bluuebunny/crossref_metadata_2025 huggingface dataset:
Stereo- and Regioselective Hydroxylation of α-Ionone by
Streptomyces
Strains
Authors: Lutz-Wahl Sabine , Fischer Peter , Schmidt-Dannert Claudia , Wohlleben Wolfgang , Hauer Bernhard , Schmid Rolf D.
Published: December 2019

A total of 215
Streptomyces
strains were screened for their capacity to regio- and stereoselectively hydroxylate β- and/or α-ionone to the respective 3-hydroxy derivatives. With β-ionone as the substrate, 15 strains showed little conversion to 4-hydroxy- and none showed conversion to the 3-hydroxy product as desired. Among these 15
Streptomyces
strains,
S. fradiae
Tü27,
S. arenae
Tü 495,
S. griseus
ATCC 13273,
S. violaceoniger
Tü 38, and
S. antibioticus
Tü 4 and Tü 46 converted α-ionone to 3-hydroxy-α-ionone with significantly higher hydroxylation activity compared to that of β-ionone. Hydroxylation of racemic α-ionone [(6
R
)-(−)/(6
S
)-(+)] resulted in the exclusive formation of only the two enantiomers (3
R
,6
R
)- and (3
S
,6
S
)-hydroxy-α-ionone. Thus, the enzymatic hydroxylation of α-ionone by the
Streptomyces
strains tested proceeds with both high regio- and stereoselectivity.
Molecular Characterization of Plasmid-Mediated Oxytetracycline Resistance in
Aeromonas salmonicida
Authors: Adams C. A. , Austin B. , Meaden P. G. , McIntosh D.
Published: December 2019

Using broth conjugation, we found that 19 of 29 (66%) oxytetracycline (OT)-resistant isolates of
Aeromonas salmonicida
transferred the OT resistance phenotype to
Escherichia coli
. The OT resistance phenotype was encoded by high-molecular-weight R-plasmids that were capable of transferring OT resistance to both environmental and clinical isolates of
Aeromonas
spp. The molecular basis for antibiotic resistance in OT-resistant isolates of
A. salmonicida
was determined. The OT resistance determinant from one plasmid (pASOT) of
A. salmonicida
was cloned and used in Southern blotting and hybridization experiments as a probe. The determinant was identified on a 5.4-kb
Eco
RI fragment on R-plasmids from the 19 OT-resistant isolates of
A. salmonicida
. Hybridization with plasmids encoding the five classes (classes A to E) of OT resistance determinants demonstrated that the OT resistance plasmids of the 19
A. salmonicida
isolates carried the class A resistance determinant. Analysis of data generated from restriction enzyme digests showed that the OT resistance plasmids were not identical; three profiles were characterized, two of which showed a high degree of homology.
Monoclonal Antibodies Raised against Native Major Capsid Proteins of Lactococcal c2-Like Bacteriophages
Authors: Chibani Azaïez Sandra R. , Fliss Ismaïl , Simard Ronald E. , Moineau Sylvain
Published: December 2019

Phage Q38, a representative member of the c2 species, was purified by CsCl gradient and used to immunize BALB/c mice. Monoclonal antibodies (MAbs) were raised and then characterized by enzyme-linked immunosorbent assay. Two MAbs of isotype immunoglobulin G2a, designated 2A5 and 6G7, reacted only with phages belonging to the c2 species and not with phages of the 936 and P335 species, with a
Lactococcus lactis
cell extract, or with phage DNA. Immunoelectron microscopy showed that both MAbs recognized only phage head proteins. They did not react with any denatured phage proteins in Western blot assays. However, when the nitrocellulose membranes were treated with a Triton-based buffer to assist in protein renaturation, MAbs 2A5 and 6G7 recognized the two major capsid proteins with molecular masses of 80 and 170 kDa. Competitive inhibition tests showed that the two MAbs bind to overlapping epitopes. These MAbs may be a useful tool for monitoring c2 bacteriophages during dairy fermentation and in genetic studies.
Physiological Diversity of the Rhizosphere Diazotroph Assemblages of Selected Salt Marsh Grasses
Authors: Bagwell Christopher E. , Piceno Yvette M. , Ashburne-Lucas Amy , Lovell Charles R.
Published: December 2019

Rhizosphere diazotroph assemblages of salt marsh grasses are thought to be influenced by host plant species and by a number of porewater geochemical parameters. Several geochemical variables can adversely affect plant productivity and spatial distributions, resulting in strong zonation of plant species and growth forms. This geochemically induced stress may also influence the species compositions and distributions of rhizosphere diazotroph assemblages, but little is currently known about these organisms. The diversity and key physiological features of culturable, O
2
-tolerant rhizosphere diazotrophs associated with the tall and short growth forms of
Spartina alterniflora
and with
Juncus roemerianus
were examined. A total of 339 gram-negative strains were isolated by a root stab culture approach and morphologically and physiologically characterized by using API and BIOLOG tests. Eighty-six distinct groups composed of physiologically similar strains were identified. Of these groups, 72% were shown to be capable of N
2
fixation through molecular analyses, and a representative strain was chosen from each diazotroph group for further characterization. Cluster and principal-components analysis of BIOLOG data allowed the designation of physiologically distinct strain groupings. Most of these groups were dominated by strains that were not identifiable to species on the basis of API or BIOLOG testing. Representatives of several families including the
Enterobacteriaceae
,
Vibrionaceae
,
Azotobacteraceae
,
Spirillaceae
,
Pseudomonadaceae
, and
Rhizobiaceae
were recovered, as well as strains with no clear taxonomic affiliations. This study identifies numerous potentially important physiological groups of the salt marsh diazotroph assemblage.
Phylogenetic Analysis of Nonthermophilic Members of the Kingdom
Crenarchaeota
and Their Diversity and Abundance in Soils
Authors: Buckley Daniel H. , Graber Joseph R. , Schmidt Thomas M.
Published: December 2019

Within the last several years, molecular techniques have uncovered numerous 16S rRNA gene (rDNA) sequences which represent a unique and globally distributed lineage of the kingdom
Crenarchaeota
that is phylogenetically distinct from currently characterized crenarchaeotal species. rDNA sequences of members of this novel crenarchaeotal group have been recovered from low- to moderate-temperature environments (−1.5 to 32°C), in contrast to the high-temperature environments (temperature, >80°C) required for growth of the currently recognized crenarchaeotal species. We determined the diversity and abundance of the nonthermophilic members of the
Crenarchaeota
in soil samples taken from cultivated and uncultivated fields located at the Kellogg Biological Station’s Long-Term Ecological Research site (Hickory Corners, Mich.). Clones were generated from 16S rDNA that was amplified by using broad-specificity archaeal PCR primers. Twelve crenarchaeotal sequences were identified, and the phylogenetic relationships between these sequences and previously described crenarchaeotal 16S rDNA sequences were determined. Phylogenetic analyses included nonthermophilic crenarchaeotal sequences found in public databases and revealed that the nonthermophilic
Crenarchaeota
group is composed of at least four distinct phylogenetic clusters. A 16S rRNA-targeted oligonucleotide probe specific for all known nonthermophilic crenarchaeotal sequences was designed and used to determine their abundance in soil samples. The nonthermophilic
Crenarchaeota
accounted for as much as 1.42% ± 0.42% of the 16S rRNA in the soils analyzed.
Whole-Cell Kinetics of Trichloroethylene Degradation by Phenol Hydroxylase in a
Ralstonia eutropha
JMP134 Derivative
Authors: Ayoubi Patricia J. , Harker Alan R.
Published: December 2019

The rate, progress, and limits of trichloroethylene (TCE) degradation by
Ralstonia eutropha
AEK301/pYK3021 whole cells were examined in the absence of aromatic induction. At TCE concentrations up to 800 μM, degradation rates were sustained until TCE was no longer detectable. The

K
s

and
V
max
for TCE degradation by AEK301/pYK3021 whole cells were determined to be 630 μM and 22.6 nmol/min/mg of total protein, respectively. The sustained linear rates of TCE degradation by AEK301/pYK3021 up to a concentration of 800 μM TCE suggest that solvent effects are limited during the degradation of TCE and that this construct is little affected by the formation of toxic intermediates at the TCE levels and assay duration tested. TCE degradation by this strain is subject to carbon catabolite repression.
Ammonium Removal by the Oxygen-Limited Autotrophic Nitrification-Denitrification System
Authors: Kuai Linping , Verstraete Willy
Published: December 2019

The present lab-scale research reveals the potential of implementation of an oxygen-limited autotrophic nitrification-denitrification (OLAND) system with normal nitrifying sludge as the biocatalyst for the removal of nitrogen from nitrogen-rich wastewater in one step. In a sequential batch reactor, synthetic wastewater containing 1 g of NH
4
+
-N liter
−1
and minerals was treated. Oxygen supply to the reactor was double-controlled with a pH controller and a timer. At a volumetric loading rate (

B
v

) of 0.13 g of NH
4
+
-N liter
−1
day
−1
, about 22% of the fed NH
4
+
-N was converted to NO
2
−
-N or NO
3
−
-N, 38% remained as NH
4
+
-N, and the other 40% was removed mainly as N
2
. The specific removal rate of nitrogen was on the order of 50 mg of N liter
−1
day
−1
, corresponding to 16 mg of N g of volatile suspended solids
−1
day
−1
. The microorganisms which catalyzed the OLAND process are assumed to be normal nitrifiers dominated by ammonium oxidizers. The loss of nitrogen in the OLAND system is presumed to occur via the oxidation of NH
4
+
to N
2
with NO
2
−
as the electron acceptor. Hydroxylamine stimulated the removal of NH
4
+
and NO
2
−
. Hydroxylamine oxidoreductase (HAO) or an HAO-related enzyme might be responsible for the loss of nitrogen.
Humic Acid Reduction by
Propionibacterium freudenreichii
and Other Fermenting Bacteria
Authors: Benz Marcus , Schink Bernhard , Brune Andreas
Published: December 2019

Iron-reducing bacteria have been reported to reduce humic acids and low-molecular-weight quinones with electrons from acetate or hydrogen oxidation. Due to the rapid chemical reaction of amorphous ferric iron with the reduced reaction products, humic acids and low-molecular-weight redox mediators may play an important role in biological iron reduction. Since many anaerobic bacteria that are not able to reduce amorphous ferric iron directly are known to transfer electrons to other external acceptors, such as ferricyanide, 2,6-anthraquinone disulfonate (AQDS), or molecular oxygen, we tested several physiologically different species of fermenting bacteria to determine their abilities to reduce humic acids.
Propionibacterium freudenreichii
,
Lactococcus lactis
, and
Enterococcus cecorum
all shifted their fermentation patterns towards more oxidized products when humic acids were present;
P. freudenreichii
even oxidized propionate to acetate under these conditions. When amorphous ferric iron was added to reoxidize the electron acceptor, humic acids were found to be equally effective when they were added in substoichiometric amounts. These findings indicate that in addition to iron-reducing bacteria, fermenting bacteria are also capable of channeling electrons from anaerobic oxidations via humic acids towards iron reduction. This information needs to be considered in future studies of electron flow in soils and sediments.
Methanotrophs and Methanogens in Masonry
Authors: Kussmaul Martin , Wilimzig Markus , Bock Eberhard
Published: December 2019

Methanotrophs were present in 48 of 225 stone samples which were removed from 19 historical buildings in Germany and Italy. The average cell number of methanotrophs was 20 CFU per g of stone, and their activities ranged between 11 and 42 pmol of CH
4
g of stone
−1
day
−1
. Twelve strains of methane-oxidizing bacteria were isolated. They belonged to the type II methanotrophs of the genera
Methylocystis
,
Methylosinus
, and
Methylobacterium
. In masonry, growth substrates like methane or methanol are available in very low concentrations. To determine if methane could be produced by the stone at rates sufficient to support growth of methanotrophs, methane production by stone samples under nonoxic conditions was examined. Methane production of 0.07 to 215 nmol of CH
4
g of stone
−1
day
−1
was detected in 23 of 47 stone samples examined. This indicated the presence of the so-called “mini-methane”-producing bacteria and/or methanogenic archaea. Methanotrophs occurred in nearly all samples which showed methane production. This finding indicated that methanotrophs depend on biogenic methane production in or on stone surfaces of historical buildings.
Antagonistic Activity against
Helicobacter
Infection In Vitro and In Vivo by the Human
Lactobacillus acidophilus
Strain LB
Authors: Coconnier Marie-Helene , Lievin Vanessa , Hemery Elisabeth , Servin Alain L.
Published: December 2019

The purpose of the present study was to examine the activity of the human
Lactobacillus acidophilus
strain LB, which secretes an antibacterial substance(s) against
Helicobacter pylori
in vitro and in vivo. The spent culture supernatant (SCS) of the strain LB (LB-SCS) dramatically decreased the viability of
H. pylori
in vitro independent of pH and lactic acid levels. Adhesion of
H. pylori
to the cultured human mucosecreting HT29-MTX cells decreased in parallel with the viability of
H. pylori
. In conventional mice, oral treatment with the LB-SCS protected against infection with
Helicobacter felis
. Indeed, at both 8 and 49 days post-LB-SCS treatment (29 and 70 days postinfection), inhibition of stomach colonization by
H. felis
was observed, and no evidence of gastric histopathological lesions was found. LB-SCS treatment inhibits the
H. pylori
urease activity in vitro and in
H. pylori
that remained associated with the cultured human mucosecreting HT29-MTX cells. Moreover, a decrease in urease activity was detected in the stomach of the mice infected with
H. felis
and treated with LB-SCS.
A Novel Screening Method for Isolating Exopolysaccharide-Deficient Mutants
Authors: Liu Minghsun , González Juan E. , Willis Laura B. , Walker Graham C.
Published: December 2019

A screening method based on differential staining of the wild type and exopolysaccharide-deficient mutants of
Rhizobium
(
Sinorhizobium
)
meliloti
by the lipophilic dye Sudan Black B is described. Mutants defective in the production of either succinoglycan or EPS II (galactoglucan) were isolated by using this method, which might also prove useful for isolating exopolysaccharide-defective derivatives of other bacteria.
Enzymatic Recovery of Elemental Palladium by Using Sulfate-Reducing Bacteria
Authors: Lloyd Jon R. , Yong Ping , Macaskie Lynne E.
Published: December 2019

Worldwide usage of platinum group metals is increasing, prompting new recovery technologies. Resting cells of
Desulfovibrio desulfuricans
reduced soluble Pd
2+
to elemental, cell-bound Pd
0
supported by pyruvate, formate, or H
2
as the electron donor without biochemical cofactors. Pd reduction was O
2
insensitive, opening the way for recycling and recovery of Pd under oxic conditions.
Bacterial Oxidation of Dibromomethane and Methyl Bromide in Natural Waters and Enrichment Cultures
Authors: Goodwin K. D. , Schaefer J. K. , Oremland R. S.
Published: December 2019

Bacterial oxidation of14CH2Br2and14CH3Br was measured in freshwater, estuarine, seawater, and hypersaline-alkaline samples. In general, bacteria from the various sites oxidized similar amounts of14CH2Br2and comparatively less14CH3Br. Bacterial oxidation of14CH3Br was rapid in freshwater samples compared to bacterial oxidation of14CH3Br in more saline waters. Freshwater was also the only site in which methyl fluoride-sensitive bacteria (e.g., methanotrophs or nitrifiers) governed brominated methane oxidation. Half-life calculations indicated that bacterial oxidation of CH2Br2was potentially significant in all of the waters tested. In contrast, only in freshwater was bacterial oxidation of CH3Br as fast as chemical removal. The values calculated for more saline sites suggested that bacterial oxidation of CH3Br was relatively slow compared to chemical and physical loss mechanisms. However, enrichment cultures demonstrated that bacteria in seawater can rapidly oxidize brominated methanes. Two distinct cultures of nonmethanotrophic methylotrophs were recovered; one of these cultures was able to utilize CH2Br2as a sole carbon source, and the other was able to utilize CH3Br as a sole carbon source.
Pathway of Glucose Catabolism by Strain VeGlc2, an Anaerobe Belonging to the
Verrucomicrobiales
Lineage of Bacterial Descent
Authors: Janssen Peter H.
Published: December 2019

Strain VeGlc2, an anaerobic ultramicrobacterium belonging to the
Verrucomicrobiales
lineage of bacterial descent, fermented glucose to acetate, propionate, succinate, and CO
2
. The distribution of radiolabel in the fermentation end products produced from position-labelled glucose and in vitro measurements of enzyme activities in crude cell extracts prepared from glucose-grown cells showed that glucose was metabolized via the Embden-Meyerhof-Parnas pathway. The 6-phosphofructokinase (EC
2.7.1.90
) activity required pyrophosphate as the phosphoryl donor, and ATP could not replace pyrophosphate. The other enzyme activities were those of a classical Embden-Meyerhof-Parnas pathway.
14
CO
2
was incorporated into propionate and succinate, suggesting that a carboxylation reaction rather than a transcarboxylation reaction was involved in the reductive pathway leading to succinate and propionate. Difference spectra showed that a type
b
cytochrome was present, which could be involved in electron transport in the reductive pathway.
Characterization of the Critical Amino Acids of an
Aspergillus parasiticus
Cytochrome P-450 Monooxygenase Encoded by
ordA
That Is Involved in the Biosynthesis of Aflatoxins B
1
, G
1
, B
2
, and G
2
Authors: Yu Jiujiang , Chang Perng-Kuang , Ehrlich Kenneth C. , Cary Jeffrey W. , Montalbano Beverly , Dyer John M. , Bhatnagar Deepak , Cleveland Thomas E.
Published: December 2019

The conversion of
O
-methylsterigmatocystin (OMST) and dihydro-
O
-methylsterigmatocystin to aflatoxins B
1
, G
1
, B
2
, and G
2
requires a cytochrome P-450 type of oxidoreductase activity.
ordA
, a gene adjacent to the
omtA
gene, was identified in the aflatoxin-biosynthetic pathway gene cluster by chromosomal walking in
Aspergillus parasiticus
. The
ordA
gene was a homolog of the
Aspergillus flavus ord1
gene, which is involved in the conversion of OMST to aflatoxin B
1
. Complementation of
A. parasiticus
SRRC 2043, an OMST-accumulating strain, with the
ordA
gene restored the ability to produce aflatoxins B
1
, G
1
, B
2
, and G
2
. The
ordA
gene placed under the control of the
GAL1
promoter converted exogenously supplied OMST to aflatoxin B
1
in
Saccharomyces cerevisiae
. In contrast, the
ordA
gene homolog in
A. parasiticus
SRRC 2043,
ordA1
, was not able to carry out the same conversion in the yeast system. Sequence analysis revealed that the
ordA1
gene had three point mutations which resulted in three amino acid changes (His-400→Leu-400, Ala-143→Ser-143, and Ile-528→Tyr-528). Site-directed mutagenesis studies showed that the change of His-400 to Leu-400 resulted in a loss of the monooxygenase activity and that Ala-143 played a significant role in the catalytic conversion. In contrast, Ile-528 was not associated with the enzymatic activity. The involvement of the
ordA
gene in the synthesis of aflatoxins G
1
, and G
2
in
A. parasiticus
suggests that enzymes required for the formation of aflatoxins G
1
and G
2
are not present in
A. flavus
. The results showed that in addition to the conserved heme-binding and redox reaction domains encoded by
ordA
, other seemingly domain-unrelated amino acid residues are critical for cytochrome P-450 catalytic activity. The
ordA
gene has been assigned to a new cytochrome P-450 gene family named CYP64 by The Cytochrome P450 Nomenclature Committee.
A New Approach To Utilize PCR–Single-Strand-Conformation Polymorphism for 16S rRNA Gene-Based Microbial Community Analysis
Authors: Schwieger Frank , Tebbe Christoph C.
Published: December 2019

Single-strand-conformation polymorphism (SSCP) of DNA, a method widely used in mutation analysis, was adapted to the analysis and differentiation of cultivated pure-culture soil microorganisms and noncultivated rhizosphere microbial communities. A fragment (approximately 400 bp) of the bacterial 16S rRNA gene (V-4 and V-5 regions) was amplified by PCR with universal primers, with one primer phosphorylated at the 5′ end. The phosphorylated strands of the PCR products were selectively digested with lambda exonuclease, and the remaining strands were separated by electrophoresis with an MDE polyacrylamide gel, a matrix specifically optimized for SSCP purposes. By this means, reannealing and heteroduplex formation of DNA strands during electrophoresis could be excluded, and the number of bands per organism was reduced. PCR products from 10 of 11 different bacterial type strains tested could be differentiated from each other. With template mixtures consisting of pure-culture DNAs from 5 and 10 bacterial strains, most of the single strains could be detected from such model communities after PCR and SSCP analyses. Purified bands amplified from pure cultures and model communities extracted from gels could be reamplified by PCR, but by this process, additional products were also generated, as detected by further SSCP analysis. Profiles generated with DNAs of rhizosphere bacterial communities, directly extracted from two different plant species grown in the same field site, could be clearly distinguished. This study demonstrates the potential of the selected PCR–single-stranded DNA approach for microbial community analysis.
Genetic Diversity and Phylogeny of Rhizobia That Nodulate
Acacia
spp. in Morocco Assessed by Analysis of rRNA Genes
Authors: Khbaya Bouchaib , Neyra Marc , Normand Philippe , Zerhari Karim , Filali-Maltouf Abdelkarim
Published: December 2019

Forty rhizobia nodulating four
Acacia
species (
A. gummifera
,
A. raddiana
,
A. cyanophylla
, and
A. horrida
) were isolated from different sites in Morocco. These rhizobia were compared by analyzing both the 16S rRNA gene (rDNA) and the 16S-23S rRNA spacer by PCR with restriction fragment length polymorphism (RFLP) analysis. Analysis of the length of 16S-23S spacer showed a considerable diversity within these microsymbionts, but RFLP analysis of the amplified spacer revealed no additional heterogeneity. Three clusters were identified when 16S rDNA analysis was carried out. Two of these clusters include some isolates which nodulate, nonspecifically, the four
Acacia
species. These clusters, A and B, fit within the
Sinorhizobium
lineage and are closely related to
S. meliloti
and
S. fredii
, respectively. The third cluster appeared to belong to the
Agrobacterium-Rhizobium galegae
phylum and is more closely related to the
Agrobacterium tumefaciens
species. These relations were confirmed by sequencing a representative strain from each cluster.
Analysis of the Dynamics of Bacterial Communities in the Rhizosphere of the Chrysanthemum via Denaturing Gradient Gel Electrophoresis and Substrate Utilization Patterns
Authors: Duineveld Bernadette M. , Rosado Alexandre S. , van Elsas Jan Dirk , van Veen Johannes A.
Published: December 2019

In order to gain a better understanding of the spatial and temporal dynamics of bacterial communities of the rhizosphere of the chrysanthemum, two complementary methods were used: a molecular bacterial community profiling method, i.e., 16S rRNA gene-based PCR followed by denaturing gradient gel electrophoresis (DGGE), and an agar plate method in which 11 sole-carbon-source utilization tests were used. The DGGE patterns showed that the bacterial communities as determined from direct rhizosphere DNA extracts were largely stable along developing roots of the chrysanthemum, with very little change over time or between root parts of different ages. The patterns were also similar to those produced with DNA extracts obtained from bulk soil samples. The DGGE patterns obtained by using microbial colonies from dilution plates as the source of target DNA were different from those found with the direct DNA extracts. Moreover, these patterns showed differences among plant replicates but also among replicate plates. Results obtained with the sole-carbon-source utilization tests indicated that the metabolic profile of the bacterial communities in the rhizosphere of the root tip did not change substantially during plant growth. This suggests selective development of specific bacterial populations by the presence of a root tip. On the other hand, the metabolic profile of bacterial communities in the rhizosphere of the root base changed during plant growth. With eight sole-carbon-source utilization tests, a significant effect of the development stage of the plant on the number of bacteria which were able to grow on these carbon sources was observed.
Indole-3-Acetic Acid Biosynthesis in
Colletotrichum gloeosporioides
f. sp.
aeschynomene
Authors: Robinson M. , Riov J. , Sharon A.
Published: December 2019

We characterized the biosynthesis of indole-3-acetic acid by the mycoherbicide
Colletotrichum gloeosporioides
f. sp.
aeschynomene
. Auxin production was tryptophan dependent. Compounds from the indole-3-acetamide and indole-3-pyruvic acid pathways were detected in culture filtrates. Feeding experiments and in vitro assay confirmed the presence of both pathways. Indole-3-acetamide was the major pathway utilized by the fungus to produce indole-3-acetic acid in culture.
Induction of the
tod
Operon by Trichloroethylene in
Pseudomonas putida
TVA8
Authors: Shingleton Justin T. , Applegate Bruce M. , Nagel Aaron C. , Bienkowski Paul R. , Sayler Gary S.
Published: December 2019

Bioluminescence, mRNA levels, and toluene degradation rates in
Pseudomonas putida
TVA8 were measured as a function of various concentrations of toluene and trichloroethylene (TCE). TVA8 showed an increasing bioluminescence response to increasing TCE and toluene concentrations. Compared to uninduced TVA8 cultures,
todC1
mRNA levels increased 11-fold for TCE-treated cultures and 13-fold for toluene-treated cultures. Compared to uninduced
P. putida
F1 cultures,
todC1
mRNA levels increased 4.4-fold for TCE-induced cultures and 4.9-fold for toluene-induced cultures. Initial toluene degradation rates were linearly correlated with specific bioluminescence in TVA8 cultures.
Cloning and Targeted Disruption of
MLG1
, a Gene Encoding Two of Three Extracellular Mixed-Linked Glucanases of
Cochliobolus carbonum
Authors: Görlach Jenifer M. , Van Der Knaap Esther , Walton Jonathan D.
Published: December 2019

Mixed-linked glucanases (MLGases), which are extracellular enzymes able to hydrolyze β1,3-1,4-glucans (also known as mixed-linked glucans or cereal β-glucans), were identified in culture filtrates of the plant-pathogenic fungus
Cochliobolus carbonum
. Three peaks of MLGase activity, designated Mlg1a, Mlg1b, and Mlg2, were resolved by cation-exchange and hydrophobic-interaction high-performance liquid chromatography (HPLC). Mlg1a and Mlg1b also hydrolyze β1,3-glucan (laminarin), whereas Mlg2 does not degrade β1,3-glucan but does degrade β1,4-glucan to a slight extent. Mlg1a, Mlg1b, and Mlg2 have monomer molecular masses of 33.5, 31, and 29.5 kDa, respectively. The N-terminal amino acid sequences of Mlg1a and Mlg1b are identical (AAYNLI). Mlg1a is glycosylated, whereas Mlg1b is not. The gene encoding Mlg1b,
MLG1
, was isolated by using PCR primers based on amino acid sequences of Mlg1b. The product of
MLG1
has no close similarity to any known protein but does contain a motif (EIDI) that occurs at the active site of MLGases from several prokaryotes. An internal fragment of
MLG1
was used to create
mlg1
mutants by transformation-mediated gene disruption. The total MLGase and β1,3-glucanase activities in culture filtrates of the mutants were reduced by approximately 50 and 40%, respectively. When analyzed by cation-exchange HPLC, the mutants were missing the two peaks of MLGase activity corresponding to Mlg1a and Mlg1b. Together, the data indicate that Mlg1a and Mlg1b are products of the same gene,
MLG1
. The growth of
mlg1
mutants in culture medium supplemented with macerated maize cell walls or maize bran and the disease symptoms on maize were identical to the growth and disease symptoms of the wild type.
Expression and Regulation of the Arsenic Resistance Operon of
Acidiphilium multivorum
AIU 301 Plasmid pKW301 in
Escherichia coli
Authors: Suzuki Katsuhisa , Wakao Norio , Kimura Tetsuya , Sakka Kazuo , Ohmiya Kunio
Published: December 2019

The arsenic resistance (
ars
) operon from plasmid pKW301 of
Acidiphilium multivorum
AIU 301 was cloned and sequenced. This DNA sequence contains five genes in the following order:
arsR
,
arsD
,
arsA
,
arsB
,
arsC
. The predicted amino acid sequences of all of the gene products are homologous to the amino acid sequences of the
ars
gene products of
Escherichia coli
plasmid R773 and IncN plasmid R46. The
ars
operon cloned from
A. multivorum
conferred resistance to arsenate and arsenite on
E. coli
. Expression of the
ars
genes with the bacteriophage T7 RNA polymerase-promoter system allowed
E. coli
to overexpress ArsD, ArsA, and ArsC but not ArsR or ArsB. The apparent molecular weights of ArsD, ArsA, and ArsC were 13,000, 64,000, and 16,000, respectively. A primer extension analysis showed that the
ars
mRNA started at a position 19 nucleotides upstream from the
arsR
ATG in
E. coli
. Although the
arsR
gene of
A. multivorum
AIU 301 encodes a polypeptide of 84 amino acids that is smaller and less homologous than any of the other ArsR proteins, inactivation of the
arsR
gene resulted in constitutive expression of the
ars
genes, suggesting that ArsR of pKW301 controls the expression of this operon.
Osmoregulatory Responses of Fungi Inhabiting Standing Litter of the Freshwater Emergent MacrophyteJuncus effusus
Authors: Kuehn Kevin A. , Churchill Perry F. , Suberkropp Keller
Published: December 2019

Standing litter of emergent macrophytes often forms a major portion of the detrital mass in wetland habitats. Microbial assemblages inhabiting this detritus must adapt physiologically to daily fluctuations in temperature and water availability. We examined the effects of various environmental conditions on the concentrations of osmoregulatory solutes (polyols and trehalose) and the respiratory activities of fungal assemblages inhabiting standing litter of the freshwater emergent macrophyteJuncus effusus. Under field conditions, the concentrations of osmolytes (polyols plus trehalose) in fungal decomposers were negatively correlated with plant litter water potentials (r= −0.75,P< 0.001) and rates of microbial respiration (r= −0.66,P< 0.001). The highest concentration of osmolytes (polyols plus trehalose) occurred in standing litter exposed to desiccating conditions (range from wet to dry, 0.06 to 0.68 μmol · mg of fungal biomass−1). Similar fluctuations in polyol and trehalose concentrations were observed in standing litter wetted and dried under laboratory conditions and for four predominant fungal decomposers ofJ. effususgrown individually on sterilizedJuncusleaves. These studies suggest that fungal inhabitants associated with standing litter of emergent macrophytes can adjust their intracellular solute concentrations in response to daily fluctuations in water availability.
Identification of Bacterial Isolates Obtained from Intestinal Contents Associated with 12,000-Year-Old Mastodon Remains
Authors: Rhodes A. N. , Urbance J. W. , Youga H. , Corlew-Newman H. , Reddy C. A. , Klug M. J. , Tiedje J. M. , Fisher D. C.
Published: December 2019

Mastodon (Mammut americanum) remains unearthed during excavation of ancient sediments usually consist only of skeletal material, due to postmortem decomposition of soft tissues by microorganisms. Two recent excavations of skeletal remains in anoxic sediments in Ohio and Michigan, however, have uncovered organic masses which appear to be remnants of the small and large intestines, respectively. Macrobotanical examinations of the composition of these masses revealed assemblages of plant material radiocarbon dated to approximately 11,500 years before the present and thought to be incompletely digested food remains from this extinct mammal. We attempted to cultivate and identify bacteria from the intestinal contents, bone-associated sediments, and sediments not in proximity to the remains using a variety of general and selective media. In all, 295 isolates were cultivated, and 38 individual taxa were identified by fatty acid-methyl ester (FAME) profiles and biochemical characteristics (API-20E). The taxonomic positions of selected enteric and obligately anaerobic bacteria were confirmed by 16S ribosomal DNA (rDNA) sequencing. Results indicate that the intestinal and bone-associated samples contained the greatest diversity of bacterial taxa and that members of the familyEnterobacteriaceaerepresented 41% of all isolates and were predominant in the intestinal masses and sediments in proximity to the skeleton but were uncommon in the background sediments.Enterobacter cloacaewas the most commonly identified isolate, and partial rDNA sequencing revealed thatRahnella aquatiliswas the correct identity of strains suggested by FAME profiles to beYersinia enterocolitica. NoBacteroidesspp. or expected intestinal anaerobes were recovered. The only obligate anaerobes recovered were clostridia, and these were not recovered from the small intestinal masses. Microbiological evidence from this study supports other, macrobotanical data indicating the intestinal origin of these masses. Whether these organisms are direct descendants of the original intestinal microbiota, however, cannot be established.
A Nested Reverse Transcriptase PCR Assay for Detection of Small Round-Structured Viruses in Environmentally Contaminated Molluscan Shellfish
Authors: Green J. , Henshilwood K. , Gallimore C. I. , Brown D. W. G. , Lees D. N.
Published: December 2019

We describe the evaluation of a nested reverse transcriptase PCR (RT-PCR) procedure for the detection of small round-structured viruses (SRSVs) in molluscan shellfish and the application of this assay for the detection of SRSVs in commercially produced shellfish and in shellfish implicated in outbreaks of gastroenteritis. The range of virus strains detected and the sensitivity of detection were evaluated by using a representative panel of 21 well-characterized SRSV strains. The nested RT-PCR detected 15 of 21 SRSVs, demonstrating that the assay detects a broad range of SRSVs including strains from both genogroup I and genogroup II. Seeding experiments showed the nested RT-PCR assay to be 10 to 1,000 times more sensitive than the single-round RT-PCR assay for the detection of SRSV in shellfish. SRSV-contaminated samples were identified by nested RT-PCR for shellfish grown in polluted harvesting areas and for shellfish associated with outbreaks of gastroenteritis which were negative by a previously described single-round RT-PCR. The assay was shown to be effective for investigation of virus elimination during commercial shellfish processing procedures such as depuration and relaying and has potential applications for monitoring at-risk shellfish harvesting areas, for investigation of SRSV contamination in shellfish from producers linked to gastroenteritis outbreaks, and for the direct detection of virus in shellfish implicated in outbreaks.
Purification and Characterization of Exo-β-
d
-Glucosaminidase from a Cellulolytic Fungus,
Trichoderma reesei
PC-3-7
Authors: Nogawa Masahiro , Takahashi Hiroya , Kashiwagi Aya , Ohshima Kenji , Okada Hirofumi , Morikawa Yasushi
Published: December 2019

Chitosan-degrading activities induced by glucosamine (GlcN) or
N
-acetylglucosamine (GlcNAc) were found in a culture filtrate of
Trichoderma reesei
PC-3-7. One of the chitosan-degrading enzymes was purified to homogeneity by precipitation with ammonium sulfate followed by anion-exchange and hydrophobic-interaction chromatographies. The enzyme was monomeric, and its molecular mass was 93 kDa. The optimum pH and temperature of the enzyme were 4.0 and 50°C, respectively. The activity was stable in the pH range 6.0 to 9.0 and at a temperature below 50°C. Reaction product analysis from the viscosimetric assay and thin-layer chromatography and
1
H nuclear magnetic resonance spectroscopy clearly indicated that the enzyme was an exo-type chitosanase, exo-β-
d
-glucosaminidase, that releases GlcN from the nonreducing end of the chitosan chain.
1
H nuclear magnetic resonance spectroscopy also showed that the exo-β-
d
-glucosaminidase produced a β-form of GlcN, demonstrating that the enzyme is a retaining glycanase. Time-dependent liberation of the reducing sugar from partially acetylated chitosan with exo-β-
d
-glucosaminidase and the partially purified exo-β-
d
-
N
-acetylglucosaminidase from
T. reesei
PC-3-7 suggested that the exo-β-
d
-glucosaminidase cleaves the glycosidic link of either GlcN-β(1→4)-GlcN or GlcN-β(1→4)-GlcNAc.
Fermentation, Purification, and Characterization of Protective Antigen from a Recombinant, Avirulent Strain of
Bacillus anthracis
Authors: Farchaus J. W. , Ribot W. J. , Jendrek S. , Little S. F.
Published: December 2019

Bacillus anthracis
, the etiologic agent for anthrax, produces two bipartite, AB-type exotoxins, edema toxin and lethal toxin. The B subunit of both exotoxins is an
M
r
83,000 protein termed protective antigen (PA). The human anthrax vaccine currently licensed for use in the United States consists primarily of this protein adsorbed onto aluminum oxyhydroxide. This report describes the production of PA from a recombinant, asporogenic, nontoxigenic, and nonencapsulated host strain of
B. anthracis
and the subsequent purification and characterization of the protein product. Fermentation in a high-tryptone, high-yeast-extract medium under nonlimiting aeration produced 20 to 30 mg of secreted PA per liter. Secreted protease activity under these fermentation conditions was low and was inhibited more than 95% by the addition of EDTA. A purity of 88 to 93% was achieved for PA by diafiltration and anion-exchange chromatography, while greater than 95% final purity was achieved with an additional hydrophobic interaction chromatography step. The purity of the PA product was characterized by reversed-phase high-pressure liquid chromatography, sodium dodecyl sulfate (SDS)-capillary electrophoresis, capillary isoelectric focusing, native gel electrophoresis, and SDS-polyacrylamide gel electrophoresis. The biological activity of the PA, when combined with excess lethal factor in the macrophage cell lysis assay, was comparable to previously reported values.
Differential Transmission of the Genospecies of
Borrelia burgdorferi
Sensu Lato by Game Birds and Small Rodents in England
Authors: Kurtenbach Klaus , Peacey Mick , Rijpkema Sjoerd G. T. , Hoodless Andrew N. , Nuttall Patricia A. , Randolph Sarah E.
Published: December 2019

The genetic diversity of
Borrelia burgdorferi
sensu lato was assessed in a focus of Lyme borreliosis in southern Britain dominated by game birds. Ticks, rodents, and pheasants were analyzed for spirochete infections by PCR targeting the 23S-5S rRNA genes, followed by genotyping by the reverse line blot method. In questing
Ixodes ricinus
ticks, three genospecies of
B. burgdorferi
sensu lato were detected, with the highest prevalences found for
Borrelia garinii
and
Borrelia valaisiana. B. burgdorferi
sensu stricto was rare (<1%) in all tick stages.
Borrelia afzelii
was not detected in any of the samples. More than 50% of engorged nymphs collected from pheasants were infected with borreliae, mainly
B. garinii
and/or
B. valaisiana
. Although 19% of the rodents harbored
B. burgdorferi
sensu stricto and/or
B. garinii
in internal organs, only
B. burgdorferi
sensu stricto was transmitted to xenodiagnostic tick larvae (it was transmitted to 1% of the larvae). The data indicate that different genospecies of
B. burgdorferi
sensu lato can be maintained in nature by distinct transmission cycles involving the same vector tick species but different vertebrate host species. Wildlife management may have an influence on the relative risk of different clinical forms of Lyme borreliosis.
Enhancement of Exopolysaccharide Production by
Lactobacillus delbrueckii
subsp.
bulgaricus
NCFB 2772 with a Simplified Defined Medium
Authors: Grobben G. J. , Chin-Joe I. , Kitzen V. A. , Boels I. C. , Boer F. , Sikkema J. , Smith M. R. , de Bont J. A. M.
Published: December 2019

The aim of this work was to investigate the medium requirements for growth and production of exopolysaccharides by
Lactobacillus delbrueckii
subsp.
bulgaricus
NCFB 2772. The strain was grown in batch cultures on a chemically defined medium, and the technique of single omission of medium components was applied to determine the nutritional requirements. The omission of aspartic acid, glutamic acid, or glycine affected growth only slightly, and the omission of glutamine, asparagine, or threonine resulted in a stronger reduction of the growth. All the other amino acids were essential. Multiple omissions of amino acids caused an almost complete loss of growth.
L. delbrueckii
subsp.
bulgaricus
required only riboflavin, calcium pantothenate, and nicotinic acid as individual vitamins. Surprisingly, when only these vitamins were present in the medium and other vitamins were not, less growth was observed than in the complete medium but the amount of exopolysaccharide produced was significantly greater. These observations were studied in more detail with a simplified defined medium in which
L. delbrueckii
subsp.
bulgaricus
was able to grow and produce exopolysaccharides. Although the final optical density in the simplified medium was lower, the production of exopolysaccharides was about twofold higher than in the complete medium.
Pichia stipitis
Genes for Alcohol Dehydrogenase with Fermentative and Respiratory Functions
Authors: Cho Jae-yong , Jeffries Thomas W.
Published: December 2019

Two genes coding for isozymes of alcohol dehydrogenase (ADH); designated
PsADH1
and
PsADH2
, have been identified and isolated from
Pichia stipitis
CBS 6054 genomic DNA by Southern hybridization to
Saccharomyces cerevisiae ADH
genes, and their physiological roles have been characterized through disruption. The amino acid sequences of the PsADH1 and PsADH2 isozymes are 80.5% identical to one another and are 71.9 and 74.7% identical to the
S. cerevisiae
ADH1 protein. They also show a high level identity with the group I ADH proteins from
Kluyveromyces lactis
. The PsADH isozymes are presumably localized in the cytoplasm, as they do not possess the amino-terminal extension of mitochondrion-targeted ADHs. Gene disruption studies suggest that PsADH1 plays a major role in xylose fermentation because
PsADH1
disruption results in a lower growth rate and profoundly greater accumulation of xylitol. Disruption of
PsADH2
does not significantly affect ethanol production or aerobic growth on ethanol as long as
PsADH1
is present. The PsADH1 and PsADH2 isozymes appear to be equivalent in the ability to convert ethanol to acetaldehyde, and either is sufficient to allow cell growth on ethanol. However, disruption of both genes blocks growth on ethanol.
P. stipitis
strains disrupted in either
PsADH1
or
PsADH2
still accumulate ethanol, although in different amounts, when grown on xylose under oxygen-limited conditions. The
PsADH
double disruptant, which is unable to grow on ethanol, still produces ethanol from xylose at about 13% of the rate seen in the parental strain. Thus, deletion of both
PsADH1
and
PsADH2
blocks ethanol respiration but not production, implying a separate path for fermentation.
Purification and Characterization of a Nylon-Degrading Enzyme
Authors: Deguchi Tetsuya , Kitaoka Yoshihisa , Kakezawa Masaaki , Nishida Tomoaki
Published: December 2019

A nylon-degrading enzyme found in the extracellular medium of a ligninolytic culture of the white rot fungus strain IZU-154 was purified by ion-exchange chromatography, gel filtration chromatography, and hydrophobic chromatography. The characteristics of the purified protein (i.e., molecular weight, absorption spectrum, and requirements for 2,6-dimethoxyphenol oxidation) were identical to those of manganese peroxidase, which was previously characterized as a key enzyme in the ligninolytic systems of many white rot fungi, and this result led us to conclude that nylon degradation is catalyzed by manganese peroxidase. However, the reaction mechanism for nylon degradation differed significantly from the reaction mechanism reported for manganese peroxidase. The nylon-degrading activity did not depend on exogenous H
2
O
2
but nevertheless was inhibited by catalase, and superoxide dismutase inhibited the nylon-degrading activity strongly. These features are identical to those of the peroxidase-oxidase reaction catalyzed by horseradish peroxidase. In addition, α-hydroxy acids which are known to accelerate the manganese peroxidase reaction inhibited the nylon-degrading activity strongly. Degradation of nylon-6 fiber was also investigated. Drastic and regular erosion in the nylon surface was observed, suggesting that nylon is degraded to soluble oligomers and that nylon is degraded selectively.
Synergism between
Bacillus thuringiensis
Spores and Toxins against Resistant and Susceptible Diamondback Moths (
Plutella xylostella
)
Authors: Liu Yong-Biao , Tabashnik Bruce E. , Moar William J. , Smith Robert A.
Published: December 2019

We studied the effects of combinations of
Bacillus thuringiensis
spores and toxins on the mortality of diamondback moth (
Plutella xylostella
) larvae in leaf residue bioassays. Spores of
B. thuringiensis
subsp.
kurstaki
increased the toxicity of crystals of
B. thuringiensis
subsp.
kurstaki
to both resistant and susceptible larvae. For
B. thuringiensis
subsp.
kurstaki
, resistance ratios were 1,200 for a spore-crystal mixture and 56,000 for crystals without spores. Treatment of a spore-crystal formulation of
B. thuringiensis
subsp.
kurstaki
with the antibiotic streptomycin to inhibit spore germination reduced toxicity to resistant larvae but not to susceptible larvae. In contrast, analogous experiments with
B. thuringiensis
subsp.
aizawai
revealed no significant effects of adding spores to crystals or of treating a spore-crystal formulation with streptomycin. Synergism occurred between Cry2A and
B. thuringiensis
subsp.
kurstaki
spores against susceptible larvae and between Cry1C and
B. thuringiensis
subsp.
aizawai
spores against resistant and susceptible larvae. The results show that
B. thuringiensis
toxins combined with spores can be toxic even though the toxins and spores have little or no independent toxicity. Results reported here and previously suggest that, for diamondback moth larvae, the extent of synergism between spores and toxins of
B. thuringiensis
depends on the strain of insect, the type of spore, the set of toxins, the presence of other materials such as formulation ingredients, and the concentrations of spores and toxins.