bluuebunny/crossref_metadata_2025_split huggingface dataset:
Reverse Transcription-PCR Differential Display Analysis of
Escherichia coli
Global Gene Regulation in Response to Heat Shock
Authors: Gill R. T. , Valdes J. J. , Bentley W. E. 
Published: December 2019

ABSTRACT

A reverse transcription (RT)-PCR technique was developed to analyze global gene regulation in
Escherichia coli
. A novel combination of primers designed specifically for the start and stop regions of
E. coli
genes (based on the findings of Fislage et al. [R. Fislage, M. Berceanu, Y. Humboldt, M. Wendt, and H. Oberender, Nucleic Acids Res. 25:1830–1835, 1997]) was used as an alternative to the poly(T) primers often used in eukaryotic RT-PCR. The validity of the technique was demonstrated by applying it to heat shock analysis. Specifically, RT-PCR-amplified total RNA from heat-shocked and non-heat-shocked cells were hybridized with slot blots of the Kohara set (U. Kohara, K. Akiyama, and K. Isono, Cell 50:495–508, 1987; S. Chuang, D. Daniels, and F. Blattner, J. Bacteriol. 175:2026–2036, 1993). The signals obtained for heat-shocked and control cultures of each clone were compared, and differences in intensity were evaluated by calculating induction ratios. Clones that were considered significantly induced were subsequently mapped by the Southern blot technique in order to determine specific gene upregulation. Also, for several genes, Northern blotting and total RNA dot blotting were performed to confirm that the transcript levels in the original RNA samples were different. This technique extended previously described methods for studying global gene regulation in
E. coli
by incorporating a PCR amplification step in which global, mRNA-specific primers were used. In addition, the method employed here can be easily extended to study
E. coli
global gene regulation in response to additional environmental stimuli.
Biochemical and Phylogenetic Analyses of a Cold-Active β-Galactosidase from the Lactic Acid Bacterium
Carnobacterium piscicola
BA
Authors: Coombs Jonna M. , Brenchley Jean E. 
Published: December 2019

ABSTRACT

We are investigating glycosyl hydrolases from new psychrophilic isolates to examine the adaptations of enzymes to low temperatures. A β-galactosidase from isolate BA, which we have classified as a strain of the lactic acid bacterium
Carnobacterium piscicola
, was capable of hydrolyzing the chromogen 5-bromo-4-chloro-3-indolyl β-
d
-galactopyranoside (X-Gal) at 4°C and possessed higher activity in crude cell lysates at 25 than at 37°C. Sequence analysis of a cloned DNA fragment encoding this activity revealed a gene cluster containing three glycosyl hydrolases with homology to an α-galactosidase and two β-galactosidases. The larger of the two β-galactosidase genes,
bgaB
, encoded the 76.8-kDa cold-active enzyme. This gene was homologous to family 42 glycosyl hydrolases, a group which contains several thermophilic enzymes but none from lactic acid bacteria. The
bgaB
gene from isolate BA was subcloned in
Escherichia coli
, and its enzyme, BgaB, was purified. The purified enzyme was highly unstable and required 10% glycerol to maintain activity. Its optimal temperature for activity was 30°C, and it was inactivated at 40°C in 10 min. The

K
m

of freshly purified enzyme at 30°C was 1.7 mM, and the
V
max
was 450 μmol · min
−1
· mg
−1
with
o
-nitrophenyl β-
d
-galactopyranoside. This cold-active enzyme is interesting because it is homologous to a thermophilic enzyme from
Bacillus stearothermophilus
, and comparisons could provide information about structural features important for activity at low temperatures.
Permeabilization of Fungal Membranes by Plant Defensins Inhibits Fungal Growth
Authors: Thevissen Karin , Terras Franky R. G. , Broekaert Willem F. 
Published: December 2019

ABSTRACT

We used an assay based on the uptake of SYTOX Green, an organic compound that fluoresces upon interaction with nucleic acids and penetrates cells with compromised plasma membranes, to investigate membrane permeabilization in fungi. Membrane permeabilization induced by plant defensins in
Neurospora crassa
was biphasic, depending on the plant defensin dose. At high defensin levels (10 to 40 μM), strong permeabilization was detected that could be strongly suppressed by cations in the medium. This permeabilization appears to rely on direct peptide-phospholipid interactions. At lower defensin levels (0.1 to 1 μM), a weaker, but more cation-resistant, permeabilization occurred at concentrations that correlated with the inhibition of fungal growth. Rs-AFP2(Y38G), an inactive variant of the plant defensin Rs-AFP2 from
Raphanus sativus
, failed to induce cation-resistant permeabilization in
N. crassa
. Dm-AMP1, a plant defensin from
Dahlia merckii
, induced cation-resistant membrane permeabilization in yeast (
Saccharomyces cerevisiae
) which correlated with its antifungal activity. However, Dm-AMP1 could not induce cation-resistant permeabilization in the Dm-AMP1-resistant
S. cerevisiae
mutant DM1, which has a drastically reduced capacity for binding Dm-AMP1. We think that cation-resistant permeabilization is binding site mediated and linked to the primary cause of fungal growth inhibition induced by plant defensins.
Regulation of the Feruloyl Esterase (faeA) Gene fromAspergillus niger
Authors: de Vries Ronald P. , Visser Jaap 
Published: December 2019

ABSTRACTFeruloyl esterases can remove aromatic residues (e.g., ferulic acid) from plant cell wall polysaccharides (xylan, pectin) and are essential for complete degradation of these polysaccharides. Expression of the feruloyl esterase-encoding gene (faeA) fromAspergillus nigerdepends ond-xylose (expression is mediated by XlnR, the xylanolytic transcriptional activator) and on a second system that responds to aromatic compounds with a defined ring structure, such as ferulic acid and vanillic acid. Several compounds were tested, and all of the inducing compounds contained a benzene ring which had a methoxy group at C-3 and a hydroxy group at C-4 but was not substituted at C-5. Various aliphatic groups occurred at C-1.faeAexpression in the presence of xylose or ferulic acid was repressed by glucose.faeAexpression in the presence of ferulic acid and xylose was greater thanfaeAexpression in the presence of either compound alone. The various inducing systems allowA. nigerto produce feruloyl esterase not only during growth on xylan but also during growth on other ferulic acid-containing cell wall polysaccharides, such as pectin.
Electrochemical Studies of a Truncated Laccase Produced inPichia pastoris
Authors: Gelo-Pujic Mirjana , Kim Hyug-Han , Butlin Nathan G. , Palmore G. Tayhas R. 
Published: December 2019

ABSTRACTThe cDNA that encodes an isoform of laccase fromTrametes versicolor(LCCI), as well as a truncated version (LCCIa), was subcloned and expressed by using the yeastPichia pastorisas the heterologous host. The amino acid sequence of LCCIa is identical to that of LCCI except that the final 11 amino acids at the C terminus of LCCI are replaced with a single cysteine residue. This modification was introduced for the purpose of improving the kinetics of electron transfer between an electrode and the copper-containing active site of laccase. The two laccases (LCCI and LCCIa) are compared in terms of their relative activity with two substrates that have different redox potentials. Results from electrochemical studies on solutions containing LCCI and LCCIa indicate that the redox potential of the active site of LCCIa is shifted to more negative values (411 mV versus normal hydrogen electrode voltage) than that found in other fungal laccases. In addition, replacing the 11 codons at the C terminus of the laccase gene with a single cysteine codon (i.e., LCCI→LCCIa) influences the rate of heterogeneous electron transfer between an electrode and the copper-containing active site (khetfor LCCIa = 1.3 × 10−4cm s−1). These results demonstrate for the first time that the rate of electron transfer between an oxidoreductase and an electrode can be enhanced by changes to the primary structure of a protein via site-directed mutagenesis.
Molecular Phylogenetic Analysis of Archaeal Intron-Containing Genes Coding for rRNA Obtained from a Deep-Subsurface Geothermal Water Pool
Authors: Takai Ken , Horikoshi Koki 
Published: December 2019

ABSTRACT

Molecular phylogenetic analysis of a naturally occurring microbial community in a deep-subsurface geothermal environment indicated that the phylogenetic diversity of the microbial population in the environment was extremely limited and that only hyperthermophilic archaeal members closely related to
Pyrobaculum
were present. All archaeal ribosomal DNA sequences contained intron-like sequences, some of which had open reading frames with repeated homing-endonuclease motifs. The sequence similarity analysis and the phylogenetic analysis of these homing endonucleases suggested the possible phylogenetic relationship among archaeal rRNA-encoded homing endonucleases.
Involvement of Manganese in Conversion of Phenylalanine to Benzaldehyde by Lactic Acid Bacteria
Authors: Nierop Groot Masja N. , de Bont Jan A. M. 
Published: December 2019

ABSTRACT

We examined the involvement of Mn(II) in the conversion of phenylalanine to benzaldehyde in cell extracts of lactic acid bacteria. Experiments performed with
Lactobacillus plantarum
demonstrated that Mn(II), present at high levels in this strain, is involved in benzaldehyde formation by catalyzing the conversion of phenylpyruvic acid. Experiments performed with various lactic acid bacterial strains belonging to different genera revealed that benzaldehyde formation in a strain was related to a high Mn(II) level.
Cooccurrence of Elevated Urea Levels and Dinoflagellate Blooms in Temperate Estuarine Aquaculture Ponds
Authors: Glibert Patricia M. , Terlizzi Daniel E. 
Published: December 2019

ABSTRACT
In hybrid striped bass aquaculture ponds, dinoflagellate blooms were found on 10 of 14 occasions to co-occur with concentrations of urea in excess of 1.5 μM nitrogen. When urea levels were <1.5 μM nitrogen, on seven occasions, no evidence of dinoflagellate blooms was observed in these ponds.
Isolation of
Terrabacter
sp. Strain DDE-1, Which Metabolizes 1,1-Dichloro-2,2-Bis(4-Chlorophenyl)Ethylene when Induced with Biphenyl
Authors: Aislabie J. , Davison A. D. , Boul H. L. , Franzmann P. D. , Jardine D. R. , Karuso P. 
Published: December 2019

ABSTRACT

Terrabacter
sp. strain DDE-1, able to metabolize 1,1-dichloro-2,2-bis(4-chlorophenyl)ethylene (DDE) in pure culture when induced with biphenyl, was enriched from a 1-1-1-trichloro-2,2-bis(4-chlorophenyl)ethane residue-contaminated agricultural soil. Gas chromatography-mass spectrometry analysis of culture extracts revealed a number of DDE catabolites, including 2-(4′-chlorophenyl)-3,3-dichloropropenoic acid, 2-(4′-chlorophenyl)-2-hydroxy acetic acid, 2-(4′-chlorophenyl) acetic acid, and 4-chlorobenzoic acid.
Regiospecific Internal Desaturation of Aliphatic Compounds by a Mutant
Rhodococcus
Strain
Authors: Koike Kenzo , Ara Katsutoshi , Adachi Shigehito , Takigawa Hirofumi , Mori Hajime , Inoue Shigeo , Kimura Yoshiharu , Ito Susumu 
Published: December 2019

ABSTRACT

A mutant
Rhodococcus
strain lacking the ability to utilize 1-chlorohexadecane was found to
cis
-desaturate aliphatic compounds, such as 1-chlorohexadecane,
n
-hexadecane, and heptadecanonitrile, yielding corresponding products with a double bond mainly at the ninth carbon from the terminal methyl groups. A new oxidative pathway involving the
cis
-desaturation step was suggested for alkane utilization by
Rhodococcus
spp.
Biochemical Characterization of Fungal Phytases (
myo
-Inositol Hexakisphosphate Phosphohydrolases): Catalytic Properties
Authors: Wyss Markus , Brugger Roland , Kronenberger Alexandra , Rémy Roland , Fimbel Rachel , Oesterhelt Gottfried , Lehmann Martin , van Loon Adolphus P. G. M. 
Published: December 2019

ABSTRACT

Supplementation with phytase is an effective way to increase the availability of phosphorus in seed-based animal feed. The biochemical characteristics of an ideal phytase for this application are still largely unknown. To extend the biochemical characterization of wild-type phytases, the catalytic properties of a series of fungal phytases, as well as
Escherichia coli
phytase, were determined. The specific activities of the fungal phytases at 37°C ranged from 23 to 196 U · (mg of protein)
−1
, and the pH optima ranged from 2.5 to 7.0. When excess phytase was used, all of the phytases were able to release five phosphate groups of phytic acid (
myo
-inositol hexakisphosphate), which left
myo
-inositol 2-monophosphate as the end product. A combination consisting of a phytase and
Aspergillus niger
pH 2.5 acid phosphatase was able to liberate all six phosphate groups. When substrate specificity was examined, the
A. niger
,
Aspergillus terreus
, and
E. coli
phytases were rather specific for phytic acid. On the other hand, the
Aspergillus fumigatus
,
Emericella nidulans
, and
Myceliophthora thermophila
phytases exhibited considerable activity with a broad range of phosphate compounds, including phenyl phosphate,
p
-nitrophenyl phosphate, sugar phosphates, α- and β-glycerophosphates, phosphoenolpyruvate, 3-phosphoglycerate, ADP, and ATP. Both phosphate liberation kinetics and a time course experiment in which high-performance liquid chromatography separation of the degradation intermediates was used showed that all of the
myo
-inositol phosphates from the hexakisphosphate to the bisphosphate were efficiently cleaved by
A. fumigatus
phytase. In contrast, phosphate liberation by
A. niger
or
A. terreus
phytase decreased with incubation time, and the
myo
-inositol tris- and bisphosphates accumulated, suggesting that these compounds are worse substrates than phytic acid is. To test whether broad substrate specificity may be advantageous for feed application, phosphate liberation kinetics were studied in vitro by using feed suspensions supplemented with 250 or 500 U of either
A. fumigatus
phytase or
A. niger
phytase (Natuphos) per kg of feed. Initially, phosphate liberation was linear and identical for the two phytases, but considerably more phosphate was liberated by the
A. fumigatus
phytase than by the
A. niger
phytase at later stages of incubation.
Degradation of Starch–Poly(β-Hydroxybutyrate-Co-β-Hydroxyvalerate) Bioplastic in Tropical Coastal Waters
Authors: Imam S. H. , Gordon S. H. , Shogren R. L. , Tosteson T. R. , Govind N. S. , Greene R. V. 
Published: December 2019

ABSTRACT
Extruded bioplastic was prepared from cornstarch or poly(β-hydroxybutyrate-co-β-hydroxyvalerate) (PHBV) or blends of cornstarch and PHBV. The blended formulations contained 30 or 50% starch in the presence or absence of polyethylene oxide (PEO), which enhances adherence of starch granules to PHBV. Degradation of these formulations was monitored for 1 year at four stations in coastal water southwest of Puerto Rico. Two stations were within a mangrove stand. The other two were offshore; one of these stations was on a shallow shoulder of a reef, and the other was at a location in deeper water. Microbial enumeration at the four stations revealed considerable flux in the populations over the course of the year. However, in general, the overall population densities were 1 order of magnitude less at the deeper-water station than at the other stations. Starch degraders were 10- to 50-fold more prevalent than PHBV degraders at all of the stations. Accordingly, degradation of the bioplastic, as determined by weight loss and deterioration of tensile properties, correlated with the amount of starch present (100% starch >50% starch > 30% starch > 100% PHBV). Incorporation of PEO into blends slightly retarded the rate of degradation. The rate of loss of starch from the 100% starch samples was about 2%/day, while the rate of loss of PHBV from the 100% PHBV samples was about 0.1%/day. Biphasic weight loss was observed for the starch-PHBV blends at all of the stations. A predictive mathematical model for loss of individual polymers from a 30% starch–70% PHBV formulation was developed and experimentally validated. The model showed that PHBV degradation was delayed 50 days until more than 80% of the starch was consumed and predicted that starch and PHBV in the blend had half-lives of 19 and 158 days, respectively. Consistent with the relatively low microbial populations, bioplastic degradation at the deeper-water station exhibited an initial lag period, after which degradation rates comparable to the degradation rates at the other stations were observed. Presumably, significant biodegradation occurred only after colonization of the plastic, a parameter that was dependent on the resident microbial populations. Therefore, it can be reasonably inferred that extended degradation lags would occur in open ocean water where microbes are sparse.
Staphylokinase as a Plasminogen Activator Component in Recombinant Fusion Proteins
Authors: Szarka S. J. , Sihota E. G. , Habibi H. R. , Wong S.-L. 
Published: December 2019

ABSTRACT

The plasminogen activator staphylokinase (SAK) is a promising thrombolytic agent for treatment of myocardial infarction. It can specifically stimulate the thrombolysis of both erythrocyte-rich and platelet-rich clots. However, SAK lacks fibrin-binding and thrombin inhibitor activities, two functions which would supplement and potentially improve its thrombolytic potency. Creating a recombinant fusion protein is one approach for combining protein domains with complementary functions. To evaluate SAK for use in a translational fusion protein, both N- and C-terminal fusions to SAK were constructed by using hirudin as a fusion partner. Recombinant fusion proteins were secreted from
Bacillus subtilis
and purified from culture supernatants. The rate of plasminogen activation by SAK was not altered by the presence of an additional N- or C-terminal protein sequence. However, cleavage at N-terminal lysines within SAK rendered the N-terminal fusion unstable in the presence of plasmin. The results of site-directed mutagenesis of lysine 10 and lysine 11 in SAK suggested that a plasmin-resistant variant cannot be created without interfering with the plasmin processing necessary for activation of SAK. Although putative plasmin cleavage sites are located at the C-terminal end of SAK at lysine 135 and lysine 136, these sites were resistant to plasmin cleavage in vitro. Therefore, C-terminal fusions represent stable configurations for developing improved thrombolytic agents based on SAK as the plasminogen activator component.
Identification and Characterization of a Lysis Module Present in a Large Proportion of Bacteriophages Infecting
Streptococcus thermophilus
Authors: Sheehan Michelle M. , Stanley Elizabeth , Fitzgerald Gerald F. , van Sinderen Douwe 
Published: December 2019

ABSTRACT

A lysis module encoded by the temperate bacteriophage φO1205 was identified. This lysis module contains a lysin gene, designated
lyt
51, and two putative holin-encoding genes, designated
lyt
49 and
lyt
50.
lyt
51 encodes a lytic enzyme specifically directed against streptococcal cell walls. Similar to other phage-encoded lysins, Lyt51 appears to have a modular design in which the N-terminal portion corresponds to its enzymatic activity while the C-terminal region is responsible for its substrate binding specificity. The two putative holin-encoding genes,
lyt
49 and
lyt
50, located immediately upstream of
lyt
51, were identified on the basis of their homology to other identified holin-encoding genes. Expression of
lyt
49 or
lyt
50 in
Escherichia coli
was shown to cause cell death and leakage of the intracellular enzyme isocitrate dehydrogenase into the growth medium without apparent lysis of the cells. Southern blotting experiments demonstrated that at least one of the three components of the identified lysis module is present in all members of a large collection of bacteriophages, indicating that components of this lysis module are widespread among bacteriophages infecting
Streptococcus thermophilus
.
Antilisterial Activity of Peptide AS-48 and Study of Changes Induced in the Cell Envelope Properties of an AS-48-Adapted Strain of
Listeria monocytogenes
Authors: Mendoza Fátima , Maqueda Mercedes , Gálvez Antonio , Martínez-Bueno Manuel , Valdivia Eva 
Published: December 2019

ABSTRACT

The peptide AS-48 is highly active on all
Listeria
species. It has a bactericidal and bacteriolytic mode of action on
Listeria monocytogenes
CECT 4032, causing depletion of the membrane electrical potential and pH gradient. The producer strain
Enterococcus faecalis
A-48-32, releases sufficient amounts of AS-48 into the growth medium to suppress
L. monocytogenes
in cocultures at enterococcus-to-listeria ratios above 1 at 37°C or above 10 at 15°C. As the temperature decreases, the bactericidal effects of AS-48 are less pronounced, but at 2.5 μg/ml it still can inhibit the growth of listeria at 6°C. AS-48 is highly active on liquid cultures, although concentrations above 0.2 μg/ml are required to avoid adaptation of listeria. AS-48-adapted cells can be selected at low (but still inhibitory) concentrations, and they can be inhibited completely by AS-48 at 0.5 μg/ml. The adaptation is lost gradually upon repeated subcultivation. AS48
ad
cells are cross-resistant to nisin and show an increased resistance to muramidases. Their fatty acid composition is modified: they show a much higher proportion of branched fatty acids as well as a higher C
15:0 An
-to-C
17:0 An
ratio. Resistance to AS-48 is also maintained by protoplasts from AS48
ad
cells. Electron microscopy observations show that the cell wall of AS48
ad
cells is thicker and less dense. The structure of wild-type cells is severely modified after AS-48 treatment: the cell wall and the cytoplasmic membrane are disorganized, and the cytoplasmic content is lost. Intracytoplasmic membrane vesicles are also observed when the wild-type strain is treated with high AS-48 concentrations.
Detection of Methanotrophs in Groundwater by PCR
Authors: Cheng Y. S. , Halsey J. L. , Fode K. A. , Remsen C. C. , Collins M. L. P. 
Published: December 2019

ABSTRACT

Methanotrophic bacteria have significant potential for bioremediation, which would require methods for monitoring the presence and activity of these organisms in environmental samples. In this study, PCR was used to detect methanotrophic bacteria. Primers were designed on the basis of a partial sequence of
pmoA
, which encodes one of the proteins of the particulate methane monooxygenase. Specific amplification of a portion of
pmoA
was obtained with template DNA isolated from lab strains of methanotrophs. A
pmoA
product was also obtained by using DNA from groundwater. The identity of the PCR product was confirmed by sequencing or by amplification with a nested primer. Reverse transcriptase PCR detected
pmoA
mRNA.