bluuebunny/crossref_metadata_2025 huggingface dataset:
Involvement of an Intracellular Oligogalacturonate Hydrolase in Metabolism of Pectin by
Clostridium thermosaccharolyticum
Authors: Van Rijssel Marion , Smidt Marten P. , Van Kouwen Gisella , Hansen Theo A.
Published: January 2020

The enzymes pectin methylesterase and polygalacturonate hydrolase, which are responsible for the initial steps of pectin degradation by
Clostridium thermosaccharolyticum
, were shown to be induced on the polymeric substrates pectin and pectate, as well as on oligogalacturonates, and to be repressed in the presence of glucose. The digalacturonate and trigalacturonate produced by the extracellular pectin methylesterase-polygalacturonate hydrolase complex were transported across the cytoplasmic membrane and hydrolyzed by an inducible oligogalacturonate hydrolase to galacturonate. The oligogalacturonate hydrolase was separated from the polygalacturonate hydrolase and characterized. Its temperature optimum was 65�C, and its pH optimum was 6. The native molecular size was 90 kDa, and the enzyme was stable for more than 1 h at 65�C. The maximum reaction rate on oligomers decreased with the increasing degree of polymerization. Galacturonate was released by hydrolysis from the nonreducing end of the oligomer. The amounts of pectinolytic enzymes produced were all strictly correlated to the amount of biomass formed. Galacturonate was metabolized via a modified Entner-Doudoroff route.
Enhanced Carotenoid Biosynthesis by Oxidative Stress in Acetate-Induced Cyst Cells of a Green Unicellular Alga,
Haematococcus pluvialis
Authors: Kobayashi Makio , Kakizono Toshihide , Nagai Shiro
Published: January 2020

In a green alga,
Haematococcus pluvialis,
a morphological change of vegetative cells into cyst cells was rapidly induced by the addition of acetate or acetate plus Fe
2+
to the vegetative growth phase. Accompanied by cyst formation, algal astaxanthin formation was more enhanced by the addition of acetate plus Fe
2+
than by the addition of acetate alone. Encystment and enhanced carotenoid biosynthesis were inhibited by either actinomycin D or cycloheximide. However, after cyst formation was induced by the addition of acetate alone, carotenoid formation could be enhanced with the subsequent addition of Fe
2+
even in the presence of the inhibitors. The Fe
2+
-enhanced carotenogenesis was inhibited by potassium iodide, a scavenger for hydroxyl radical, suggesting that hydroxyl radical formed by an iron-catalyzed Fenton reaction may be required for enhanced carotenoid biosynthesis. Moreover, it was demonstrated that four active oxygen species, singlet oxygen, superoxide anion radical, hydrogen peroxide, and peroxy radical, were capable of replacing Fe
2+
in its role in the enhanced carotenoid formation in the acetate-induced cyst. From these results, it was concluded that oxidative stress is involved in the posttranslational activation of carotenoid biosynthesis in acetate-induced cyst cells.
Hydroxydiether Lipid Structures in
Methanosarcina
spp. and
Methanococcus voltae
Authors: Sprott G. Dennis , Dicaire Chantal J. , Choquet Christian G. , Patel Girishchandra B. , Ekiel Irena
Published: January 2020

Hydroxylated diether lipids are the most abundant lipids in
Methanosarcina acetivorans, Methanosarcina thermophila
, and
Methanosarcina barkeri
MS and Fusaro, regardless of the substrate used for growth. Structural analysis of the lipid moiety freed of polar head groups revealed that the hydroxydiether lipids of all the
Methanosarcina
strains were hydroxylated at position 3 of
sn
-2 phytanyl chains. The finding that
Methanosarcina
strains synthesize the same hydroxydiether structure suggests that this is a taxonomic characteristic of the genus.
Methanococcus voltae
produced minor amounts of the 3-hydroxydiether characteristic of
Methanosarcina
spp. and also the 3′-hydroxydiether described previously for
Methanosaeta concilii
.
Comparison of chemical assay, bioassay, enzyme-linked immunosorbent assay, and dot blot hybridization for detection of aerobactin in members of the family Enterobacteriaceae
Authors: Le Roy D , Bouchet A , Saulnier P , Pecquet S , Andremont A
Published: January 2020

In order to determine the best strategy for detection of aerobactin in members of the family Enterobacteriaceae, we compared the results of three phenotypic assays, including a chemical assay, a cross-feeding bioassay, and an enzyme-linked immunosorbent assay (ELISA), with the results of a dot blot hybridization assay using a specific probe for the aerobactin genes. The sensitivity and specificity of the ELISA were better than those of the chemical and cross-feeding assays, but the results of dot blot hybridization were the most reproducible. However, none of the Serratia and Enterobacter cloacae strains which produced aerobactin hybridized with the probe. We concluded that the best strategy for aerobactin detection is a two-step procedure that combines screening by dot blot hybridization with an ELISA for negative strains.
Involvement of Genes on a Megaplasmid in the Acid-Tolerant Phenotype of
Rhizobium leguminosarum
Biovar Trifolii
Authors: Chen Hancai , Gartner Elena , Rolfe Barry G.
Published: January 2020

The acid-tolerant
Rhizobium leguminosarum
biovar trifolii strain ANU1173 exhibited several new phenotypes when cured of its symbiotic (Sym) plasmid and the second largest megaplasmid. Strain P22, which has lost these two plasmids, had reduced exopolysaccharide production and cell mobility on TY medium. The parent strain ANU1173 was able to grow easily in laboratory media at pH 4.5, whereas the derivative strain P22 was unable to grow in media at a pH of <4.7. The intracellular pH of strain ANU1173 was 6.8 when the external pH was 4.5. In contrast, strain P22 had an acidic intracellular pH of <6.4 when the external pH was <5.5. Strain P22 had a dramatically increased membrane permeability to protons and decreased proton extrusion activity. Analysis with sodium dodecyl sulfate-polyacrylamide gels showed that strain P22 lacked a slow-migrating lipopolysaccharide (LPS) banding group which was present in the parent strain. Mobilization of the second largest megaplasmid of strain ANU1173 back into strain P22 restored the altered LPS structure and physiological characteristics of strain P22. Mobilization of the Sym plasmid of strain ANU1173 into strain P22 showed that the second largest megaplasmid of strain ANU1173 was required for the establishment of nitrogen-fixing nodules on
Trifolium repens
and
Trifolium subterraneum
. Furthermore, an examination of a large number of specific exopolysaccharide- or LPS-deficient
Rhizobium
mutants did not show a positive correlation between exopolysaccharide or LPS synthesis and acid tolerance.
Dynamics, spread, and persistence of a single genotype of Pseudomonas syringae relative to those of its conspecifics on populations of snap bean leaflets
Authors: Hirano S S , Upper C D
Published: January 2020

A rifampin-resistant strain of Pseudomonas syringae (R10) was introduced onto bean plants grown in field plots to examine the processes of growth, spread, and survival of a single genotype relative to the dynamics of its conspecifics on populations of individual leaflets. R10 was applied to four plots (400, 200, 100, and 50 m2), each of which was centered in a quadrant of a bean field (90 by 90 m). Population sizes of the species P. syringae and of R10 were determined on each of 25 individual leaflets collected from the largest plot (A) at approximately weekly intervals during a 10-week period following application. The spread of R10 from all plots was monitored by leaf imprinting of individual leaflets collected at sites along four transects, each of which bisected two of the plots. The introduced strain was a dominant component of the species for about 5 weeks postinoculation on leaflets from plot A. Although the population sizes of R10 remained at about 5.0 to 5.5 log10 CFU per leaflet, the strain became a progressively minor component of the species as the population sizes of its conspecifics continued to increase during the latter part of the growing season. In general, a positive correlation was found for the population sizes of R10 and its conspecifics on individual leaflets collected throughout the growing season. This result suggests that large numbers of R10 early in the growing season did not exclude the colonization of bean leaflets by its conspecifics. It is apparent that the species pool comprised genotypes that were more fit than R10 under the conditions that prevailed during the latter part of the growing season. By 6 weeks postinoculation, R10 was detected at all sites sampled within the unsprayed areas of the field. However, it was present as a minor component of the species. The persistence of R10 throughout the winter and into the following growing season was monitored in plot A, which was plowed and replanted with wheat in the fall. R10 was detected on some of the samples (wheat seedlings or soil) taken at approximately monthly intervals from November to June of the following year. In June, the field was plowed and replanted with beans. We could not detect R10 on emerging bean seedlings in plot A. The results demonstrate that the successful spread and persistence of an introduced bacterium do not necessarily lead to the establishment of large populations of the bacterium in adjacent untreated areas or on its host plant in subsequent growing seasons.
Production of Specifically Labeled Compounds by
Methanobacterium espanolae
Grown on H
2
-CO
2
plus [
13
C]Acetate
Authors: Patel Girishchandra B. , Sprott Dennis , Ekiel Irena
Published: January 2020

Methanobacterium espanolae
, an acidiphilic methanogen, required acetate for maximal growth on H
2
-CO
2
. In the presence of 5 to 15 mM acetate, at a growth pH of 5.5, the μ
max
was 0.05 h
-1
.
M. espanolae
consumed 12.3 mM acetate during 96 h of incubation at 35°C with shaking at 100 rpm. At initial acetate levels of 2.5 to 10.0 mM, the amount of biomass produced was dependent on the amount of acetate in the medium.
13
C nuclear magnetic resonance spectra of protein hydrolysates obtained from cultures grown on [1-
13
C]- or [2-
13
C]acetate indicated that an incomplete tricarboxylic acid pathway, operating in the reductive direction, was functional in this methanogen. The amino acids were labeled with a very high degree of specificity and at greater than 90% enrichment levels. Less than 2% label randomization occurred between positions primarily labeled from either the carboxyl or methyl group of acetate, and very little label was transferred to positions primarily labeled from CO
2
. The labeling pattern of carbohydrates was typical for glucogenesis from pyruvate. This methanogen, by virtue of the properties described above and its ability to incorporate all of the available acetate (10 mM or lower) from the growth medium, has advantages over other microorganisms for use in the production of specifically labeled compounds.
Purification of Pseudomonas putida acyl coenzyme A ligase active with a range of aliphatic and aromatic substrates
Authors: Fernández-Valverde M , Reglero A , Martinez-Blanco H , Luengo J M
Published: January 2020

Acyl coenzyme A (acyl-CoA) ligase (acyl-CoA synthetase [ACoAS]) from Pseudomonas putida U was purified to homogeneity (252-fold) after this bacterium was grown in a chemically defined medium containing octanoic acid as the sole carbon source. The enzyme, which has a mass of 67 kDa, showed maximal activity at 40 degrees C in 10 mM K2PO4H-NaPO4H2 buffer (pH 7.0) containing 20% (wt/vol) glycerol. Under these conditions, ACoAS showed hyperbolic behavior against acetate, CoA, and ATP; the Kms calculated for these substrates were 4.0, 0.7, and 5.2 mM, respectively. Acyl-CoA ligase recognizes several aliphatic molecules (acetic, propionic, butyric, valeric, hexanoic, heptanoic, and octanoic acids) as substrates, as well as some aromatic compounds (phenylacetic and phenoxyacetic acids). The broad substrate specificity of ACoAS from P. putida was confirmed by coupling it with acyl-CoA:6-aminopenicillanic acid acyltransferase from Penicillium chrysogenum to study the formation of several penicillins.
Enhanced mineralization of polychlorinated biphenyls in soil inoculated with chlorobenzoate-degrading bacteria
Authors: Hickey W J , Searles D B , Focht D D
Published: January 2020

An Altamont soil containing no measurable population of chlorobenzoate utilizers was examined for the potential to enhance polychlorinated biphenyl (PCB) mineralization by inoculation with chlorobenzoate utilizers, a biphenyl utilizer, combinations of the two physiological types, and chlorobiphenyl-mineralizing transconjugants. Biphenyl was added to all soils, and biodegradation of 14C-Aroclor 1242 was assessed by disappearance of that substance and by production of 14CO2. Mineralization of PCBs was consistently greatest (up to 25.5%) in soils inoculated with chlorobenzoate degraders alone. Mineralization was significantly lower in soils receiving all other treatments: PCB cometabolizer (10.7%); chlorobiphenyl mineralizers (8.7 and 14.9%); and mixed inocula of PCB cometabolizers and chlorobenzoate utilizers (11.4 and 18.0%). However, all inoculated soils had higher mineralization than did the uninoculated control (3.1%). PCB disappearance followed trends similar to that observed with the mineralization data, with the greatest degradation occurring in soils inoculated with the chlorobenzoate-degrading strains Pseudomonas aeruginosa JB2 and Pseudomonas putida P111 alone. While the mechanism by which the introduction of chlorobenzoate degraders alone enhanced biodegradation of PCBs could not be elucidated, the possibility that chlorobenzoate inoculants acquired the ability to metabolize biphenyl and possibly PCBs was explored. When strain JB2, which does not utilize biphenyl, was inoculated into soil containing biphenyl and Aroclor 1242, the frequency of isolates able to utilize biphenyl and 2,5-dichlorobenzoate increased progressively with time from 3.3 to 44.4% between 15 and 48 days, respectively. Since this soil contained no measurable level of chlorobenzoate utilizers yet did contain a population of biphenyl utilizers, the possibility of genetic transfer between the latter group and strain JB2 cannot be excluded.
Mycoplasmalike organisms (MLOs) and distinguish them from other MLOs
Authors: Nakashima K , Kato S , Iwanami S , Murata N
Published: January 2020

DNA of 10 lines of rice yellow dwarf (RYD) mycoplasmalike organisms (MLOs) from Japan, the Phillippines, and Thailand hybridized with four probes containing chromosomal and six probes containing extrachromosomal DNA of a Tochigi (Japan) line of RYD MLO. One chromosomal probe (RYD9) and all six extrachromosomal probes hybridized with various other MLOs (sugarcane white leaf, onion yellows, cineraria witches'-broom, Japanese hornwort witches'-broom, water dropwort wiches'-broom, gentian witches'-broom, udo dwarf, tsuwabuki witches'-broom, pelargonium witches's-broom, peach western-X, and pear decline). DNA from the culturable mollicutes Spiroplasma kunkelii, Spiroplasma citri, Mycoplasma hominis, and Mycoplasma orale did not hybridize with RYD MLO probes. The extrachromosomal DNAs hybridizing with the probes showed variations in electrophoretic behavior.
Detection of naturally occurring enteroviruses in waters by reverse transcription, polymerase chain reaction, and hybridization
Authors: Kopecka H , Dubrou S , Prevot J , Marechal J , López-Pila J M
Published: January 2020

Comparison in virus-seeded mineral water of three detection methods for enteroviruses, direct hybridization, cell culture, and reverse transcription into cDNA followed by polymerase chain reaction and hybridization, showed that the last procedure was 10 to 1,000 times more sensitive than detection by cell culture and 10(5) to 10(7) times more sensitive than direct hybridization. The presence of naturally occurring enteroviruses was also demonstrated in activated sludge and in concentrated and non-concentrated surface water samples by reverse transcription-polymerase chain reaction-hybridization. However, in activated sludge and in concentrated surface waters, enzymatic amplification was sometimes inhibited by contaminants.
Impact of the ring-billed gull (Larus delawarensis) on the microbiological quality of recreational water
Authors: Benoît Leévesque  , Brousseau P , Simard P , Dewailly E , Meisels M , Ramsay D , Joly J
Published: January 2020

We evaluated the impact of the ring-billed gull (Larus delawarensis) on the microbiological quality of water. We measured fecal coliforms, Salmonella spp., and Aeromonas spp. in the gull droppings and the number of fecal coliforms in the water prior to and after attracting these birds to the beach with food. Gulls can contribute to the bacteriological degradation of recreational water.
Soluble Methane Monooxygenase Production and Trichloroethylene Degradation by a Type I Methanotroph,
Methylomonas methanica
68-1
Authors: Koh Sung-Cheol , Bowman John P. , Sayler Gary S.
Published: January 2020

A methanotroph (strain 68-1), originally isolated from a trichloroethylene (TCE)-contaminated aquifer, was identified as the type I methanotroph
Methylomonas methanica
on the basis of intracytoplasmic membrane ultrastructure, phospholipid fatty acid profile, and 16S rRNA signature probe hybridization. Strain 68-1 was found to oxidize naphthalene and TCE via a soluble methane monooxygenase (sMMO) and thus becomes the first type I methanotroph known to be able to produce this enzyme. The specific whole-cell sMMO activity of 68-1, as measured by the naphthalene oxidation assay and by TCE biodegradation, was comparatively higher than sMMO activity levels in
Methylosinus trichosporium
OB3b grown in the same copper-free conditions. The maximal naphthalene oxidation rates of
Methylomonas methanica
68-1 and
Methylosinus trichosporium
OB3b were 551 ± 27 and 321 ± 16 nmol h
-1
mg of protein
-1
, respectively. The maximal TCE degradation rates of
Methylomonas methanica
68-1 and
Methylosinus trichosporium
OB3b were 2,325 ± 260 and 995 ± 160 nmol h
-1
mg of protein
-1
, respectively. The substrate affinity of 68-1 sMMO to naphthalene (

K
m

, 70 ± 4 μM) and TCE (

K
m

, 225 ± 13 μM), however, was comparatively lower than that of the sMMO of OB3b, which had affinities of 40 ± 3 and 126 ± 8 μM, respectively. Genomic DNA slot and Southern blot analyses with an sMMO gene probe from
Methylosinus trichosporium
OB3b showed that the sMMO genes of 68-1 have little genetic homology to those of OB3b. This result may indicate the evolutionary diversification of the sMMOs.
Seasonal Dynamics of Bacterial Colonization of Cotton Fiber and Effects of Moisture on Growth of Bacteria within the Cotton Boll
Authors: Zuberer D. A. , Kenerley C. M.
Published: January 2020

A highly replicated 3-year field study was conducted to determine the seasonal patterns of bacterial colonization of cotton fiber from the time of dehiscence of the bolls (the point at which the bolls just begin to open) through harvest and commercial ginning. Bacterial numbers on fiber samples from 16 plots were determined by dilution pour plating with tryptic soy agar containing cycloheximide, and numbers of gram-negative bacteria were determined by plating on tryptic soy agar containing vancomycin and cycloheximide. Populations of bacteria varied from year to year, but in all three seasons the pattern of colonization was generally a pattern consisting of a rapid increase following opening of the bolls and a more or less stable number thereafter throughout the growing season. Gram-negative bacteria accounted for 50% or more of the recoverable bacterial population. We hypothesized that the luxuriant bacterial flora developed as a result of the availability of sufficient free water in the bolls to allow bacterial proliferation with the carbon sources remaining after fiber maturation. Therefore, laboratory experiments were conducted to determine the threshold moisture level allowing growth of bacteria on fiber in the bolls. Bacterial proliferation occurred when as little as 2% moisture was added to air-dried fiber. Using simulated bolls, we demonstrated bacterial growth resulting from dew formation on fiber held in controlled-humidity chambers.
Binding of Extracellular Carboxymethylcellulase Activity from the Marine Shipworm Bacterium to Insoluble Cellulosic Substrates
Authors: Imam S. H. , Greene R. V. , Griffin H. L.
Published: January 2020

The binding of extracellular endoglucanase, a carboxymethylcellulase (CMCase), produced by the marine shipworm bacterium to insoluble cellulose substrates was investigated. Up to 70% of CMCase activity bound to cellulosic substrates, and less than 10% bound to noncellulosic substrates. CMCase binding to cellulose was enhanced in basal salt medium or sodium phosphate buffer containing 0.5 M NaCl. Increased cellulose particle size correlated with decreased CMCase binding. Also, cellulose treated with either 5 N NaOH or commercial cellulase reduced the CMCase binding to these surfaces. Pretreatment of CMCase preparations with 0.01% sodium dodecyl sulfate, 5% β-mercaptoethanol, and 5 mM EDTA or ethylene glycol-bis(β-aminoethyl ether)-
N,N,N′,N′
-tetraacetic acid (EGTA) had little effect on binding to cellulose. While pretreatment of CMCase with trypsin, chymotrypsin, and pronase had little effect on CMCase enzymatic activity, the ability to bind to cellulose was greatly diminished by these treatments.
Detection of Listeria monocytogenes in cheese with the magnetic immuno-polymerase chain reaction assay
Authors: Fluit A C , Torensma R , Visser M J , Aarsman C J , Poppelier M J , Keller B H , Klapwijk P , Verhoef J
Published: January 2020

A new detection system, the magnetic immuno-polymerase chain reaction (PCR) assay (MIPA) has been developed to detect Listeria monocytogenes in food. This method separates Listeria cells from PCR-inhibitory factors present in enrichment broths containing food samples by using magnetic beads coated with specific monoclonal antibodies (MAbs). The separated bacteria were lysed, and the supernatant containing the bacterial DNA was subjected to the PCR. Detection of L. monocytogenes in three naturally contaminated cheese samples with two different MAbs and PCR primers specific for the gene encoding the delayed-hypersensitivity factor showed that with MAb 55 all three samples were positive whereas with MAb A two samples were positive. A further improvement of the method was obtained by using a PCR step based on the listeriolysin O gene. A MIPA employing MAb 55 and the listeriolysin O gene primer set detected L. monocytogenes after 24 h of culture in Listeria Enrichment Broth samples from Port Salut artificially contaminated with 40 CFU/25 g. We could detect 1 CFU of L. monocytogenes per g of cheese after a second enrichment for 24 h in Fraser broth. The analysis time including both enrichments is approximately 55 h.
Detection of enteroviruses in groundwater with the polymerase chain reaction
Authors: Abbaszadegan M , Huber M S , Gerba C P , Pepper I L
Published: January 2020

Standard methods for the detection of enteroviruses in environmental samples involve the use of cell culture, which is expensive and time-consuming. The polymerase chain reaction (PCR) is an attractive method for the detection of enteroviruses in water because primary cell culture is not needed and the increased sensitivity of PCR allows detection of the low numbers of target DNAs and RNAs usually found in environmental samples. However, environmental samples often contain substances that inhibit PCR amplification of target DNA and RNA. Procedures that remove substances that interfere with the amplification process need to be developed if PCR is to be successfully applied to environmental samples. An RNA-PCR assay for the detection of enteroviruses in water was developed and used to test a variety of groundwater concentrates and humic acid solutions seeded with poliovirus type 1. The groundwater samples and humic acid solutions were treated with Sephadex G-50, Sephadex G-100, Sephadex G-200, Chelex-100 resin, and a mixed bed resin to remove PCR-inhibitory material from the samples. Sephadex G-100 in combination with Chelex-100 was found to be very effective in removing inhibitory factors for the detection of enteroviruses in groundwater concentrates by PCR. Viruses were detected in two of the groundwater concentrates by the RNA-PCR assay after treatment with Sephadex G-100 plus Chelex-100. This was confirmed by tissue culture, suggesting that the treatment protocol and, subsequently, the RNA-PCR assay are applicable for the detection of enteroviruses in environmental samples.
Resistance to Toxins from
Bacillus thuringiensis
subsp.
kurstaki
Causes Minimal Cross-Resistance to
B. thuringiensis
subsp.
aizawai
in the Diamondback Moth (Lepidoptera: Plutellidae)
Authors: Tabashnik Bruce E. , Finson Naomi , Johnson Marshall W. , Moar William J.
Published: January 2020

Repeated exposure in the field followed by laboratory selection produced 1,800- to >6,800-fold resistance to formulations of
Bacillus thuringiensis
subsp.
kurstaki
in larvae of the diamondback moth,
Plutella xylostella.
Four toxins from
B. thuringiensis
subsp.
kurstaki
[CryIA(a), CryIA(b), CryIA(c), and CryIIA] caused significantly less mortality in resistant larvae than in susceptible larvae. Resistance to
B. thuringiensis
subsp.
kurstaki
formulations and toxins did not affect the response to CryIC toxin from
B. thuringiensis
subsp.
aizawai.
Larvae resistant to
B. thuringiensis
subsp.
kurstaki
showed threefold cross-resistance to formulations of
B. thuringiensis
subsp.
aizawai
containing CryIC and CryIA toxins. This minimal cross-resistance may be caused by resistance to CryIA toxins shared by
B. thuringiensis
subsp.
kurstaki
and
B. thuringiensis
subsp.
aizawai.
Novel Allylic Oxidation of α-Cedrene to
sec
-Cedrenol by a
Rhodococcus
Strain
Authors: Takigawa Hirofumi , Kubota Hiromi , Sonohara Hiroshi , Okuda Mitsuyoshi , Tanaka Shigeyoshi , Fujikura Yoshiaki , Ito Susumu
Published: January 2020

A bacterial strain, designated KSM-7358, that can use α-cedrene for growth was isolated. The strain was identified as a member of the genus
Rhodococcus
and catalyzed the novel allylic oxidation of α-cedrene regiospecifically to produce (
R
)-10-hydroxycedrene (
sec
-cedrenol) with a very high yield. α-Curcumene was also produced as a possible metabolite of
sec
-cedrenol. A possible pathway for the microbial conversion of α-cedrene to
sec
-cedrenol and α-curcumene is proposed.
Conditions of formation, purification, and characterization of an alpha-galactosidase of Trichoderma reesei RUT C-30
Authors: Zeilinger S , Kristufek D , Arisan-Atac I , Hodits R , Kubicek C P
Published: January 2020

Trichoderma reesei RUT C-30 formed an extracellular alpha-galactosidase when it was grown in a batch culture containing lactose or locust bean gum as a carbon source. Short-chain alpha-galactosides (melibiose, raffinose, stachyose), as well as the monosaccharides galactose, dulcitol, arabinose, and arabitol, also induced alpha-galactosidase activity both when they were used as carbon sources (at a concentration of 1%) in batch cultures and in resting mycelia (at concentrations in the millimolar range). The addition of 50 mM glucose did not affect the induction of alpha-galactosidase formation by galactose. alpha-Galactosidase from T. reesei RUT C-30 was purified to homogeneity from culture fluids of galactose-induced mycelia. The active enzyme was a 50 +/- 3-kDa, nonglycosylated monomer which had an isoelectric point of 5.2. It was active against several alpha-galactosides (p-nitrophenyl-alpha-D-galactoside, melibiose, raffinose, and stachyose) and galactomannan (locust bean gum) and was inhibited by the product galactose. It released galactose from locust bean gum and exhibited synergism with T. reesei beta-mannanase. Its activity was optimal at pH 4, and it displayed broad pH stability (pH 4 to 8). Its temperature stability was moderate (60 min at 50 degrees C resulted in recovery of 70% of activity), and its highest level of activity occurred at 60 degrees C. Its action on galactomannan was increased by the presence of beta-mannanase.
Detection of tetracycline resistance determinants in pig isolates from three herds with different histories of antimicrobial agent exposure
Authors: Lee C , Langlois B E , Dawson K A
Published: January 2020

A total of 114 gram-negative fecal isolates from domestic pigs in herds with different histories of antimicrobial agent exposure were screened for the presence of plasmid DNA and specific tetracycline resistance determinants. More than 84% of the isolates harbored plasmid DNA, which ranged in size from 2.1 to 186 kb. A total of 78 isolates (68.4%) were resistant to tetracycline at concentrations greater than 4 micrograms/ml. Plasmid DNAs from about 56% of the tetracycline-resistant isolates hybridized with DNA probes for class A, B, C, and D tetracycline resistance determinants. The class B determinant was the most common determinant (35% of the isolates), followed by the class C determinant (12%) and the class A determinant (1%). About 9% of the isolates contained two determinants on plasmids. None of the plasmids from isolates hybridized with the class D determinant probe. The class C determinant was the most prevalent determinant on plasmids in isolates from pigs not exposed to antimicrobial agents for more than 146 months, while the class B determinant was more prevalent on plasmids in isolates from pigs exposed to either subtherapeutic or therapeutic levels of antimicrobial agents. Most tetracycline resistance determinants were localized on plasmids which were more than 30 kb long. A great number of wild-type tetracycline-resistant Escherichia coli strains were found with the class E determinant on their chromosomes. This study revealed a high prevalence of tetracycline resistance determinants in the fecal flora of pig herds whether or not they were fed with antibiotics.
Identification of the Sources of Energy for Nitrogen Fixation and Physiological Characterization of Nitrogen-Fixing Members of a Marine Microbial Mat Community
Authors: Bebout Brad M. , Fitzpatrick Matthew W. , Paerl Hans W.
Published: January 2020

Experimental manipulations of a microbial mat community were performed to determine sources of energy and reductant used for nitrogen fixation and to physiologically characterize the responsible diazotrophs. The dominant photolithotrophic members of this community were nonheterocystous cyanobacteria, but other potential nitrogen-fixing microorganisms were also present. Pronounced diel variability in rates of acetylene reduction was observed, with nighttime rates a factor of three to four higher than daytime rates. Acetylene reduction measured at night was dependent upon the occurrence of oxygenic photosynthesis the preceding day; mats incubated in the dark during the daytime reduced acetylene at rates comparable to those of light-incubated mats but were not able to reduce acetylene at the normally high rates the following night. The addition of various exogenous carbon compounds to these dark-incubated mats did not elicit nighttime acetylene reduction. Nighttime acetylene reduction apparently proceeds under anoxic conditions in these mats; the highest rates of acetylene reduction occur late at night. Additions of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (an inhibitor of oxygenic photosynthesis) to mats resulted in a pronounced stimulation of acetylene reduction during the day, but acetylene reduction the next night proceeded at greatly reduced rates (relative to untreated mats). This daytime stimulation, under the 3-(3,4-dichlorophenyl)-1,1-dimethylurea-induced anoxic conditions in the experimentally treated mats, was light dependent. These results suggest that nitrogen fixation in these mats may be attributed to the activities of nonheterocystous cyanobacteria utilizing storage products of oxygenic photosynthesis under anoxic conditions at night.
Microbial oxidation of dimethylnaphthalene isomers
Authors: Miyachi N , Tanaka T , Suzuki T , Hotta Y , Omori T
Published: January 2020

Three bacterial strains, identified as Alcaligenes sp. strain D-59 and Pseudomonas sp. strains D-87 and D-186, capable of growing on 2,6-dimethylnaphthalene (2,6-DMN) as the sole source of carbon and energy were isolated from soil samples. 2,6-Naphthalene dicarboxylic acid was formed in the culture broths of these three strains grown on 2,6-DMN. In addition, 2-hydroxymethyl-6-methylnaphthalene and 6-methylnaphthalene-2-carboxylic acid were detected in the culture broth of strain D-87. Strain D-87 grew well on 1,2-, 1,3-, 1,4-, 1,5-, 2,3-, and 2,7-DMN as the sole source of carbon and energy and accumulated 2-methylnaphthalene-3-carboxylic acid and 2,3-naphthalene dicarboxylic acid from 2,3-DMN, 4-methylnaphthalene-1-carboxylic acid from 1,4-DMN, and 7-methylnaphthalene-2-carboxylic acid from 2,7-DMN.
Effects of chemical speciation in growth media on the toxicity of mercury(II)
Authors: Farrell R E , Germida J J , Huang P M
Published: January 2020

The toxicity of metals, including mercury, is expressed differently in different media, and the addition of soluble organics to the growth medium can have a significant impact on bioassay results. Although the effect of medium composition on metal toxicity is generally attributed to its effect on metal speciation (i.e., the chemical form in which the metal occurs), the importance of individual metal-ligand species remains largely unclear. Here, we report the results of a study that investigated, both experimentally and from a modeling perspective, the effects of complex soluble organic supplements on the acute toxicity (i.e., 50% inhibitory concentration [IC50]) of mercury to a Pseudomonas fluorescens isolate in chemically well-defined synthetic growth media (M-IIX). The media consisted of a basal inorganic salts medium supplemented with glycerol (0.1%, vol/vol) and a variety of common protein hydrolysates (0.1%, vol/vol), i.e., Difco beef extract (X = B), Casamino Acids (X = C), peptone (X = P), soytone (X = S), tryptone (X = T), and yeast extract (X = Y). These were analyzed to obtain cation, anion, and amino acid profiles and the results were used to compute the aqueous speciation of Hg(II) in the media. Respirometric bioassays were performed and IC50s were calculated. Medium components varied significantly in their effects on the acute toxicity of Hg(II) to the P. fluorescens isolate. IC50s ranged from 1.48 to 14.54 micrograms of Hg ml-1, and the acute toxicity of Hg(II) in the different media decreased in the order M-IIC >> M-IIP > M-IIB >> M-IIT > M-IIS >>> M-IIY.(ABSTRACT TRUNCATED AT 250 WORDS)
Evaluation and simplification of the assimilable organic carbon nutrient bioassay for bacterial growth in drinking water
Authors: Kaplan L A , Bott T L , Reasoner D J
Published: January 2020

A modified assimilable organic carbon (AOC) bioassay is proposed. We evaluated all aspects of the AOC bioassay technique, including inoculum, incubation water, bioassay vessel, and enumeration technique. Other concerns included eliminating the need to prepare organic carbon-free glassware and minimizing the risks of bacterial and organic carbon contamination. Borosilicate vials (40 ml) with Teflon-lined silicone septa are acceptable incubation vessels. Precleaned vials are commercially available, and the inoculum can be injected directly through the septa. Both bioassay organisms, Pseudomonas fluorescens P-17 and Spirillum sp. strain NOX, are available from the American Type Culture Collection and grow well on R2A agar, making this a convenient plating medium. Turbid raw waters need to be filtered prior to an AOC analysis. Glass fiber filters used with either a peristaltic pump or a syringe-type filter holder are recommended for this purpose. A sampling design that emphasizes replication of the highest experimental level, individual batch cultures, is the most efficacious way to reduce the total variance associated with the AOC bioassay. Quality control for the AOC bioassay includes an AOC blank and checks for organic carbon limitation and inhibition of the bioassay organisms.
Relationships among non-Acremonium sp. fungal endophytes in five grass species
Authors: An Z Q , Siegel M R , Hollin W , Tsai H F , Schmidt D , Schardl C L
Published: January 2020

Many cool-season grasses (subfamily Pooideae) possess maternally transmitted fungal symbionts which cause no known pathology and often enhance the ecological fitness and biochemical capabilities of the grass hosts. The most commonly described endophytes are the Acremonium section Albo-lanosa spp. (Acremonium endophytes), which are conidial anamorphs (strictly asexual forms) of Epichloë typhina. Other endophytes which have been noted are a Gliocladium-like fungus in perennial ryegrass (Lolium perenne L.) and a Phialophora-like fungus in tall fescue (Festuca arundinacea Schreb.). Here, we report the identification of additional non-Acremonium sp. endophytes (herein designated p-endophytes) in three more grass species: Festuca gigantea, Festuca arizonica, and Festuca pratensis. In each grass species, the p-endophyte was cosymbiotic with an Acremonium endophyte. Serological analysis and sequence determinations of variable portions of their rRNA genes indicated that the two previously identified non-Acremonium endophytes are closely related to each other and to the newly identified p-endophytes. Therefore, the p-endophytes represent a second group of widely distributed grass symbionts.
Purification and characterization of a new agarase from a marine bacterium, Vibrio sp. strain JT0107
Authors: Sugano Y , Terada I , Arita M , Noma M , Matsumoto T
Published: January 2020

A marine bacterial strain that decomposes the cell walls of some seaweeds, including a Laminaria sp. and Undaria pinnatifida, has been isolated from seawater. This strain has been classified to the genus Vibrio. One of the enzymes which the bacteria secreted into the culture medium was isolated and purified 45-fold from the culture fluid by a combination of ammonium sulfate precipitation and successive rounds of anion-exchange column chromatography. Purified protein migrated as a single band (M(r), 107,000) on sodium dodecyl sulfate-polyacrylamide gels. By amino acid sequence analysis, it was determined that this protein had a single N-terminal sequence that did not exhibit identity with the sequences of other agarases from marine bacteria. This novel enzyme was found to be an endo-type beta-agarase (EC 3.2.1.81) which hydrolyzes the beta-1,4 linkage of agarose to yield neoagarotetraose [O-3,6-anhydro-alpha-L-galactopyranosyl (1-->3)-O-beta-D-galactopyranosyl(1-->4)-O-3,6-anhydro-alpha-L-galactopy ranosyl (1-->3)-D-galactose] and neoagarobiose [O-3,6-anhydro-alpha-L-galactopyranosyl (1-->3)-D-galactose] at a pH of around 8. The optimum temperature was 30 degrees C. This enzyme did not decompose sodium alginate or lambda-, iota-, or kappa-carrageenan. This enzyme may be of practical application in gene technology in the isolation of DNA fragments from agarose gels after electrophoresis.
Development of an oligonucleotide probe targeting 16S rRNA and its application for detection and quantitation of the ruminal bacterium Synergistes jonesii in a mixed-population chemostat
Authors: McSweeney C S , Mackie R I , Odenyo A A , Stahl D A
Published: January 2020

Radiolabelled and fluorescent-dye-conjugated oligonucleotide probes which targeted rRNA sequences were developed for the enumeration of the ruminal bacterium Synergistes jonesii 78-1 in mixed culture. Two probes were tested, and both were highly specific for the respective complementary sequences of the target organism. Individual cells of S. jonesii in pure and mixed cultures were clearly visualized in situ by hybridization with the fluorescent-dye-conjugated probe but could not be detected in natural samples. Therefore the radiolabelled probe was used to monitor the population of S. jonesii introduced into a chemostat which simulated the rumen ecosystem. The S. jonesii probe did not hybridize to RNA extracted from the culture prior to inoculation with the target organism. After inoculation, S. jonesii rRNA represented 4.5% of the total bacterial rRNA and then rapidly declined to < 0.2% before increasing to about 1% of the total bacterial rRNA during the following 3 weeks. This study demonstrates that rRNA-targeted probes could be used for tracking organisms introduced into the rumen ecosystem.
Effects of Environmental and Nutritional Factors on Production of the Polyketide Phytotoxin Coronatine by
Pseudomonas syringae
pv. Glycinea
Authors: Palmer David A. , Bender Carol L.
Published: January 2020

Pseudomonas syringae
pv. glycinea PG4180 produces the polyketide phytotoxin coronatine. The effects of environmental, nutritional, and host factors on growth and coronatine production by PG4180 were examined by varying the components of a defined basal medium which contained the following nutrients per liter: glucose (10 g), NH
4
Cl (1 g), MgSO
4
· 7H
2
O (0.2 g), KH
2
PO
4
(4.1 g), K
2
HPO
4
· 3H
2
O (3.6 g), and FeCl
3
(2 μM). Bacterial growth was recorded as dry weight, and coronatine production was measured by high-performance liquid chromatography. Both growth and the quantity of coronatine synthesized were significantly affected by carbon source, nutrient levels (glucose, NH
4
Cl, phosphate, Mg, and SO
4
), amino acid supplements, and the presence of complex carbon and nitrogen sources. The yield of coronatine generally declined when conditions were varied from those in the basal medium. Coronatine production and growth were not affected when the pH was adjusted from 6.5 to 7.8. Increases in the osmolarity of the basal medium significantly decreased coronatine production without affecting growth. The addition of plant extracts, plant-derived secondary metabolites, or zinc did not affect growth or coronatine production, while the addition of millimolar levels of KNO
3
or micromolar levels of FeCl
3
significantly enhanced coronatine production. The yield of coronatine was maximized after a 7-day incubation at 18°C and 280 rpm. The results of the present study were used to formulate a medium which allowed for enhanced coronatine production in nearly all strains of
P. syringae
tested. A rapid method for extracting coronatine from small volumes of culture supernatant was also developed.
Similarity between Copper Resistance Genes from
Xanthomonas campestris
and
Pseudomonas syringae
Authors: Voloudakis Andreas E. , Bender Carol L. , Cooksey Donald A.
Published: January 2020

Plasmid-borne copper resistance genes from copper-resistant strains of
Xanthomonas campestris
pv. vesicatoria from California, Florida, and Oklahoma shared structural similarities. A strain of
X. campestris
pv. campestris also contained plasmid-borne copper resistance genes similar to the resistance genes from
X. campestris
pv. vesicatoria. Furthermore, a region of the copper resistance genes from
X. campestris
pv. vesicatoria 07882 hybridized with
copA,
the first gene of the copper resistance operon (
cop
) of
Pseudomonas syringae
pv. tomato. A copper-inducible protein of similar size to CopA was detected by Western blot (immunoblot) analysis from the wild-type strain 07882 and from the cloned copper resistance genes of 07882 introduced into a copper-sensitive strain of
X. campestris
pv. vesicatoria. A low level of hybridization was observed with chromosomal DNA from other xanthomonads when the copper resistance genes from strain 07882 were used as probes.
Physiological factors affecting carbon tetrachloride dehalogenation by the denitrifying bacterium Pseudomonas sp. strain KC
Authors: Lewis T A , Crawford R L
Published: January 2020

Pseudomonas sp. strain KC was grown on a medium with a low content of transition metals in order to examine the conditions for carbon tetrachloride (CT) transformation. Several carbon sources, including acetate, glucose, glycerol, and glutamate, were able to support CT transformation. The chelators 2,2'-dipyridyl and 1,10-phenanthroline stimulated CT transformation in a rich medium that otherwise did not support this activity. Low (< 10 microM) additions of dissolved iron(II), iron(III), and cobalt(II), as well as an insoluble iron(III) compound, ferric oxyhydroxide, inhibited CT transformation. The addition of 50 microM iron to actively growing cultures resulted in delayed inhibition of CT transformation. CT transformation was seen in aerobic cultures of KC, but with reduced efficiency compared with denitrifying cultures. Inhibition of CT transformation by iron was also seen in aerobically grown cultures. Optimal conditions were used in searching for effective CT transformation activity among denitrifying enrichments grown from samples of aquifer material. No activity comparable to that of Pseudomonas sp. strain KC was found among 16 samples tested.
Alternative methods for production and staining of Phanerochaete chrysosporium basidiospores
Authors: Dhawale S S , Kessler K
Published: January 2020

Homokaryotic isolates of Phanerochaete chrysosporium are generally obtained by stimulating the production of basidiospores. The most commonly used method requires a special incubator that is maintained at 28 degrees C with continuous illumination. Here we report an alternate method which permits the production of basidiopores with common laboratory incubators and requires no special illumination conditions. This alternate method gives reproducible results and yields basidiospores that are not contaminated with conidia. We also report a detailed optimized method for staining basidiospores for visualizing nuclei.
Screening by polymerase chain reaction of Bacillus thuringiensis serotypes for the presence of cryV-like insecticidal protein genes and characterization of a cryV gene cloned from B. thuringiensis subsp. kurstaki
Authors: Gleave A P , Williams R , Hedges R J
Published: January 2020

Polymerase chain reaction screening using cryV-specific oligonucleotides, designed to amplify the 5' half of cryV-type genes, revealed the presence of such genes in 7 of 21 Bacillus thuringiensis serotypes examined. Restriction analysis and hybridization studies indicated that these putative genes fall into at least three subclasses. The nucleotide sequence of the cryV-type gene cloned from B. thuringiensis subsp. kurstaki DSIR732 revealed an open reading frame coding for a protein of 719 amino acids, and lysates of Escherichia coli cells expressing the 81.2-kDa CryV732 protein were toxic to Epiphyas postvittana (Lepidoptera: Tortricidae).
Whole-Cell Hybridization of
Frankia
Strains with Fluorescence- or Digoxigenin-Labeled, 16S rRNA-Targeted Oligonucleotide Probes
Authors: Hahn Dittmar , Amann Rudolf I. , Zeyer Josef
Published: January 2020

Whole-cell hybridization with non-radioactively labeled oligonucleotide probes was used to detect and identify
Frankia
strains in pure cultures and in nodules. Digoxigenin-labeled probes, which were detected with antibody-alkaline phosphatase conjugates, were more suitable for in situ detection of
Frankia
strains than fluorescent probes since the sensitivity of the former was higher and problems arising from the autofluorescence of cells and plant material were avoided. Successful detection of
Frankia
strains in paraformaldehyde-fixed cell material with digoxigenin-labeled oligonucleotide probes depended on pretreatments to permeabilize the cells. Specific hybridization signals on vesicles were obtained after lysozyme pretreatment (1 mg ml
-1
for 30 min at 20°C). Reliable penetration of the antibody-enzyme conjugate into hyphae required additional washing with the detergent Nonidet P-40 (0.1%) and toluene (1% in ethanol) after lysozyme treatment. Identification of
Frankia
vesicles in nodule homogenates was possible only after the removal of the polysaccharide capsule surrounding the vesicles. Incubation with H
2
O
2
(15% in water for 1 h at room temperature) before lysozyme and detergent treatments was found to facilitate specific hybridization. No filaments or spores could be detected in nodule homogenates. This technique should be a powerful tool in the identification of
Frankia
isolates, in the characterization of as-yet-uncultured nodule populations, and in the confirmation of the origin of unusual
Frankia
isolates.