bluuebunny/crossref_metadata_2025 huggingface dataset:
Regiospecificity of Chlorophenol Reductive Dechlorination by Vitamin B
12s
Authors: Smith Mark H. , Woods Sandra L.
Published: January 2020

Vitamin B
12
, reduced by titanium (III) citrate to vitamin B
12s
, catalyzes the reductive dechlorination of chlorophenols. Reductive dechlorination of pentachlorophenol and of all tetrachlorophenol and trichlorophenol isomers was observed. Reaction of various chlorophenols with vitamin B
12
favored reductive dechlorination at positions adjacent to another chlorinated carbon, but chlorines
ortho
to the hydroxyl group of a phenol were particularly resistant to reductive dechlorination, even if they were also
ortho
to a chlorine. This resulted in a reductive dechlorination pattern favoring removal of
para
and
meta
chlorines, which differs substantially from the pattern exhibited by anaerobic microbial consortia.
Isolation, characterization, and expression of a second beta-tubulin-encoding gene from Colletotrichum gloeosporioides f. sp. aeschynomene
Authors: Buhr T L , Dickman M B
Published: January 2020

Colletotrichum gloeosporioides f. sp. aeschynomene is a fungal plant pathogen of Aeschynomene virginica. A beta-tubulin-encoding gene (TUB2) from this pathogen was cloned and sequenced. The deduced amino acid sequence of TUB2 had a high degree of homology to other fungal beta-tubulins. A portion of TUB2 from a benomyl-resistant C. gloeosporioides f. sp. aeschynomene mutant was also cloned and sequenced. A point mutation resulting in a glutamic acid-to-lysine substitution at amino acid 198 likely confers benomyl resistance. The mutation is relevant for use as a selectable marker in developing a gene transfer system in C. gloeosporioides f. sp. aeschynomene. Northern (RNA) hybridizations with C. gloeosporioides f. sp. aeschynomene TUB2 and another C. gloeosporioides f. sp. aeschynomene beta-tubulin-encoding gene (TUB1) as probes showed differential expression of these genes in different cell types.
A Simple and Widely Applicable Method for Preparing Homogeneous and Stable Quality Control Samples in Water Microbiology
Authors: Schijven Jack F. , Havelaar Arie H. , Bahar Mahdieh
Published: January 2020

Test strains suspended in skim milk, quickly frozen in dry ice-ethanol, and stored at - 70°C can be used as quality control samples that are immediately available by quickly thawing at 37°C. The samples remain homogeneous and stable for at least 1 year, except for
Aeromonas hydrophila
, which decreases 20 to 30% in 1 year.
Alteration of Substrate Specificity and Optimum pH of Sarcosine Oxidase by Random and Site-Directed Mutagenesis
Authors: Nishiya Yoshiaki , Imanaka Tadayuki
Published: January 2020

The sarcosine oxidase gene was randomly mutated, and F103V, which showed altered specificity, was screened with the plate assay. Five different mutants at position 103 were also constructed. Each mutant exhibited a rather striking change in substrate specificity, except for F103W. The optimum pH was also shifted, depending on the substrate.
Production of some extracellular enzymes by a lignin peroxidase-producing brown rot fungus, Polyporus ostreiformis, and its comparative abilities for lignin degradation and dye decolorization
Authors: Dey S , Maiti T K , Bhattacharyya B C
Published: January 2020

Polyporus ostreiformis produced Mn peroxidase, acid protease, alpha-amylase, and lignin peroxidase, with maximum activities of 40, 8,300, and 4,200 U liter-1 and 50 nkat liter-1, respectively, in nitrogen-limited liquid media. The fungus removed only 18.6% lignin from rice straw in 3 weeks but effected 99% decolorization of Congo red dye in 9 days.
Induction of Chitin-Binding Proteins during the Specific Attachment of the Marine Bacterium
Vibrio harveyi
to Chitin
Authors: Montgomery Michael T. , Kirchman David L.
Published: January 2020

Previous work has shown that attachment of
Vibrio harveyi
to chitin is specific and involves at least two chitin-binding peptides. However, the roles and regulation of these chitin-binding peptides in attachment are still unclear. Here we show that preincubation with the oligomeric sugars composing chitin stimulated chitinase activity, cellular attachment to chitin, and production of chitin-binding peptides. One of these peptides, a 53-kDa peptide, is produced constitutively and appears to mediate initial attachment to chitin. Synthesis of another peptide, a 150-kDa chitin-binding peptide, is induced by chitin and thus may be involved in time-dependent attachment. Coordinated regulation of attachment and degradation of chitin may give bacteria like
V. harveyi
a selective advantage over other bacteria in nutrient-poor aquatic environments.
Isolation of Insertion Sequence IS
RLd
TAL1145-1 from a
Rhizobium
sp. (
Leucaena diversifolia
) and Distribution of Homologous Sequences Identifying Cross-Inoculation Group Relationships
Authors: Rice Douglas J. , Somasegaran Padma , MacGlashan Kathryn , Bohlool B. Ben
Published: January 2020

Insertion sequence (IS) element IS
RLd
TAL1145-1 from
Rhizobium
sp. (
Leucaena diversifolia
) strain TAL 1145 was entrapped in the
sacB
gene of the positive selection vector pUCD800 by insertional inactivation. A hybridization probe prepared from the whole 2.5-kb element was used to determine the distribution of homologous sequences in a diverse collection of 135
Rhizobium
and
Bradyrhizobium
strains. The IS probe hybridized strongly to Southern blots of genomic DNAs from 10 rhizobial strains that nodulate both
Phaseolus vulgaris
(beans) and
Leucaena leucocephala
(leguminous trees), 1
Rhizobium
sp. that nodulates
Leucaena
spp., 9
R. meliloti
(alfalfa) strains, 4
Rhizobium
spp. that nodulate
Sophora chrysophylla
(leguminous trees), and 1 nonnodulating bacterium associated with the nodules of
Pithecellobium dulce
from the
Leucaena
cross-inoculation group, producing distinguishing IS patterns for each strain. Hybridization analysis revealed that IS
RLd
TAL1145-1 was strongly homologous with and closely related to a previously isolated element, IS
Rm
USDA1024-1 from
R. meliloti
, while restriction enzyme analysis found structural similarities and differences between the two IS homologs. Two internal segments of these IS elements were used to construct hybridization probes of 1.2 kb and 380 bp that delineate a structural similarity and a difference, respectively, of the two IS homologs. The internal segment probes were used to analyze the structures of homologous IS elements in other strains. Five types of structural variation in homolog IS elements were found. The predominate IS structural type naturally occurring in a strain can reasonably identify the strain's cross-inoculation group relationships. Three IS structural types were found in
Rhizobium
species that nodulate beans and
Leucaena
species, one of which included the designated type IIB strain of
R. tropici
(CIAT 899). Weak homology to the whole IS probe, but not with the internal segments, was found with two
Bradyrhizobium japonicum
strains. The taxonomic and ecological implications of the distribution of IS
RLd
TAL1145-1 are discussed.
A reporter gene analysis of penicillin biosynthesis gene expression in Penicillium chrysogenum and its regulation by nitrogen and glucose catabolite repression
Authors: Feng B , Friedlin E , Marzluf G A
Published: January 2020

Vectors which possess a truncated niaD gene encoding nitrate reductase were developed to allow targeted gene integration during transformation of an niaD mutant Penicillium chrysogenum host. The Penicillium genes pcbC and penAB are immediately adjacent to each other and are divergently transcribed, with an intergenic control region serving as their promoters. Gene fusions were constructed with a reporter gene, uidA, which encodes beta-glucuronidase. The pcbC-penAB intergenic region was fused to the uidA gene in both orientations so that regulated expression of each structural gene could be investigated. These fusion genes were targeted to the chromosomal site of the niaD locus of P. chrysogenum, and their expression was examined under different growth conditions. The expression of each of these penicillin biosynthesis genes was found to be regulated by nitrogen repression, glucose repression, and growth stage control.
Effect of Mineral and Organic Soil Constituents on Microbial Mineralization of Organic Compounds in a Natural Soil
Authors: Knaebel David B. , Federle Thomas W. , McAvoy Drew C. , Vestal J. Robie
Published: January 2020

This research addressed the effect of mineral and organic soil constituents on the fate of organic compounds in soils. Specifically, it sought to determine how the associations between organic chemicals and different soil constituents affect their subsequent biodegradation in soil. Four
14
C-labeled surfactants were aseptically adsorbed to montmorillonite, kaolinite, illite, sand, and humic acids. These complexes were mixed with a woodlot soil, and
14
CO
2
production was measured over time. The mineralization data were fitted to various production models by nonlinear regression, and a mixed (3/2)-order model was found to most accurately describe the mineralization patterns. Different mineralization patterns were observed as a function of the chemical and soil constituents. Surfactants that had been preadsorbed to sand or kaolinite usually showed similar mineralization kinetics to the control treatments, in which the surfactants were added to the soil as an aqueous solution. Surfactants that had been bound to illite or montmorillonite were typically degraded to lesser extents than the other forms, while surfactant-humic acid complexes were degraded more slowly than the other forms. The desorption coefficients (

K
d

) of the soil constituent-bound surfactants were negatively correlated with the initial rates of degradation (
k
1
) and estimates of
14
CO
2
yield (
P
o
) as well as actual total yields of
14
CO
2
. However, there was no relationship between

K
d

and second-stage zero-order rates of mineralization (
k
o
). Microbial community characteristics (biomass and activity) were not correlated with any of the mineralization kinetic parameters. Overall, this study showed that environmental form had a profound effect on the ultimate fate of biodegradable chemicals in soil. This form is defined by the physicochemical characteristics of the chemical, the composition and mineralogy of the soil, and the mode of entry of the chemical into the soil environment.
Pseudomonas sp. strain HBP1 Prp degrades 2-isopropylphenol (ortho-cumenol) via meta cleavage
Authors: Reichlin F , Kohler H P
Published: January 2020

Pseudomonas sp. strain HBP1 Prp grew on 2-isopropylphenol as the sole carbon and energy source with a maximal specific growth rate of 0.14 h-1 and transient accumulation of isobutyric acid. Oxygen uptake experiments with resting cells and enzyme assays with crude-cell extracts showed that 2-isopropylphenol was catabolized via a broad-spectrum meta cleavage pathway. These findings were confirmed by experiments with partially purified enzymes. Identification of 3-isopropylcatechol and 2-hydroxy-6-oxo-7-methylocta-2,4-dienoic acid as the products of the initial monooxygenase reaction and the subsequent extradiol ring cleavage dioxygenase reaction, respectively, was based on gas chromatography-mass spectrometry analysis of the corresponding trimethylsilyl derivatives. The meta cleavage product hydrolase hydrolyzed 2-hydroxy-6-oxo-7-methylocta-2,4-dienoic acid (meta cleavage product of 2-isopropylphenol) to isobutyric acid and 2-hydroxypent-2,4-dienoic acid.
Transformation of the Fungal Soybean Pathogen
Cercospora kikuchii
with the Selectable Marker
bar
Authors: Upchurch Robert G. , Meade Maura J. , Hightower Robin C. , Thomas Robert S. , Callahan Terrence M.
Published: January 2020

An improved transformation protocol, utilizing selection for resistance to the herbicide bialaphos, has been developed for the plant pathogenic fungus
Cercospora kikuchii
. Stable, bialaphos-resistant transformants are recovered at frequencies eight times higher than those achieved with the previous system that was based on selection for benomyl resistance. In addition to
C. kikuchii
, this improved method can also be used to transform other species of
Cercospora
.
Symbiotic Characteristics of
Rhizobium leguminosarum
bv. trifolii Isolates Which Represent Major and Minor Nodule-Occupying Chromosomal Types of Field-Grown Subclover (
Trifolium subterraneum
L.)
Authors: Leung K. , Wanjage F. N. , Bottomley P. J.
Published: January 2020

The symbiotic effectiveness and nodulation competitiveness of
Rhizobium leguminosarum
bv. trifolii soil isolates were evaluated under nonsoil greenhouse conditions. The isolates which we used represented both major and minor nodule-occupying chromosomal types (electrophoretic types [ETs]) recovered from field-grown subclover (
Trifolium subterraneum
L.). Isolates representing four ETs (ETs 2, 3, 7, and 8) that were highly successful field nodule occupants fixed between 2- and 10-fold less nitrogen and produced lower herbage dry weights and first-harvest herbage protein concentrations than isolates that were minor nodule occupants of field-grown plants. Despite their equivalent levels of abundance in nodules on field-grown subclover plants, ET 2 and 3 isolates exhibited different competitive nodulation potentials under nonsoil greenhouse conditions. ET 3 isolates generally occupied more subclover nodules than isolates belonging to other ETs when the isolates were mixed in 1:1 inoculant ratios and inoculated onto seedlings. In contrast, ET 2 isolates were less successful at nodulating under these conditions. In many cases, ET 2 isolates required a numerical advantage of at least 6:1 to 11:1 to occupy significantly more nodules than their competitors. We identified highly effective isolates that were as competitive as the ET 3 isolates despite representing serotypes that were rarely recovered from nodules of field-grown plants. When one of the suboptimally effective isolates (ET2-1) competed with an effective and competitive isolate (ET31-5) at several different inoculant ratios, the percentages of nodules occupied by the former increased as its numerical advantage increased. Although subclover yields declined as nodule occupancy by ET2-1 increased, surprisingly, this occurred at inoculant ratios at which large percentages of nodules were still occupied by ET31-5.
Multicellular Organization in a Degradative Biofilm Community
Authors: Wolfaardt G. M. , Lawrence J. R. , Robarts R. D. , Caldwell S. J. , Caldwell D. E.
Published: January 2020

Diclofop methyl, a commercial herbicide, was used as the sole carbon source to cultivate diclofop-degrading biofilms in continuous-flow slide culture. The biofilms were analyzed by using scanning confocal laser microscopy and image analysis. Spatial relationships among members of the community were distinctive to diclofop-grown biofilms. These relationships did not develop when the biofilms were grown on more labile substrates but were conserved when the biofilms were cultivated with other chlorinated ring compounds. The structures included conical bacterial consortia rising to 30 μm above the surrounding biofilm, grape-like clusters of cocci embedded in a matrix of perpendicularly oriented bacilli, and other highly specific patterns of intra- and intergeneric cellular coaggregation and growth. These unique consortial relationships indicated that syntrophic interactions may be necessary for optimal degradation of diclofop methyl and other chlorinated ring compounds.
Integrative Cloning, Expression, and Stability of the
cryIA(c)
Gene from
Bacillus thuringiensis
subsp.
kurstaki
in a Recombinant Strain of
Clavibacter xyli
subsp.
cynodontis
Authors: Lampel Jay S. , Canter Gayle L. , Dimock Michael B. , Kelly Jeffrey L. , Anderson James J. , Uratani Brenda B. , Foulke James S. , Turner John T.
Published: January 2020

A bacterial endophyte was engineered for insecticidal activity against the European corn borer. The
cryIA(c)
gene from
Bacillus thuringiensis
subsp.
kurstaki
was introduced into the chromosome of
Clavibacter xyli
subsp.
cynodontis
by using an integrative plasmid vector. The integration vectors pCG740 and pCG741 included the replicon pGEM5Zf(+), which is maintained in
Escherichia coli
but not in
C. xyli
subsp.
cynodontis; tetM
as a marker for selection in
C. xyli
subsp.
cynodontis
; and a chromosomal fragment of
C. xyli
subsp.
cynodontis
to allow for homologous recombination between the vector and the bacterial chromosome. Insertion of vector DNA into the chromosome was demonstrated by DNA hybridization. Recombinant strains MDR1.583 and MDR1.586 containing the
cryIA(c)
gene were shown to produce the 133,000-kDa protoxin and several smaller immunoreactive proteins. Both strains were equally toxic to insect larvae in bioassays. Significant insecticidal activity was demonstrated in planta. The
cryIA(c)
gene and the
tetM
gene introduced into strain MDR1.586 were shown to be deleted from some cells, thereby giving rise to a noninsecticidal segregant population. In DNA hybridization experiments and insect bioassays, these segregants were indistinguishable from the wild-type strain. Overall, these results demonstrate the plausibility of genetically engineered bacterial endophytes for insect control.
Mass Spectrometric Studies of the Effect of pH on the Accumulation of Intermediates in Denitrification by
Paracoccus denitrificans
Authors: Thomsen Jens K. , Geest Torben , Cox Raymond P.
Published: January 2020

We have used a quadrupole mass spectrometer with a gas-permeable membrane inlet for continuous measurements of the production of N
2
O and N
2
from nitrate or nitrite by cell suspensions of
Paracoccus denitrificans
. The use of nitrate and nitrite labeled with
15
N was shown to simplify the interpretation of the results when these gases were measured. This approach was used to study the effect of pH on the production of denitrification intermediates from nitrate and nitrite under anoxic conditions. The kinetic patterns observed were quite different at acidic and alkaline pH values. At pH 5.5, first nitrate was converted to nitrite, then nitrite was converted to N
2
O, and finally N
2
O was converted to N
2
. At pH 8.5, nitrate was converted directly to N
2
, and the intermediates accumulated to only low steady-state concentrations. The sequential usage of nitrate, nitrite, and nitrous oxide observed at pH 5.5 was simulated by using a kinetic model of a branched electron transport chain in which alternative terminal reductases compete for a common reductant.
Aerobic Mineralization of Trichloroethylene, Vinyl Chloride, and Aromatic Compounds by
Rhodococcus
Species
Authors: Malachowsky K. J. , Phelps T. J. , Teboli A. B. , Minnikin D. E. , White D. C.
Published: January 2020

Two
Rhodococcus
strains which were isolated from a trichloroethylene (TCE)-degrading bacterial mixture and
Rhodococcus rhodochrous
ATCC 21197 mineralized vinyl chloride (VC) and TCE. Greater than 99.9% of a 1-mg/liter concentration of VC was degraded by cell suspensions. [1,2-
14
C]VC was degraded by cell suspensions, with the production of greater than 66%
14
CO
2
and 20%
14
C-aqueous phase products and incorporation of 10% of the
14
C into the biomass. Cultures that utilized propane as a substrate were able to mineralize greater than 28% of [1,2-
14
C]TCE to
14
CO
2
, with approximately 40% appearing in
14
C-aqueous phase products and another 10% of
14
C incorporated into the biomass. VC degradation was oxygen dependent and occurred at a pH range of 5 to 10 and temperatures of 4 to 35°C. Cell suspensions degraded up to 5 mg of TCE per liter and up to 40 mg of VC per liter. Propane competitively inhibited TCE degradation. Resting cell suspensions also degraded other chlorinated aliphatic hydrocarbons, such as chloroform, 1,1-dichloroethylene, and 1,1,1-trichloroethane. The isolates degraded a mixture of aromatic and chlorinated aliphatic solvents and utilized benzene, toluene, sodium benzoate, naphthalene, biphenyl, and
n
-alkanes ranging in size from propane to hexadecane as carbon and energy sources. The environmental isolates appeared more catabolically versatile than
R. rhodochrous
ATCC 21197. The data report that environmental isolates of
Rhodococcus
species and
R. rhodochrous
ATCC 21197 have the potential to degrade TCE and VC in addition to a variety of aromatic and chlorinated aliphatic compounds either individually or in mixtures.
A Selective Medium for the Isolation and Quantification of
Bradyrhizobium japonicum
and
Bradyrhizobium elkanii
Strains from Soils and Inoculants
Authors: Tong Zhaokun , Sadowsky Michael J.
Published: January 2020

The ecological examination of members of the family
Rhizobiaceae
has been hampered by the lack of a selective medium for isolation of root nodule bacteria from soil. A novel non-antibiotic-containing medium has been developed which allows selective isolation of
Bradyrhizobium japonicum
and
B. elkanii
strains from soil and inoculants. The medium, BJSM, is based on the resistance of
B.japonicum
and
B. elkanii
strains to more than 40 μg of the metals ions Zn
2+
and Co
2+
per ml. BJSM does not allow growth of
Rhizobium
sp. strains. We used BJSM to isolate bacteria from a Hubbard soil and from several commercially prepared soybean inoculants. Ninety-eight percent of the isolates obtained from Hubbard soil nodulated
Glycine max
cv. Kasota, and between 55 and 95% of the isolates from the commercial inoculants had the ability to nodulate soybeans. Numbers of bradyrhizobia obtained by using BJSM, strain-specific fluorescent antibodies, and the most-probable-number plant infection assay indicated that the three techniques were comparable in quantifying
B. japonicum
strains in soils and inoculants, although most-probable-number counts were generally 0.5 order of magnitude greater than those obtained by using BJSM. Results of our studies indicate that BJSM is useful for direct isolation and quantification of
B. japonicum
and
B. elkanii
from natural soils and inoculants. This medium may prove to be an important tool for autecological and enumeration studies of diverse populations of bradyrhizobia and as a quality control method for soybean inoculants.
Use of the Escherichia coli beta-glucuronidase (gusA) gene as a reporter gene for analyzing promoters in lactic acid bacteria
Authors: Platteeuw C , Simons G , de Vos W M
Published: January 2020

A transcriptional fusion vector, designated pNZ272, based on the promoterless beta-glucuronidase gene (gusA) of Escherichia coli as a reporter gene, has been constructed for lactic acid bacteria. The replicon of pNZ272 was derived from the Lactococcus lactis plasmid pSH71, allowing replication in a wide range of gram-positive bacteria and E. coli. The applicability of pNZ272 and the expression of the gusA gene in L. lactis was demonstrated in shotgun cloning experiments with lactococcal chromosomal and bacteriophage DNA. In addition, three defined lactococcal promoters were inserted in pNZ272: the plasmid-derived lacA promoter, the chromosomal usp45 promoter, and a promoter from bacteriophage phi SK11G. The three resulting plasmids showed beta-glucuronidase activity in a gusA-deficient E. coli strain and in four species of lactic acid bacteria belonging to the genera Lactobacillus, Lactococcus, and Leuconostoc. The copy numbers of the gusA-expressing plasmids were similar within a single species of lactic acid bacteria. However, the specific beta-glucuronidase activity and the gusA mRNA levels varied considerably both within a single species and among different species of lactic acid bacteria. The transcriptional start site of all three promoters was determined and found to be identical in the different species. The results of this comparative promoter analysis indicate that the requirements for efficient transcription initiation differ among the lactic acid bacteria studied.
Cloning and targeted gene disruption of EXG1, encoding exo-beta 1, 3-glucanase, in the phytopathogenic fungus Cochliobolus carbonum
Authors: Schaeffer H J , Leykam J , Walton J D
Published: January 2020

The phytopathogenic fungus Cochliobolus carbonum produces an extracellular enzyme capable of degrading beta 1,3-glucan in an exolytic manner. On the basis of partial amino acid sequences of the purified enzyme, two degenerate oligonucleotides were synthesized and used as PCR primers to amplify a 1.1-kb fragment of corresponding genomic DNA. The PCR product was used to isolate the genomic copy of the gene, called EXG1. Partial sequencing of the genomic DNA confirmed that the PCR product corresponded to EXG1. A strain of the fungus specifically mutated in the EXG1 gene was constructed by homologous integration of an internal fragment of EXG1. In the mutant, enzymatic activity and the corresponding peak of UV absorption during high-pressure liquid chromatography purification were reduced by at least 98%. However, crude culture filtrates of the mutant retained 44% of the wild-type beta 1,3-glucanase activity. This residual activity was due to two additional activities which were chromatographically separable from the product of EXG1 and which were coeluted with beta 1,3-beta 1,4-glucanase activity. Growth of the EXG1 mutant was normal on sucrose and oat bran but was reduced by 65% on pure beta 1,3-glucan. The EXG1 mutant was still pathogenic to maize.
Manganese peroxidases of the white rot fungus Phanerochaete sordida
Authors: Rüttimann-Johnson C , Cullen D , Lamar R T
Published: January 2020

The ligninolytic enzymes produced by the white rot fungus Phanerochaete sordida in liquid culture were studied. Only manganese peroxidase (MnP) activity could be detected in the supernatant liquid of the cultures. Lignin peroxidase (LiP) and laccase activities were not detected under a variety of different culture conditions. The highest MnP activity levels were obtained in nitrogen-limited cultures grown under an oxygen atmosphere. The enzyme was induced by Mn(II). The initial pH of the culture medium did not significantly affect the MnP production. Three MnP isozymes were identified (MnPI, MnPII, and MnPIII) and purified to homogeneity by anion-exchange chromatography followed by hydrophobic chromatography. The isozymes are glycoproteins with approximately the same molecular mass (around 45 kDa) but have different pIs. The pIs are 5.3, 4.2, and 3.3 for MnPI, MnPII, and MnPIII, respectively. The three isozymes are active in the same range of pHs (pHs 3.0 to 6.0) and have optimal pHs between 4.5 and 5.0. Their amino-terminal sequences, although highly similar, were distinct, suggesting that each is the product of a separate gene.
Reduction of Chromate by
Desulfovibrio vulgaris
and Its
c
3
Cytochrome
Authors: Lovley Derek R. , Phillips Elizabeth J. P.
Published: January 2020

Washed cell suspensions of
Desulfovibrio vulgaris
rapidly reduced Cr(VI) to Cr(III) with H
2
as the electron donor. The
c
3
cytochrome from this organism functioned as a Cr(VI) reductase.
D. vulgaris
may have advantages over previously described Cr(VI) reducers for the bioremediation of Cr(VI)-contaminated waters.
Rapid detection and identification of Vibrio anguillarum by using a specific oligonucleotide probe complementary to 16S rRNA
Authors: Martínez-Picado J , Blanch A R , Jofre J
Published: January 2020

Partial 16S rDNA from Vibrio collection type strains and recent isolates of Vibrio-related strains were sequenced and compared with previously published sequences. A 24-base DNA oligonucleotide (VaV3) was designed and used as a specific probe for detection and identification of Vibrio anguillarum. Its specificity was tested against collection type strains and environmental isolates and no cross-reaction was found. The probe detected 8 of the 10 V. anguillarum serovars. It was applied to screen different Vibrio-related strains isolated from marine hatcheries and fish farms. The detection limit in DNA-DNA slot blot hybridization was 150 pg.
Isolation of Helicobacter strains from wild bird and swine feces
Authors: Seymour C , Lewis R G , Kim M , Gagnon D F , Fox J G , Dewhirst F E , Paster B J
Published: January 2020

We report the first isolations of Helicobacter strains from wild birds and swine. Genus-specific oligonucleotide probes identified nine Cape Cod isolates from gull, tern, house sparrow, and pig feces as Helicobacter spp. and not Campylobacter spp. Antibiotic sensitivity and urease tests distinguished three phenotypes. Strains examined rapidly lost culturability under simulated natural conditions.
Expression of the lysostaphin gene of Staphylococcus simulans in a eukaryotic system
Authors: Williamson C M , Bramley A J , Lax A J
Published: January 2020

The lysostaphin gene of Staphylococcus simulans was cloned into Escherichia coli. The 5' end of the gene was modified to include a eukaryotic start codon, the Kozak expression start site consensus sequence, and an enzyme site to facilitate manipulation of the gene. Transcription of the modified gene in vitro yielded an RNA transcript which, when added to a rabbit reticulocyte cell-free translation system, directed the synthesis of several products. The largest product, migrating at approximately 93 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was probably preprolysostaphin, since it was cleaved in the presence of an S. simulans culture supernatant to yield a polypeptide of a size similar to that of mature lysostaphin. When canine pancreatic microsomal vesicles were added to the translation system, translocation of the newly synthesized polypeptides occurred, as judged by protection from proteolysis. The gene was also expressed transiently from the human cytomegalovirus promoter in COS-7 cells. Active enzyme could be detected in the cell lysate, and the prokaryotic signal appeared to target secretion of active enzyme to the culture medium. The successful expression of the lysostaphin gene and processing of the precursor to produce active secreted enzyme open up the possibility of controlling staphylococcal mastitis by targeting expression of this gene to the mammary glands of transgenic animals.
Characteristics and Diversity of β-
d
-Glucosidase (EC 3.2.1.21) Activity in Marine Snow
Authors: Rath Johannes , Herndl Gerhard J.
Published: January 2020

Large amorphous aggregates commonly described as marine snow were sampled in the water column of the northern Adriatic Sea in August 1991. β-
d
-Glucosidase activity associated with these aggregates was characterized. Enzymatic activity was measured with the fluorogenic substrate analog 4-methylumbelliferyl-β-
d
-glucoside and found to be mainly associated with particles; on average, only 24% of the whole β-
d
-glucosidase activity remained in the supernatant after centrifugation. Although the temperature optimum for β-
d
-glucosidase activity was ≈ 40°C, incubation of the previously liberated particle-attached enzyme at 50°C for 20 min caused a >90% reduction of enzymatic activity relative to the activity at 40°C. The level of inactivation of β-
d
-glucosidase was much lower, however, when whole marine snow was incubated, indicating qualitative modifications of β-
d
-glucosidase in marine snow. Separation of β-
d
-glucosidase by different approaches indicated that the diversity of isoenzymes is restricted. In samples taken from the pycnocline, only one major isoenzyme was present in noticeable amounts. This isoenzyme contributed up to 70% of the whole β-
d
-glucosidase activity detectable by two different chromatographic separations (anion-exchange chromatography and size exclusion chromatography). Although the same isoenzyme was dominant in marine snow taken from surface waters (0.5-m depth), we found a second peak of activity which eluted at lower NaCl concentrations from the anion-exchange column. Generally, the diversity of isoenzymes exhibiting β-
d
-glucosidase activity seems to be surprisingly small in amorphous pelagic aggregates.
Isolation and Characterization of Sexual Spore Pigments from
Aspergillus nidulans
Authors: Brown Daren W. , Salvo Joseph J.
Published: January 2020

The homothallic ascomycete
Aspergillus nidulans
produces two types of pigmented spores: conidia and ascospores. The synthesis and localization of the spore pigments is developmentally regulated and occurs in specialized cell types. On the basis of spectroscopic evidence, we propose that the major ascospore pigment of
A. nidulans
(ascoquinone A) is a novel dimeric hydroxylated anthraquinone. The structure of ascoquinone A, as well as a comparison to model compounds, suggests that it is the product of a polyketide synthase. Previous studies have revealed that the conidial pigments from
A. nidulans
and a related
Aspergillus
species (
A. parasiticus
) also appear to be produced via polymerization of polyketide precursors (D. W. Brown, F. M. Hauser, R. Tommasi, S. Corlett, and J. J. Salvo, Tetrahedron Lett. 34:419-422, 1993; M. E. Mayorga and W. E. Timberlake, Mol. Gen. Genet. 235:205-212, 1992). The structural similarity between the ascospore pigment and the toxic anthraquinone norsolorinic acid, the first stable intermediate in the aflatoxin pathway, suggests an evolutionary relationship between the respective polyketide synthase systems.
Metabolism of both 4-chlorobenzoate and toluene under denitrifying conditions by a constructed bacterial strain
Authors: Coschigano P W , Häggblom M M , Young L Y
Published: January 2020

T1, a dentrifying bacterium originally isolated for its ability to grow on toluene, can also metabolize 4-hydroxybenzoate and other aromatic compounds under denitrifying conditions. A cosmid clone carrying the three genes that code for the 4-chlorobenzoate dehalogenase enzyme complex isolated from the aerobic bacterium Pseudomonas sp. strain CBS3 was successfully conjugated into strain T1. The cloned enzyme complex catalyzes the hydrolytic dechlorination of 4-chlorobenzoate to 4-hydroxybenzoate. Since molecular oxygen is not required for the dehalogenation reaction, the transconjugate strain of T1 (T1-pUK45-10C) was able to grow on 4-chlorobenzoate in the absence of O2 under denitrifying conditions. 4-Chlorobenzoate was dehalogenated to 4-hydroxybenzoate, which was then further metabolized by strain T1. The dehalogenation and metabolism of 4-chlorobenzoate were nitrate dependent and were coupled to the production of nitrite and nitrogen gas. 4-Bromobenzoate was also degraded by this strain, while 4-iodobenzoate was not. Additionally, when T1-pUK45-10C was presented with a mixture of 4-chlorobenzoate and toluene, simultaneous degradation of the compounds was observed. These results illustrate that dechlorination and degradation of aromatic xenobiotics can be mediated by a pure culture in the absence of oxygen. Furthermore, it is possible to expand the range of xenobiotic substrates degradable by an organism, and it is possible that concurrent metabolism of these substrates can occur.
Genetic analysis of hrp-related DNA sequences of Xanthomonas campestris strains causing diseases of citrus
Authors: Leite R P , Egel D S , Stall R E
Published: January 2020

The hrp gene cluster of strains of Xanthomonas campestris that cause diseases of citrus was examined by Southern hybridization of genomic DNA and by restriction endonuclease analysis of enzymatically amplified DNA fragments of the hrp gene cluster. The hrp genes were present in all strains of the pathovars of X. campestris tested in this study, including strains of the three aggressiveness groups of the citrus bacterial spot pathogen, X. campestris pv. citrumelo. X. campestris pv. citri strains in groups A, B, and C, which cause citrus canker A, B, and C, respectively, each produced characteristic restriction banding patterns of amplified hrp fragments. The restriction banding patterns of all strains within each group were identical. In contrast, restriction fragment length polymorphism was evident among strains of the moderately and weakly aggressive groups of X. campestris pv. citrumelo. X. campestris pv. citrumelo strains in the highly aggressive group had a homogeneous restriction banding pattern. The characteristic banding patterns obtained for each bacterial group indicate that X. campestris strains causing disease in citrus can be reliably differentiated and identified by restriction analysis of amplified DNA fragments of the hrp gene cluster. In addition, the phylogenetic analysis based on the restriction banding patterns of amplified fragments suggests a polyphyletic relationship of the hrp genes among the strains of X. campestris that cause disease in citrus.
Blood agar to detect virulence factors in tap water heterotrophic bacteria
Authors: Payment P , Coffin E , Paquette G
Published: January 2020

Cytolytic colonies were found in 57% of tap water samples, and up to 6% of samples were found to contain bacteria having three or more virulence factors. The factors evaluated were cytotoxicity, hemolysis, cell adherence, and cell invasiveness. Overall, 17% of the samples contained cytolytic colonies that were adherent and hemolytic. Among the media tested, tryptic soy agar with sheep blood (incubated at 35 degrees C for 48 h) was the best medium for the detection of cytolytic colonies. Of the colonies growing on this medium, 13% were cytolytic, whereas on medium R2A, less than 3% were cytolytic. Furthermore, when tryptic soy agar with blood was used, 24% of the samples contained colonies with at least three virulence factors whereas only 5% were positive with R2A. Routine monitoring by using tryptic soy agar with sheep blood is suggested as an appropriate procedure for the detection of bacteria with pathogenic potential in drinking water.
Cloning and DNA sequencing of bgaA, a gene encoding an endo-beta-1,3-1,4-glucanase, from an alkalophilic Bacillus strain (N137)
Authors: Tabernero C , Coll P M , Fernández-Abalos J M , Pérez P , Santamaría R I
Published: January 2020

The gene bgaA encoding an alkaline endo-beta-1,3-1,4-glucanase (lichenase) from an alkalophilic Bacillus sp. strain N137, isolated in our laboratory, was cloned and expressed from its own promoter in Escherichia coli. The nucleotide sequence of a 1,416-bp DNA fragment containing bgaA was determined and revealed an open reading frame of 828 nucleotides. The deduced protein sequence consists of 276 amino acids and has a 31-amino-acid putative signal peptide which is functional in E. coli, in which the BgaA protein is located mainly in the periplasmic space. The lichenase activity of BgaA is stable between pH 6 and 12, it shows optimal activity at a temperature between 60 and 70 degrees C, and it retains 65% of its activity after incubation at 70 degrees C for 1 h. This protein is similar to some other lichenases from Bacillus species such as B. amyloliquefaciens, B. brevis, B. licheniformis, B. macerans, B. polymyxa, and B. subtilis. However, it has a lysine-rich region at the carboxy terminus which is not found in any other published lichenase sequence and might be implicated in the unusual biochemical properties of this enzyme. The location of the mRNA 5' end was determined by primer extension and corresponds to nucleotide 235. A typical Bacillus sigma A promoter precedes the transcription start site.
Quantification of methanogenic groups in anaerobic biological reactors by oligonucleotide probe hybridization
Authors: Raskin L , Poulsen L K , Noguera D R , Rittmann B E , Stahl D A
Published: January 2020

The microbial community structure of anaerobic biological reactors was evaluated by using oligonucleotide probes complementary to conserved tracts of the 16S rRNAs of phylogenetically defined groups of methanogens. Phylogenetically defined groups of methanogens were quantified and visualized, respectively, by hybridization of 32P- and fluorescent-dye-labeled probes to the 16S rRNAs from samples taken from laboratory acetate-fed chemostats, laboratory municipal solid waste digestors, and full-scale sewage sludge digestors. Methanosarcina species, members of the order Methanobacteriales, and Methanosaeta species were the most abundant methanogens present in the chemostats, the solid-waste digestors, and the sewage sludge digestors, respectively.
A Diffusion Gradient Chamber for Studying Microbial Behavior and Separating Microorganisms
Authors: Emerson David , Worden R. Mark , Breznak John A.
Published: January 2020

The natural habitats of most microbes are dynamic and include spatial gradients of growth substrates, electron acceptors, pH, salts, and inhibitory compounds. To mimic this diffusive aspect of nature, we developed an analytical diffusion gradient chamber (DGC) that can be used to separate, enrich for, isolate, and study the behavior of microorganisms. The chamber is a polycarbonate box containing an arena (5 by 5 by 2 cm) into which is cast a semisolid growth medium. Continuously replenished solute reservoirs positioned on each side of the arena but separated from it by a porous membrane enable the formation throughout the gel of multiple, intersecting gradients of solutes in two dimensions. With glucose as the solute, a gradient which spanned a 100-fold range in concentration was established across the arena in about 4 days. The shape of the glucose gradient was accurately predicted by a mathematical model based on Fickian diffusion. The growth and migratory behavior of
Escherichia coli
in response to imposed gradients of attractants (aspartate, α-methyl aspartate, and serine) and a repellent (valine) were examined. Cells responded in predictable ways to such gradients by forming distinctive growth and migration patterns in the DGC. This was true for wild-type
E. coli
as well as specific chemotaxis and motility mutants. The patterns yielded information about the threshold concentration of chemoeffectors needed to elicit a response as well as their saturating concentration. It was also evident that the metabolism of attractants significantly affected the gradients and, hence, the movement of cells. Finally, it was possible to separate
E. coli
and
Pseudomonas fluorescens
in the DGC on the basis of their differential responses to gradients of various chemoeffectors.
Luminescence-based detection of activity of starved and viable but nonculturable bacteria
Authors: Duncan S , Glover L A , Killham K , Prosser J I
Published: January 2020

A naturally luminescent bacterium, Vibrio harveyi, and two bacteria, Escherichia coli and Pseudomonas fluorescens, which had been genetically marked with luminescence were starved in liquid medium at 4 and 30 degrees C for 54 days. Total cell concentrations and concentrations of culturable and viable cells were determined by acridine orange staining, dilution plate counting, and direct viable counting, respectively, and population activity was measured by luminometry. V. harveyi became nonculturable but maintained viability during starvation at 4 degrees C and maintained both culturability and viability at 30 degrees C. In contrast, E. coli became viable but nonculturable during starvation at 30 degrees C but not at 4 degrees C. Luminescence of nonculturable cells of both strains, and culturable cells of V. harveyi, decreased to background levels during starvation. Luminescence of starved culturable cells of E. coli also fell below background levels but occasionally increased to detectable values. Viable, nonculturable forms of P. fluorescens were not detected at either temperature, and cells starved at 4 degrees C showed no decrease in luminescence measured during incubation of samples at 25 degrees C. Following incubation of late-log-phase cells with yeast extract and nalidixic acid, changes in light output directly paralleled changes in cell length, as observed during direct viable counting. Quantification of changes in luminescence following incubation of starved cells with yeast extract enabled measurement of the activity of both culturable and viable but nonculturable cells. Measurement of luminescence was significantly more sensitive, rapid, and convenient in quantifying activity following nutrient amendment than measurement of changes in cell length.(ABSTRACT TRUNCATED AT 250 WORDS)
Molecular characterization of Frankia microsymbionts from spore-positive and spore-negative nodules in a natural alder stand
Authors: Simonet P , Bosco M , Chapelon C , Moiroud A , Normand P
Published: January 2020

Among the Frankia strains capable of establishing a nitrogen-fixing symbiosis with actinorrhizal plants, in planta sporangial formation is not universal and has led to the distinction between spore-positive (Sp+) and spore-negative (Sp-) nodules. Numerous Frankia strains have been isolated in pure culture from Sp+ nodules of different host plants, but, although they were able to reinfect their respective host plant, none of them was able to differentiate endophytic sporangia under laboratory conditions. The first step of this study was to demonstrate, at the molecular level, the existence of specific Sp+ strain genotypes differing from Sp- strain genotypes in a single alder stand. In a second step, by way of PCR amplification and sequencing of the PCR products, we have characterized oligonucleotide primers specific for the genus Frankia and for each of the two types of Frankia microsymbionts able, or not, to differentiate sporangia inside natural green alder nodules. These primers applied in PCRs with DNA extracted from nodules confirmed the morphological identification and revealed the presence of nodules colonized by both types of actinomycetes. Finally, a preliminary PCR study was conducted with DNA extracted directly from soil samples which permitted checking the rhizosphere of Sp+ and Sp- nodules for the presence of the corresponding strains.
A reporter gene construct for studying the regulation of manganese peroxidase gene expression
Authors: Godfrey B J , Akileswaran L , Gold M H
Published: January 2020

The orotidylate decarboxylase (ODase) gene (ura1) from Schizophyllum commune was utilized as a reporter for studying Mn regulation of the manganese peroxidase (MnP) gene (mnp) from the lignin-degrading basidiomycete Phanerochaete chrysosporium. A 1,500-bp fragment of the mnp1 promoter was fused upstream of the coding region of the ODase gene in a plasmid (pAMO) containing the S. commune ade5 gene as a selectable marker. pAMO was used to transform a P. chrysosporium ade1 ura11 mutant lacking endogenous ODase activity. When the P. chrysosporium transformant was grown in nitrogen-limited, Mn(II)-sufficient cultures, ODase activity was detected only during secondary metabolic growth and the pattern of ODase expression was similar to that of endogenous MnP. When Mn was added to 6-day-old nitrogen-limited, Mn-deficient cultures, both ODase activity and MnP activity were induced synchronously with maximal activity at 30 h. Growth in high-nitrogen-concentration medium suppressed the induction of both the ODase and endogenous MnP. These results indicate that this promoter-reporter construct can be used to study the regulation of the mnp gene.
Composition and Rheological Properties of Extracellular Polysaccharide 105-4 Produced by
Pseudomonas
sp. Strain ATCC 53923
Authors: Dasinger Bruce L. , McArthur Hamish A. I. , Lengen James P. , Smogowicz Adam A. , Miller James W. , O'Neill John J. , Horton Derek , Costa Jill B.
Published: January 2020

A soil isolate produced a novel extracellular polysaccharide (EPS) with unusually potent thickening powers. The EPS contained
d
-mannose,
d
-glucose,
d
-galactose, and
d
-glucuronic acid in the unique molar ratio 1:4:1:2 and 10 to 15% acetate. Viscosities of a 1-g/liter aqueous solution were 1 × 10
6
and 14 × 10
3
cP at shear rates of 0.01 and 0.1 s
-1
, respectively. The EPS was insensitive to high concentrations of NaCl and CaCl
2
.
Acetate Synthesis in Soil from a Bavarian Beech Forest
Authors: Küsel Kirsten , Drake Harold L.
Published: January 2020

Soil obtained from a beech forest formed significant amounts of acetate when incubated in a bicarbonate-buffered, mineral salt solution under anaerobic conditions at both 5 and 20°C (21 and 38 g of acetate per kg [dry weight] of soil, respectively). At 20°C, following an 18-day lag period, rates of 0.07 mmol of acetate synthesized per g (dry weight) of soil per day were observed. Acetate was not subject to immediate turnover; methane and hydrogen were not formed during the time intervals (5°C, 335 days; 20°C, 95 days) evaluated. The synthesis of acetate from endogenous materials was coincident with acetogenic potentials, i.e., the capacity to catalyze the H
2
-dependent synthesis of acetate. Hydrogen consumption was not directed towards the synthesis of methane. Collectively, these results suggest that acetogenesis may be an underlying microbial activity of this forest soil.