bluuebunny/crossref_metadata_2025 huggingface dataset:
A Transposable Partitioning Locus Used To Stabilize Plasmid-Borne Hydrogen Oxidation and Trifolitoxin Production Genes in a
Sinorhizobium
Strain
Authors: Kent Angela D. , Wojtasiak Michelle L. , Robleto Eduardo A. , Triplett Eric W.
Published: December 2019

Improved nitrogen-fixing inoculum strains for leguminous crops must be able to effectively compete with indigenous strains for nodulation, enhance legume productivity compared to the productivity obtained with indigenous strains, and maintain stable expression of any added genes in the absence of selection pressure. We constructed a transposable element containing the
tfx
region for expression of increased nodulation competitiveness and the
par
locus for plasmid stability. The transposon was inserted into
tetA
of pHU52, a broad-host-range plasmid conferring the H
2
uptake phenotype. The resulting plasmid, pHUTFXPAR, conferred the plasmid stability, trifolitoxin production, and H
2
uptake phenotypes in the broad-host-range organism
Sinorhizobium
sp. strain ANU280. The broad applications of a transposon conferring plasmid stability are discussed.
A Flow Cytometric Method for Rapid Selection of Novel Industrial Yeast Hybrids
Authors: Bell P. J. L. , Deere D. , Shen J. , Chapman B. , Bissinger P. H. , Attfield P. V. , Veal D. A.
Published: December 2019

We rapidly produced and isolated novel yeast hybrids by using two-color flow cytometric cell sorting. We labeled one parent strain with a fluorescent green stain and the other parent with a fluorescent orange stain, and hybrids were selected based on their dual orange and green fluorescence. When this technique was applied to the production of hybrids by traditional mating procedures, more than 96% of the isolates were hybrids. When it was applied to rare mating, three hybrids were identified among 50 isolates enriched from a population containing 2 × 10
6
cells. This technology is not dependent on genetic markers and has applications in the development of improved industrial yeast strains.
Improved Isolation of
Vibrio vulnificus
from Seawater and Sediment with Cellobiose-Colistin Agar
Authors: Høi Lise , Dalsgaard Inger , Dalsgaard Anders
Published: December 2019

An improved selective medium, cellobiose-colistin (CC) agar, gave a significantly higher (
P
< 0.05) isolation rate of
Vibrio vulnificus
from water and sediment samples than did modified cellobiose-polymyxin B-colistin (mCPC) agar. In a total of 446 alkaline peptone water preenrichments amended with polymyxin B,
V. vulnificus
was isolated from 154 preenrichments (35%) with mCPC agar and from 179 preenrichments (40%) with CC agar. CC agar gave a higher plating efficiency of
V. vulnificus
cells than did cellobiose-polymyxin B-colistin (CPC) agar, mCPC agar, or thiosulfate-citrate-bile salts-sucrose (TCBS) agar; the only significant difference was observed with TCBS agar, which gave much lower plating efficiencies than the other selective media. Determination of MICs demonstrated that the concentrations of colistin and polymyxin B in CPC agar inhibit growth of a proportion of
V. vulnificus
strains.
Cloning of the
xynB
Gene from
Dictyoglomus thermophilum
Rt46B.1 and Action of the Gene Product on Kraft Pulp
Authors: Morris Daniel D. , Gibbs Moreland D. , Chin Charles W. J. , Koh Mei-Hsien , Wong Ken K. Y. , Allison Robert W. , Nelson Peter J. , Bergquist Peter L.
Published: December 2019

A two-step PCR protocol was used to identify and sequence a family 11 xylanase gene from
Dictyoglomus thermophilum
Rt46B.1. Family 11 xylanase consensus fragments (GXCFs) were amplified from Rt46B.1 genomic DNA by using different sets of consensus PCR primers that exhibited broad specificity for conserved motifs within fungal and/or bacterial family 11 xylanase genes. On the basis of the sequences of a representative sample of the GXCFs a single family 11 xylanase gene (
xynB
) was identified. The entire gene sequence was obtained in the second step by using genomic walking PCR to amplify Rt46B.1 genomic DNA fragments upstream and downstream of the
xynB
GXCF region. The putative XynB peptide (
M
r
, 39,800) encoded by the Rt46B.1
xynB
open reading frame was a multidomain enzyme comprising an N-terminal catalytic domain (
M
r
, 22,000) and a possible C-terminal substrate-binding domain (
M
r
, 13,000) that were separated by a short serine-glycine-rich 23-amino-acid linker peptide. Seven xylanases which differed at their N and C termini were produced from different
xynB
expression plasmids. All seven xylanases exhibited optimum activity at pH 6.5. However, the temperature optima of the XynB xylanases varied from 70 to 85°C. Pretreatment of
Pinus radiata
and eucalypt kraft-oxygen pulps with XynB resulted in moderate xylan solubilization and a substantial improvement in the bleachability of these pulps.
Ammonia-Hyperproducing Bacteria from New Zealand Ruminants
Authors: Attwood Graeme T. , Klieve Athol V. , Ouwerkerk Diane , Patel Bharat K. C.
Published: December 2019

Pasture-grazed dairy cows, deer, and sheep were tested for the presence of ammonia-hyperproducing (HAP) bacteria in roll tubes containing a medium in which tryptone and Casamino Acids were the sole nitrogen and energy sources. Colonies able to grow on this medium represented 5.2, 1.3, and 11.6% of the total bacterial counts of dairy cows, deer, and sheep, respectively. A total of 14 morphologically distinct colonies were purified and studied further. Restriction fragment length polymorphisms of 16S rRNA genes indicated that all isolates differed from the previously described HAP bacteria,
Clostridium aminophilum
,
Clostridium sticklandii
, and
Peptostreptococcus anaerobius
. Carbon source utilization experiments showed that five isolates (C2, D1, D4, D5, and S1) were unable to use any, or very few, of the carbon sources tested. Biochemical tests and phylogenetic analyses of 16S ribosomal DNA sequences indicated that all isolates were monensin sensitive; that D1 and S1 belonged to the genus
Peptostreptococcus
, that D4 and D5 belonged to the family
Bacteroidaceae
, where D4 was similar to
Fusobacterium necrophorum
; and that C2 was most similar to an unidentified species from the genus
Eubacterium
. Growth on liquid medium containing tryptone and Casamino Acids as the sole nitrogen and energy source showed that D1, D4, and S1 grew rapidly (specific growth rates of 0.40, 0.35, and 0.29 h
−1
, respectively), while C2 and D5 were slow growers (0.25 and 0.10 h
−1
, respectively). Ammonia production rates were highest in D1 and D4, which produced 945.5 and 748.3 nmol/min per mg of protein, respectively. Tests of individual nitrogen sources indicated that D1 and D4 grew best on tryptone, S1 grew equally well on Casamino Acids or tryptone, and C2 and D5 grew poorly on all nitrogen sources. The intact proteins casein and gelatin did not support significant growth of any of the isolates. These isolates extend the diversity of known HAP rumen bacteria and indicate the presence of significant HAP bacterial populations in pasture-grazed New Zealand ruminants.
Maximizing Plasmid Stability and Production of Released Proteins in
Yersinia enterocolitica
Authors: Li Huaiyu , Bhaduri Saumya , Magee Wayne E.
Published: December 2019

Virulent serotypes of
Yersinia enterocolitica
carry a plasmid (pYV) encoding a family of proteins that are released into the medium and whose expression is temperature and calcium regulated. The plasmid is easily lost from cells during their growth in the laboratory. We have used sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting with a monoclonal antibody (3.2C) that is specific for a 25-kDa released protein to show that 32°C is the lowest temperature at which plasmid-encoded proteins are expressed in quantity. The highest calcium concentration allowing full expression of these proteins was 445 to 545 μM at 32°C. Calcium concentrations of 745 μM and above at 37°C completely prevented the loss of pYV during multiple subcultures, while at 32°C, calcium concentrations of 245 μM and greater were sufficient to stabilize the plasmid. Growth of
Y. enterocolitica
at pH 5.5 was slower than at neutral pH values, but it also resulted in greatly increased stability of pYV. These studies showed that bacterial growth, retention of pYV, and expression of plasmid-encoded proteins may be maximized at 32°C with 445 μM calcium and that pYV stability is enhanced by growth at low pH. These observations suggest new approaches for isolation of plasmid-bearing virulent strains of
Y. enterocolitica
from samples contaminated with this organism and also may improve our understanding of pYV retention in vivo.
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

The 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

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

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

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

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

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

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

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

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

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

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

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

Pseudomonassp. 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

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.
Growth on Urea Can Trigger Death and Peroxidation of the Cyanobacterium
Synechococcus
sp. Strain PCC 7002
Authors: Sakamoto Toshio , Delgaizo Victoria B. , Bryant Donald A.
Published: December 2019

Laboratory conditions have been identified that cause the rapid death of cultures of cyanobacteria producing urease. Once the death phase had initiated in the stationary growth phase, cells were rapidly bleached of all pigmentation. Null mutations in the
ureC
gene, encoding the alpha subunit of urease, were constructed, and these mutants were no longer sensitive to growth in the presence of urea. High levels of peroxides, including lipid peroxides, were detected in the bleaching cells. Exogenously added polyunsaturated fatty acids triggered a similar death response. Vitamin E suppressed the formation of peroxides and delayed the onset of cell bleaching. The results suggest that these cyanobacterial cells undergo a metabolic imbalance that ultimately leads to oxidative stress and lipid peroxide formation. These observations may provide insights into the mechanism of sudden cyanobacterial bloom disappearance in nature.
Flow Cytometry Detection of Infectious Rotaviruses in Environmental and Clinical Samples
Authors: Abad F. Xavier , Pintó Rosa M. , Bosch Albert
Published: December 2019

A method for the detection of infectious human rotaviruses based on infection of CaCo-2 cells and detection of infected cells by indirect immunofluorescence and flow cytometry (IIF-FC) has been developed. The technique was validated by performing a seminested reverse transcription-PCR assay with sorted cell populations. The efficiency of the procedure has been compared with that of the standard method of infection of MA104 cells and ulterior detection by IIF and optical microscopy (IIF-OM) and with that of infection of MA104 cells and detection by IIF-FC. The limit of sensitivity for the detection of the cell-adapted strain Ito
r
P13, expressed as the most probable number of cytopathogenic units, was established as 200 and 2 for MA104 and CaCo-2 cells, respectively, by the IIF-FC method. The ratio of infectious virus particles to total virus particles for a wild-type rotavirus was determined to be 1/2 × 10
6
and 1/2 × 10
4
for IIF-OM with MA104 cells and IIF-FC with CaCo-2 cells, respectively. The use of IIF-FC with CaCo-2 cells was tested with fecal and water samples and proved to be more effective than the standard procedure for rotavirus detection.
Differentiation of
Lactobacillus
Species by Molecular Typing
Authors: Zhong Wei , Millsap Kevin , Bialkowska-Hobrzanska Hanna , Reid Gregor
Published: December 2019

A total of 64 type, reference, clinical, health food, and stock isolates of microaerophilic
Lactobacillus
species were examined by restriction fragment length polymorphisms. Of particular interest were members of six of the eight species most commonly recovered from the vaginas of healthy premenopausal women, namely,
Lactobacillus jensenii
,
L. casei
,
L. rhamnosus
,
L. acidophilus
,
L. plantarum
, and
L. fermentum
. Six main groupings were identified on the basis of ribotyping. This technique was able to classify fresh isolates to the species level. In the case of the ribotype A grouping for
L. rhamnosus
, differences between strains were evident by chromosome typing (chromotyping). Many isolates did not possess plasmids. Six
L. rhamnosus
strains isolated from four different health food products appeared to be identical to
L. rhamnosus
ATCC 21052. The molecular typing system is useful for identifying and differentiating
Lactobacillus
isolates. Studies of strains of potential importance to the urogenital flora should include molecular characterization as a means of comparing genetic traits with those of strains whose characteristics associated with colonization and antagonism against pathogens have been defined.
A Two-Component Monooxygenase Catalyzes Both the Hydroxylation of
p
-Nitrophenol and the Oxidative Release of Nitrite from 4-Nitrocatechol in
Bacillus sphaericus
JS905
Authors: Kadiyala Venkateswarlu , Spain Jim C.
Published: December 2019

Bacteria that metabolize
p
-nitrophenol (PNP) oxidize the substrate to 3-ketoadipic acid via either hydroquinone or 1,2,4-trihydroxybenzene (THB); however, initial steps in the pathway for PNP biodegradation via THB are unclear. The product of initial hydroxylation of PNP could be either 4-nitrocatechol or 4-nitroresorcinol. Here we describe the complete pathway for aerobic PNP degradation by
Bacillus sphaericus
JS905 that was isolated by selective enrichment from an agricultural soil in India. Washed cells of PNP-grown JS905 released nitrite in stoichiometric amounts from PNP and 4-nitrocatechol. Experiments with extracts obtained from PNP-grown cells revealed that the initial reaction is a hydroxylation of PNP to yield 4-nitrocatechol. 4-Nitrocatechol is subsequently oxidized to THB with the concomitant removal of the nitro group as nitrite. The enzyme that catalyzed the two sequential monooxygenations of PNP was partially purified and separated into two components by anion-exchange chromatography and size exclusion chromatography. Both components were required for NADH-dependent oxidative release of nitrite from PNP or 4-nitrocatechol. One of the components was identified as a reductase based on its ability to catalyze the NAD(P)H-dependent reduction of 2,6-dichlorophenolindophenol and nitroblue tetrazolium. Nitrite release from either PNP or 4-nitrocatechol was inhibited by the flavoprotein inhibitor methimazole. Our results indicate that the two monooxygenations of PNP to THB are catalyzed by a single two-component enzyme system comprising a flavoprotein reductase and an oxygenase.
Glutamate Biosynthesis in
Lactococcus lactis
subsp.
lactis
NCDO 2118
Authors: Lapujade P. , Cocaign-Bousquet M. , Loubiere P.
Published: December 2019

Unlike other lactic acid bacteria,
Lactococcus lactis
subsp.
lactis
NCDO 2118 was able to grow in a medium lacking glutamate and the amino acids of the glutamate family. Growth in such a medium proceeded after a lag phase of about 2 days and with a reduced growth rate (0.11 h
−1
) compared to that in the reference medium containing glutamate (0.16 h
−1
). The enzymatic studies showed that a phosphoenolpyruvate carboxylase activity was present, while the malic enzyme and the enzymes of the glyoxylic shunt were not detected. As in most anaerobic bacteria, no α-ketoglutarate dehydrogenase activity could be detected, and the citric acid cycle was restricted to a reductive pathway leading to succinate formation and an oxidative branch enabling the synthesis of α-ketoglutarate. The metabolic bottleneck responsible for the limited growth rate was located in this latter pathway. As regards the synthesis of glutamate from α-ketoglutarate, no glutamate dehydrogenase was detected. While the glutamate synthase-glutamine synthetase system was detected at a low level, high transaminase activity was measured. The conversion of α-ketoglutarate to glutamate by the transaminase, the reverse of the normal physiological direction, operated with different amino acids as nitrogen donor. All of the enzymes assayed were shown to be constitutive.
Increase in Endogenous and Exogenous Cyclic AMP Levels Inhibits Sclerotial Development in
Sclerotinia sclerotiorum
Authors: Rollins Jeffrey A. , Dickman Martin B.
Published: December 2019

Growth and development of a wild-type
Sclerotinia sclerotiorum
isolate were examined in the presence of various pharmacological compounds to investigate signal transduction pathways that influence the development of sclerotia. Compounds known to increase endogenous cyclic AMP (cAMP) levels in other organisms by inhibiting phosphodiesterase activity (caffeine and 3-isobutyl-1-methyl xanthine) or by activating adenylate cyclase (NaF) reduced or eliminated sclerotial development in
S. sclerotiorum
. Growth in the presence of 5 mM caffeine correlated with increased levels of endogenous cAMP in mycelia. In addition, incorporation of cAMP into the growth medium decreased or eliminated the production of sclerotia in a concentration-dependent manner and increased the accumulation of oxalic acid. Inhibition of sclerotial development was cAMP specific, as exogenous cyclic GMP, AMP, and ATP did not influence sclerotial development. Transfer of developing cultures to cAMP-containing medium at successive time points demonstrated that cAMP inhibits development prior to or during sclerotial initiation. Together, these results indicate that cAMP plays a role in the early transition between mycelial growth and sclerotial development.
Biodegradation of Variable-Chain-Length Alkanes at Low Temperatures by a Psychrotrophic
Rhodococcus
sp
Authors: Whyte Lyle G. , Hawari Jalal , Zhou Edward , Bourbonnière Luc , Inniss William E. , Greer Charles W.
Published: December 2019

The psychrotroph
Rhodococcus
sp. strain Q15 was examined for its ability to degrade individual
n
-alkanes and diesel fuel at low temperatures, and its alkane catabolic pathway was investigated by biochemical and genetic techniques. At 0 and 5°C, Q15 mineralized the short-chain alkanes dodecane and hexadecane to a greater extent than that observed for the long-chain alkanes octacosane and dotriacontane. Q15 utilized a broad range of aliphatics (C
10
to C
21
alkanes, branched alkanes, and a substituted cyclohexane) present in diesel fuel at 5°C. Mineralization of hexadecane at 5°C was significantly greater in both hydrocarbon-contaminated and pristine soil microcosms seeded with Q15 cells than in uninoculated control soil microcosms. The detection of hexadecane and dodecane metabolic intermediates (1-hexadecanol and 2-hexadecanol and 1-dodecanol and 2-dodecanone, respectively) by solid-phase microextraction–gas chromatography-mass spectrometry and the utilization of potential metabolic intermediates indicated that Q15 oxidizes alkanes by both the terminal oxidation pathway and the subterminal oxidation pathway. Genetic characterization by PCR and nucleotide sequence analysis indicated that Q15 possesses an aliphatic aldehyde dehydrogenase gene highly homologous to the
Rhodococcus erythropolis thcA
gene.
Rhodococcus
sp. strain Q15 possessed two large plasmids of approximately 90 and 115 kb (shown to mediate Cd resistance) which were not required for alkane mineralization, although the 90-kb plasmid enhanced mineralization of some alkanes and growth on diesel oil at both 5 and 25°C.
Distribution of
Clostridium perfringens
and Fecal Sterols in a Benthic Coastal Marine Environment Influenced by the Sewage Outfall from McMurdo Station, Antarctica
Authors: Edwards Diane D. , McFeters Gordon A. , Venkatesan M. Indira
Published: December 2019

The spatial distribution, movement, and impact of the untreated wastewater outfall from McMurdo Station, Antarctica, were investigated under early austral summer conditions. The benthic environment was examined to determine the distribution of
Clostridium perfringens
in sediment cores and the intestinal contents of native invertebrates and fish along a transect of stations. These stations extended ca. 411 m south of the outfall. The findings revealed that the concentration of
C. perfringens
decreased with depth in the sediment and distance from the outfall. High percentages of tunicates and sea urchins were colonized with this bacterium along the transect. Coprostanol concentrations were also measured in sediment samples taken from each of the transect stations, and a similar trend was observed. These results are in agreement with the findings of previous studies performed with the water column and collectively provide evidence that the disposal of domestic wastes deserves special consideration in polar marine environments.
phnE
and
glpT
Genes Enhance Utilization of Organophosphates in
Escherichia coli
K-12
Authors: Elashvili Ilya , DeFrank Joseph J. , Culotta Valeria C.
Published: December 2019

Wild-type
Escherichia coli
K-12 strain JA221 grows poorly on low concentrations (≤1 mM) of diisopropyl fluorophosphate and its hydrolysis product, diisopropyl phosphate (DIPP), as sole phosphorus sources. Spontaneous organophosphate utilization (OPU) mutants were isolated that efficiently utilized these alternate sources of phosphate. A genomic library was constructed from one such OPU mutant, and two genes were isolated that conferred the OPU phenotype to strain JA221 upon transformation. These genes were identified as
phnE
and
glpT
. The original OPU mutation represented
phnE
gene activation and corresponded to the same 8-bp unit deletion from the cryptic wild-type
E. coli
K-12
phnE
gene that has been shown previously to result in
phnE
activation. In comparison, sequence analysis revealed that the observed OPU phenotype conferred by the
glpT
gene was not the result of a mutation. PCR clones of
glpT
from both the mutant and the wild type were found to confer the OPU phenotype to JA221 when they were present on the high-copy-number pUC19 plasmid but not when they were present on the low-copy-number pWSK29 plasmid. This suggests that the OPU phenotype associated with the
glpT
gene is the result of amplification and overproduction of the
glpT
gene product. Both the active
phnE
and multicopy
glpT
genes facilitated effective metabolism of low concentrations of DIPP, whereas only the active
phnE
gene could confer the ability to break down a chromogenic substrate, 5-bromo-4-chloro-3-indoxyl phosphate-
p
-toluidine (X-P
i
). This result indicates that in
E. coli
, X-P
i
is transported exclusively by the Phn system, whereas DIPP (or its metabolite) may be transported by both Phn and Glp systems.
Effect of Insertion Site and Metabolic Load on the Environmental Fitness of a Genetically Modified
Pseudomonas fluorescens
Isolate
Authors: De Leij Frans A. A. M. , Thomas Catherine E. , Bailey Mark J. , Whipps John M. , Lynch James M.
Published: December 2019

An isolate of
Pseudomonas fluorescens
(SBW25) was modified with different marker genes (
lacZY
,
aph-1
, and
xylE
). These marker genes were inserted singly or in combination into two separate (1 Mbp apart) and presumably nonessential sites (-6- and Ee) on the chromosome of SBW25. This allowed the production of a range of genetically modified SBW25 variants that differed with respect to insertion site of the marker genes and metabolic burden. The environmental fitness of the different SBW25 variants was tested in soil, in the rhizosphere of wheat and pea, and on the phylloplane of wheat. Reduced environmental fitness of the different variants was mainly attributed to the extra metabolic burden of novel gene expression, whereas choice of insertion site was of little significance. Changes in environmental fitness were dependent on the environmental conditions; an environment, such as soil, with a low microbial carrying capacity had a negative effect on the environmental fitness of variants with a large metabolic load. In environments with a larger carrying capacity, such as the rhizosphere of pea, environmental fitness of variants with a large metabolic load was not significantly different from that of variants with a smaller metabolic burden.