bluuebunny/crossref_metadata_2025_split huggingface dataset:
Grazing Pressure by a Bacterivorous Flagellate Reverses the Relative Abundance ofComamonas acidovoransPX54 andVibrioStrain CB5 in Chemostat Cocultures
Authors: Hahn Martin W. , Höfle Manfred G. 
Published: December 2019

ABSTRACTThe response of the bacterial strainsComamonas acidovoransPX54 (β subclass of the classProteobacteria) andVibriostrain CB5 (γ subclass of the classProteobacteria) to grazing by the bacterivorous flagellateOchromonassp. was examined in one-stage chemostat experiments under conditions of low growth rates with a complex carbon source. The two bacterial strains were cultured together; they were cultured without flagellates in the first phase of the experiments and in the presence of the flagellates in the second phase. Monoclonal and polyclonal antibodies were used to determine the numbers and sizes ofC. acidovoransPX54 andVibriostrain CB5 cells. The flagellates caused strong changes in total bacterial cell numbers, in the relative abundances of the individual bacterial strains, and in bacterial cell size distribution.Vibriostrain CB5 dominated the total bacterial cell numbers during the flagellate-free phase of the experiments with a relative abundance of 93%, but this declined to 33% after inoculation with the flagellate. In contrast toVibriostrain CB5,C. acidovoransPX54 responded to grazing with a strong expansion of cell length distribution toward large, filamentous cells. These changes in cell morphology resulted in a high percentage of inedible cells in theC. acidovoransPX54 population but not in theVibriostrain CB5 population, which caused the observed change in the relative abundances of the strains. Batch culture experiments without the flagellate demonstrated that the elongation ofC. acidovoransPX54 cells was dependent on their growth rate. This indicates that the occurrence of filamentousC. acidovoransPX54 cells is not a direct response to chemical stimuli released by the flagellates but rather a response to increased growth rates due to flagellate grazing.
A PCR Detection Method for Rapid Identification of
Melissococcus pluton
in Honeybee Larvae
Authors: Govan V. A. , Brözel V. , Allsopp M. H. , Davison S. 
Published: December 2019

ABSTRACT

Melissococcus pluton
is the causative agent of European foulbrood, a disease of honeybee larvae. This bacterium is particularly difficult to isolate because of its stringent growth requirements and competition from other bacteria. PCR was used selectively to amplify specific rRNA gene sequences of
M. pluton
from pure culture, from crude cell lysates, and directly from infected bee larvae. The PCR primers were designed from
M. pluton
16S rRNA sequence data. The PCR products were visualized by agarose gel electrophoresis and confirmed as originating from
M. pluton
by sequencing in both directions. Detection was highly specific, and the probes did not hybridize with DNA from other bacterial species tested. This method enabled the rapid and specific detection and identification of
M. pluton
from pure cultures and infected bee larvae.
Comparison of Gas Chromatography and Mineralization Experiments for Measuring Loss of Selected Polychlorinated Biphenyl Congeners in Cultures of White Rot Fungi
Authors: Beaudette Lee A. , Davies Stephen , Fedorak Phillip M. , Ward Owen P. , Pickard Michael A. 
Published: December 2019

ABSTRACT

Two methods were used to compare the biodegradation of six polychlorinated biphenyl (PCB) congeners by 12 white rot fungi. Four fungi were found to be more active than
Phanerochaete chrysosporium
ATCC 24725. Biodegradation of the following congeners was monitored by gas chromatography: 2,3-dichlorobiphenyl, 4,4′-dichlorobiphenyl, 2,4′,5-trichlorobiphenyl (2,4′,5-TCB), 2,2′,4,4′-tetrachlorobiphenyl, 2,2′,5,5′-tetrachlorobiphenyl, and 2,2′,4,4′,5,5′-hexachlorobiphenyl. The congener tested for mineralization was 2,4′,5-[U-
14
C]TCB. Culture supernatants were also assayed for lignin peroxidase and manganese peroxidase activities. Of the fungi tested, two strains of
Bjerkandera adusta
(UAMH 8258 and UAMH 7308), one strain of
Pleurotus ostreatus
(UAMH 7964), and
Trametes versicolor
UAMH 8272 gave the highest biodegradation and mineralization.
P. chrysosporium
ATCC 24725, a strain frequently used in studies of PCB degradation, gave the lowest mineralization and biodegradation activities of the 12 fungi reported here. Low but detectable levels of lignin peroxidase and manganese peroxidase activity were present in culture supernatants, but no correlation was observed among any combination of PCB congener biodegradation, mineralization, and lignin peroxidase or manganese peroxidase activity. With the exception of
P. chrysosporium
, congener loss ranged from 40 to 96%; however, these values varied due to nonspecific congener binding to fungal biomass and glassware. Mineralization was much lower, ≤11%, because it measures a complete oxidation of at least part of the congener molecule but the results were more consistent and therefore more reliable in assessment of PCB biodegradation.
Towards a Biocatalyst for (
S
)-Styrene Oxide Production: Characterization of the Styrene Degradation Pathway of
Pseudomonas
sp. Strain VLB120
Authors: Panke Sven , Witholt Bernard , Schmid Andreas , Wubbolts Marcel G. 
Published: December 2019

ABSTRACT

In order to design a biocatalyst for the production of optically pure styrene oxide, an important building block in organic synthesis, the metabolic pathway and molecular biology of styrene degradation in
Pseudomonas
sp. strain VLB120 was investigated. A 5.7-kb
Xho
I fragment, which contained on the same strand of DNA six genes involved in styrene degradation, was isolated from a gene library of this organism in
Escherichia coli
by screening for indigo formation. T7 RNA polymerase expression experiments indicated that this fragment coded for at least five complete polypeptides, StyRABCD, corresponding to five of the six genes. The first two genes encoded the potential carboxy-terminal part of a sensor, named StySc, and the complete response regulator StyR. Fusion of the putative
styAp
promoter to a
lacZ
reporter indicated that StySc and StyR together regulate expression of the structural genes at the transcriptional level. Expression of
styS
c
R
also alleviated a block that prevented translation of
styA
mRNA when a heterologous promoter was used. The structural genes
styA
and
styB
produced a styrene monooxygenase that converted styrene to styrene oxide, which was then converted to phenylacetaldehyde by StyC. Sequence homology analysis of StyD indicated a probable function as a phenylacetaldehyde dehydrogenase. To assess the usefulness of the enzymes for the production of enantiomerically pure styrene oxide, we investigated the enantiospecificities of the reactions involved. Kinetic resolution of racemic styrene oxide by styrene oxide isomerase was studied with
E. coli
recombinants carrying
styC
, which converted styrene oxide at a very high rate but with only a slight preference for the
S
enantiomer. However, recombinants producing styrene monooxygenase catalyzed the formation of (
S
)-styrene oxide from inexpensive styrene with an excellent enantiomeric excess of more than 99% at rates up to 180 U g (dry weight) of cells
−1
.
Spreadsheet Method for Evaluation of Biochemical Reaction Rate Coefficients and Their Uncertainties by Weighted Nonlinear Least-Squares Analysis of the Integrated Monod Equation
Authors: Smith Laurence H. , McCarty Perry L. , Kitanidis Peter K. 
Published: December 2019

ABSTRACT
A convenient method for evaluation of biochemical reaction rate coefficients and their uncertainties is described. The motivation for developing this method was the complexity of existing statistical methods for analysis of biochemical rate equations, as well as the shortcomings of linear approaches, such as Lineweaver-Burk plots. The nonlinear least-squares method provides accurate estimates of the rate coefficients and their uncertainties from experimental data. Linearized methods that involve inversion of data are unreliable since several important assumptions of linear regression are violated. Furthermore, when linearized methods are used, there is no basis for calculation of the uncertainties in the rate coefficients. Uncertainty estimates are crucial to studies involving comparisons of rates for different organisms or environmental conditions. The spreadsheet method uses weighted least-squares analysis to determine the best-fit values of the rate coefficients for the integrated Monod equation. Although the integrated Monod equation is an implicit expression of substrate concentration, weighted least-squares analysis can be employed to calculate approximate differences in substrate concentration between model predictions and data. An iterative search routine in a spreadsheet program is utilized to search for the best-fit values of the coefficients by minimizing the sum of squared weighted errors. The uncertainties in the best-fit values of the rate coefficients are calculated by an approximate method that can also be implemented in a spreadsheet. The uncertainty method can be used to calculate single-parameter (coefficient) confidence intervals, degrees of correlation between parameters, and joint confidence regions for two or more parameters. Example sets of calculations are presented for acetate utilization by a methanogenic mixed culture and trichloroethylene cometabolism by a methane-oxidizing mixed culture. An additional advantage of application of this method to the integrated Monod equation compared with application of linearized methods is the economy of obtaining rate coefficients from a single batch experiment or a few batch experiments rather than having to obtain large numbers of initial rate measurements. However, when initial rate measurements are used, this method can still be used with greater reliability than linearized approaches.
Effects of Pyruvate Decarboxylase Overproduction on Flux Distribution at the Pyruvate Branch Point in
Saccharomyces cerevisiae
Authors: van Hoek Pim , Flikweert Marcel T. , van der Aart Quirina J. M. , Steensma H. Yde , van Dijken Johannes P. , Pronk Jack T. 
Published: December 2019

ABSTRACT

A multicopy plasmid carrying the
PDC1
gene (encoding pyruvate decarboxylase; Pdc) was introduced in
Saccharomyces cerevisiae
CEN.PK113-5D. The physiology of the resulting prototrophic strain was compared with that of the isogenic prototrophic strain CEN.PK113-7D and an empty-vector reference strain. In glucose-grown shake-flask cultures, the introduction of the
PDC1
plasmid caused a threefold increase in the Pdc level. In aerobic glucose-limited chemostat cultures growing at a dilution rate of 0.10 h
−1
, Pdc levels in the overproducing strain were 14-fold higher than those in the reference strains. Levels of glycolytic enzymes decreased by ca. 15%, probably due to dilution by the overproduced Pdc protein. In chemostat cultures, the extent of Pdc overproduction decreased with increasing dilution rate. The high degree of overproduction of Pdc at low dilution rates did not affect the biomass yield. The dilution rate at which aerobic fermentation set in decreased from 0.30 h
−1
in the reference strains to 0.23 h
−1
in the Pdc-overproducing strain. In the latter strain, the specific respiration rate reached a maximum above the dilution rate at which aerobic fermentation first occurred. This result indicates that a limited respiratory capacity was not responsible for the onset of aerobic fermentation in the Pdc-overproducing strain. Rather, the results indicate that Pdc overproduction affected flux distribution at the pyruvate branch point by influencing competition for pyruvate between Pdc and the mitochondrial pyruvate dehydrogenase complex. In respiratory cultures (dilution rate, <0.23 h
−1
), Pdc overproduction did not affect the maximum glycolytic capacity, as determined in anaerobic glucose-pulse experiments.
Cometabolism of 1,1-Dichloro-2,2-Bis(4-Chlorophenyl)Ethylene by
Pseudomonas acidovorans
M3GY Grown on Biphenyl
Authors: Hay Anthony G. , Focht Dennis D. 
Published: December 2019

ABSTRACT

1,1-Dichloro-2,2-bis(4-chlorophenyl)ethylene (DDE), a toxic breakdown product of 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT), has traditionally been viewed as a dead-end metabolite: there are no published reports detailing enzymatic ring fission of DDE by bacteria in either soil or pure culture. In this study, we investigated the ability of
Pseudomonas acidovorans
M3GY to transform DDE and its unchlorinated analog, 1,1-diphenylethylene (DPE). While strain M3GY could grow on DPE, cells grown on DPE as a sole carbon source could not degrade DDE. Cells grown on biphenyl, however, did degrade DDE. Mass balance analysis of [
14
C]DDE showed transformation of more than 40% of the recoverable radioactivity. Nine chlorinated metabolites produced from DDE were identified by gas chromatography-mass spectrometry–Fourier-transform infrared spectrometry (GC-MS-FTIR) from cultures grown on biphenyl. Recovery of these metabolites demonstrates that biphenyl-grown cells degrade DDE through a
meta
-fission pathway. This study provides a possible model for biodegradation of DDE in soil by biphenyl-utilizing bacteria.
Effects of Midgut-Protein-Preparative and Ligand Binding Procedures on the Toxin Binding Characteristics of BT-R
1
, a Common High-Affinity Receptor in
Manduca sexta
for Cry1A
Bacillus thuringiensis
Toxins
Authors: Keeton Timothy P. , Francis Brian R. , Maaty Walid S. A. , Bulla Lee A. 
Published: December 2019

ABSTRACT

The identity of the physiologically important Cry1A receptor protein(s) in the lepidopteran
Manduca sexta
has been a matter of dispute due to the multiple proteins which bind the Cry1Ac toxin. Cry1Aa, Cry1Ab, and Cry1Ac exhibit essentially identical toxicities toward
M. sexta
larvae and show a high degree of sequence and presumed structural identities. These similarities make it likely that there is a common mechanism of toxicity in these lepidopteran-specific toxins in terms of both mode of action and the receptor proteins through which these toxins exert their lepidopteran-specific toxicity. Investigators in our laboratory previously demonstrated that the cloned 210-kDa glycoprotein BT-R
1
binds all three Cry1A toxins (T. P. Keeton and L. A. Bulla, Jr., Appl. Environ. Microbiol. 63:3419–3425, 1997). This protein remains a common binding protein even after being subjected to various midgut membrane preparation and processing protocols. The method used to isolate proteins from the
M. sexta
larval midgut in no significant way affects the results of ligand binding and vacuum blotting experiments, and we have been unable to detect specific, high-affinity binding of any Cry1A toxin to Cry1Ac binding proteins other than BT-R
1
. Alterations in blot substrate and blocking, hybridization, and washing buffers support these conclusions. Collectively, these results indicate that in
M. sexta
the cadherin-like BT-R
1
protein is a common high-affinity receptor protein for the Cry1A family of toxins.
Reduction of Soluble Iron and Reductive Dissolution of Ferric Iron-Containing Minerals by Moderately Thermophilic Iron-Oxidizing Bacteria
Authors: Bridge Toni A. M. , Johnson D. Barrie 
Published: December 2019

ABSTRACT

Five moderately thermophilic iron-oxidizing bacteria, including representative strains of the three classified species (
Sulfobacillus thermosulfidooxidans
,
Sulfobacillus acidophilus
, and
Acidimicrobium ferrooxidans
), were shown to be capable of reducing ferric iron to ferrous iron when they were grown under oxygen limitation conditions. Iron reduction was most readily observed when the isolates were grown as mixotrophs or heterotrophs with glycerol as an electron donor; in addition, some strains were able to couple the oxidation of tetrathionate to the reduction of ferric iron. Cycling of iron between the ferrous and ferric states was observed during batch culture growth in unshaken flasks incubated under aerobic conditions, although the patterns of oxidoreduction of iron varied in different species of iron-oxidizing moderate thermophiles and in strains of a single species (
S. acidophilus
). All three bacterial species were able to grow anaerobically with ferric iron as a sole electron acceptor; the growth yields correlated with the amount of ferric iron reduced when the isolates were grown in the absence of oxygen. One of the moderate thermophiles (identified as a strain of
S. acidophilus
) was able to bring about the reductive dissolution of three ferric iron-containing minerals (ferric hydroxide, jarosite, and goethite) when it was grown under restricted aeration conditions with glycerol as a carbon and energy source. The significance of iron reduction by moderately thermophilic iron oxidizers in both environmental and applied contexts is discussed.
Development of a Probiotic Cheddar Cheese Containing Human-Derived
Lactobacillus paracasei
Strains
Authors: Gardiner G. , Ross R. P. , Collins J. K. , Fitzgerald G. , Stanton C. 
Published: December 2019

ABSTRACT

Cheddar cheese was manufactured with either
Lactobacillus salivarius
NFBC 310, NFBC 321, or NFBC 348 or
L. paracasei
NFBC 338 or NFBC 364 as the dairy starter adjunct. These five strains had previously been isolated from the human small intestine and have been characterized extensively with respect to their probiotic potential. Enumeration of these strains in mature Cheddar cheese, however, was complicated by the presence of high numbers (>10
7
CFU/g of cheese) of nonstarter lactic acid bacteria, principally composed of lactobacilli which proliferate as the cheese ripens. Attempts to differentiate the adjunct lactobacilli from the nonstarter lactobacilli based on bile tolerance and growth temperature were unsuccessful. In contrast, the randomly amplified polymorphic DNA method allowed the generation of discrete DNA fingerprints for each strain which were clearly distinguishable from those generated from the natural flora of the cheeses. Using this approach, it was found that both
L. paracasei
strains grew and sustained high viability in cheese during ripening, while each of the
L. salivarius
species declined over the ripening period. These data demonstrate that Cheddar cheese can be an effective vehicle for delivery of some probiotic organisms to the consumer.
Effect of Temperature Oscillation on Insect Cell Growth and Baculovirus Replication
Authors: Shao-Hua Chang , Hong-Liang Sun , Zuo-Hu Li 
Published: December 2019

ABSTRACT
Temperature oscillation can enhance cell viability of sf9 insect cells and baculovirus production of occlusion bodies (OB) and extracellular virus (ECV) compared with constant temperature in stationary culture and suspension culture. The optimal oscillation range was 24 to 28°C. At this temperature oscillation, the viability of uninfected and infected sf9 cells can be maintained much longer than at 28°C. Although the rate of virus infection was a little low at 24 to 28°C, the final cell infectivity was similar to that at a constant temperature of 28°C. The production of OB was increased from 13.4 to 17.4/cell in stationary culture and from 13.9/cell to 18.1/cell in suspension culture. The titer of ECV was increased from 87 to 114 PFU/cell in stationary culture and from 79 to 114 PFU/cell in suspension culture.
New Unstable Variants of Green Fluorescent Protein for Studies of Transient Gene Expression in Bacteria
Authors: Andersen Jens Bo , Sternberg Claus , Poulsen Lars Kongsbak , Bjørn Sara Petersen , Givskov Michael , Molin Søren 
Published: December 2019

ABSTRACT

Use of the green fluorescent protein (Gfp) from the jellyfish
Aequorea victoria
is a powerful method for nondestructive in situ monitoring, since expression of green fluorescence does not require any substrate addition. To expand the use of Gfp as a reporter protein, new variants have been constructed by the addition of short peptide sequences to the C-terminal end of intact Gfp. This rendered the Gfp susceptible to the action of indigenous housekeeping proteases, resulting in protein variants with half-lives ranging from 40 min to a few hours when synthesized in
Escherichia coli
and
Pseudomonas putida
. The new Gfp variants should be useful for in situ studies of temporal gene expression.
TheatzABCGenes Encoding Atrazine Catabolism Are Located on a Self-Transmissible Plasmid inPseudomonassp. Strain ADP
Authors: de Souza Mervyn L. , Wackett Lawrence P. , Sadowsky Michael J. 
Published: December 2019

ABSTRACTPseudomonassp. strain ADP initiates atrazine catabolism via three enzymatic steps, encoded byatzA, -B, and -C, which yield cyanuric acid, a nitrogen source for many bacteria. In-well lysis, Southern hybridization, and plasmid transfer studies indicated that theatzA, -B, and -Cgenes are localized on a 96-kb self-transmissible plasmid, pADP-1, inPseudomonassp. strain ADP. High-performance liquid chromatography analyses showed that cyanuric acid degradation was not encoded by pADP-1. pADP-1 was transferred toEscherichia colistrains at a frequency of 4.7 × 10−2. This suggests a potential molecular mechanism for the dispersion of theatzABCgenes to other soil bacteria.
Degradation and Fate of Carbon Tetrachloride in Unadapted Methanogenic Granular Sludge
Authors: Van Eekert Miriam H. A. , Schröder Thomas J. , Stams Alfons J. M. , Schraa Gosse , Field Jim A. 
Published: December 2019

ABSTRACT

The potential of granular sludge from upflow anaerobic sludge blanket (UASB) reactors for bioremediation of chlorinated pollutants was evaluated by using carbon tetrachloride (CT) as a model compound. Granular sludges cultivated in UASB reactors on methanol, a volatile fatty acid mixture, or sucrose readily degraded CT supplied at a concentration of 1,500 nmol/batch (approximately 10 μM) without any prior exposure to organohalogens. The maximum degradation rate was 1.9 μmol of CT g of volatile suspended solids
−1
day
−1
. The main end products of CT degradation were CO
2
and Cl
−
, and the yields of these end products were 44 and 68%, respectively, of the initial amounts of [
14
C]CT and CT-Cl. Lower chlorinated methanes accumulated in minor amounts temporarily. Autoclaved (dead) sludges were capable of degrading CT at rates two- to threefold lower than those for living sludges, indicating that abiotic processes (mediated by cofactors or other sludge components) played an important role in the degradation observed. Reduced components in the autoclaved sludge were vital for CT degradation. A major part (51%) of the CT was converted abiotically to CS
2
. The amount of CO
2
produced (23%) was lower and the amount of Cl
−
produced (86%) was slightly higher with autoclaved sludge than with living sludge. Both living and autoclaved sludges could degrade chloroform. However, only living sludge degraded dichloromethane and methylchloride. These results indicate that reductive dehalogenation, which was mediated better by living sludge than by autoclaved sludge, is only a minor pathway for CT degradation. The main pathway involves substitutive and oxidative dechlorination reactions that lead to the formation of CO
2
. Granular sludge, therefore, has outstanding potential for gratuitous dechlorination of CT to safe end products.