcus populations problematic.
In Situ Localization of Azospirillum brasilense in the Rhizosphere of Wheat with Fluorescently Labeled, rRNA-Targeted Oligonucleotide Probes and Scanning Confocal Laser Microscopy
Authors: Assmus B , Hutzler P , Kirchhof G , Amann R , Lawrence J R , Hartmann A
Published: January 2020

The colonization of wheat roots by Azospirillum brasilense was used as a model system to evaluate the utility of whole-cell hybridization with fluorescently labeled, rRNA-targeted oligonucleotide probes for the in situ monitoring of rhizosphere microbial communities. Root samples of agar- or soil-grown 10- and 30-day-old wheat seedlings inoculated with different strains of A. brasilense were hybridized with a species-specific probe for A. brasilense, a probe hybridizing to alpha subclass proteobacteria, and a probe specific for the domain Bacteria to identify and localize the target bacteria. After hybridization, about 10 to 25% of the rhizosphere bacteria as visualized with 4(prm1),6-diamidino-2-phenylindole (DAPI) gave sufficient fluorescence signals to be detected with rRNA-targeted probes. Scanning confocal laser microscopy was used to overcome disturbing effects arising from autofluorescence of the object or narrow depth of focus in thick specimens. This technique also allowed high-resolution analysis of the spatial distribution of bacteria in the rhizosphere. Occurrence of cells of A. brasilense Sp7 and Wa3 was restricted to the rhizosphere soil, mainly to the root hair zone. C-forms of A. brasilense were demonstrated to be physiologically active forms in the rhizosphere. Strain Sp245 also was found repeatedly at high density in the interior of root hair cells. In general, the combination of fluorescently labeled oligonucleotide probes and scanning confocal laser microscopy provided a very suitable strategy for detailed studies of rhizosphere microbial ecology.
Ability of laboratory methods to predict in-use efficacy of antimicrobial preservatives in an experimental cosmetic
Authors: Farrington J K , Martz E L , Wells S J , Ennis C C , Holder J , Levchuk J W , Avis K E , Hoffman P S , Hitchins A D , Madden J M
Published: January 2020

Volume 60, no. 12, p. 4553: a present address for S. J. Wells should be given, as follows: (dag) Present address: Cadbury Beverages North America, Trumbull, CT 06611. [This corrects the article on p. 4553 in vol. 60.].
Reverse transcription PCR to detect enteroviruses in surface water
Authors: Gilgen M , Wegmüller B , Burkhalter P , Bühler H P , Müller U , Lüthy J , Candrian U
Published: January 2020

We have developed a simple, fast, and efficient procedure to detect enteroviruses in water samples. Aliquots of water are subjected to two-step filtration, with the second filter containing a positively charged nylon membrane that holds back virus particles. Viruses thus adsorbed are directly lysed, and RNA is isolated by hybridization to specific oligonucleotides bound to magnetic beads. The solution used contains guanidine thiocyanate, which lyses virus particles, inactivates enzymes, e.g., RNases, allows mild hybridization conditions, and does not influence biotin-streptavidin interaction on magnetic beads. Detection and specific identification are accomplished by reverse transcription PCR of the highly conserved noncoding region at the 5' end of virus RNA combined with Southern hybridization. The system was tested with tap water artificially spiked with poliovirus vaccine and yielded a detection limit of 20 50% tissue culture infective doses per liter. We used the same procedure to investigate the water quality of surface water at public beaches by rivers and lakes. Of 40 samples tested, 7 were positive for enteroviruses. A comparison with enterobacterial contamination determined by PCR and classical microbiological methods in parallel showed that enteroviruses were found only in samples also positive for Escherichia coli. In conclusion, this procedure can easily be adapted to test large water samples and is simple enough to be used for routine determinations of water quality in terms of virus contamination.
Effect of impact stress on microbial recovery on an agar surface
Authors: Stewart S L , Grinshpun S A , Willeke K , Terzieva S , Ulevicius V , Donnelly J
Published: January 2020

Microbial stress due to the impaction of microorganisms onto an agar collection surface was studied experimentally. The relative recovery rates of aerosolized Pseudomonas fluorescens and Micrococcus luteus were determined as a function of the impaction velocity by using a moving agar slide impactor operating over a flow rate range from 3.8 to 40 liters/min yielding impaction velocities from 24 to 250 m/s. As a reference, the sixth stage of the Andersen Six-Stage Viable Particle Sizing Sampler was used at its operating flow rate of 28.3 liters/min (24 m/s). At a collection efficiency of close to 100% for the agar slide impactor, an increase in sampling flow rate and, therefore, in impaction velocity produced a significant decline in the percentage of microorganisms recovered. Conversely, when the collection efficiency was less than 100%, greater recovery and lower injury rates occurred. The highest relative rate of recovery (approximately 51% for P. fluorescens and approximately 62% for M. luteus) was obtained on the complete (Trypticase soy agar) medium at 40 and 24 m/s (6.4 and 3.8 liters/min), respectively. M. luteus demonstrated less damage than P. fluorescens, suggesting the hardy nature of the gram-positive strain versus that of the gram-negative microorganism. Comparison of results from the agar slide and Andersen impactors at the same sampling velocity showed that recovery and injury due to collection depends not only on the magnitude of the impaction velocity but also on the degree to which the microorganisms may be embedded in the collection medium.(ABSTRACT TRUNCATED AT 250 WORDS)
Anaerobic oxidation of ammonium is a biologically mediated process
Authors: van de Graaf A A , Mulder A , de Bruijn P , Jetten M S , Robertson L A , Kuenen J G
Published: January 2020

A newly discovered process by which ammonium is converted to dinitrogen gas under anaerobic conditions (the Anammox process) has now been examined in detail. In order to confirm the biological nature of this process, anaerobic batch culture experiments were used. All of the ammonium provided in the medium was oxidized within 9 days. In control experiments with autoclaved or raw wastewater, without added sludge or with added sterilized (either autoclaved or gamma irradiated) sludge, no changes in the ammonium and nitrate concentrations were observed. Chemical reactions could therefore not be responsible for the ammonium conversion. The addition of chloramphenicol, ampicillin, 2,4-dinitrophenol, carbonyl cyanide m-chlorophenyl-hydrazone (CCCP), and mercuric chloride (HgIICl2) completely inhibited the activity of the ammonium-oxidizing sludge. Furthermore, the rate of ammonium oxidation was proportional to the initial amount of sludge used. It was therefore concluded that anaerobic ammonium oxidation was a microbiological process. As the experiments were carried out in an oxygen-free atmosphere, the conversion of ammonium to dinitrogen gas did not even require a trace of O2. That the end product of the reaction was nitrogen gas has been confirmed by using 15NH4+ and 14NO3-. The dominant product was 14-15N2. Only 1.7% of the total labelled nitrogen gas produced was 15-15N2. It is therefore proposed that the N2 produced by the Anammox process is formed from equimolar amounts of NH4+ and NO3-.
Primer sets developed to amplify conserved genes from filamentous ascomycetes are useful in differentiating fusarium species associated with conifers
Authors: Donaldson G C , Ball L A , Axelrood P E , Glass N L
Published: January 2020

We examined the usefulness of primer sets designed to amplify introns within conserved genes in filamentous ascomycetes to differentiate 35 isolates representing six different species of Fusarium commonly found in association with conifer seedlings. We analyzed restriction fragment length polymorphisms (RFLP) in five amplified PCR products from each Fusarium isolate. The primers used in this study were constructed on the basis of sequence information from the H3, H4, and (beta)-tubulin genes in Neurospora crassa. Primers previously developed for the intergenic transcribed spacer region of the ribosomal DNA were also used. The degree of interspecific polymorphism observed in the PCR products from the six Fusarium species allowed differentiation by a limited number of amplifications and restriction endonuclease digestions. The level of intraspecific RFLP variation in the five PCR products was low in both Fusarium proliferatum and F. avenaceum but was high in a population sample of F. oxysporum isolates. Clustering of the 35 isolates by statistical analyses gave similar dendrograms for H3, H4, and (beta)-tubulin RFLP analysis, but a dendrogram produced by intergenic transcribed spacer analysis varied in the placement of some F. oxysporum isolates.