bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of Fatty Acid Composition, Spore Germination, and Thermal Resistance in a Nisin-Resistant Mutant of
Clostridium botulinum
169B and in the Wild-Type Strain
Authors: Mazzotta Alejandro S. , Montville Thomas J. 
Published: December 2019

ABSTRACT

The membrane fatty acids, thermal resistance, and germination of a nisin-resistant (Nis
r
) mutant of
Clostridium botulinum
169B were compared with those of the wild-type (WT) strain. In the membranes of WT cells, almost 50% of the total fatty acids were unsaturated, but in those of Nis
r
cells, only 23% of the fatty acids were unsaturated. WT and Nis
r
spores contained similar amounts (approximately 23%) of unsaturated fatty acids, but the saturated straight-chain/branched-chain ratio was significantly higher in Nis
r
spores than in WT spores. These fatty acid differences suggest that Nis
r
cell and spore membranes may be more rigid, a characteristic which would interfere with the pore-forming ability of nisin. Nis
r
C. botulinum
did not produce an extracellular nisin-degrading enzyme, nor were there any differences in the sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns of coat proteins extracted from WT and Nis
r
spores, eliminating these as possible reasons for nisin resistance. Nis
r
spores had thermal resistance parameters similar to those of WT spores. In WT spores, but not in Nis
r
spores, nisin caused a 40% reduction in thermal resistance and a twofold increase in the germination rate. Because the nisin-induced increase in the germination rate of WT spores occurred only in the presence of a germinant (a molecule that triggers germination), nisin can be classified as a progerminant (a molecule that stimulates germination only in the presence of a germinant).
The Chlorocatechol-Catabolic Transposon Tn
5707
of
Alcaligenes eutrophus
NH9, Carrying a Gene Cluster Highly Homologous to That in the 1,2,4-Trichlorobenzene-Degrading Bacterium
Pseudomonas
sp. Strain P51, Confers the Ability To Grow on 3-Chlorobenzoate
Authors: Ogawa Naoto , Miyashita Kiyotaka 
Published: December 2019

ABSTRACT

Alcaligenes eutrophus
(
Ralstonia eutropha
) NH9, isolated in Japan, utilizes 3-chlorobenzoate as its sole source of carbon and energy. Sequencing of the relevant region of plasmid pENH91 from strain NH9 revealed that the genes for the catabolic enzymes were homologous to the genes of the modified
ortho
-cleavage pathway. The genes from strain NH9 (
cbnR-ABCD
) showed the highest homology (89 to 100% identity at the nucleotide level) to the
tcbR-CDEF
genes on plasmid pP51 of the 1,2,4-trichlorobenzene-degrading bacterium
Pseudomonas
sp. strain P51, which was isolated in The Netherlands. The structure of the operon, including the lengths of open reading frames and intervening sequences, was completely conserved between the
cbn
and
tcb
genes. Most nucleotide substitutions were localized within and proximal to the
cbnB
(
tcbD
) gene. The difference in the chloroaromatics that the two strains could use as growth substrates seemed to be due to differences in enzymes that convert substrates to chlorocatechols. The restriction map of plasmid pENH91 was clearly different from that of pP51 except in the regions that contained the
cbnR-ABCD
and
tcbR-CDEF
genes, respectively, suggesting that the chlorocatechol gene clusters might have been transferred as units. Two homologous sequences, present as direct repeats in both flanking regions of the
cbnR-ABCD
genes on pENH91, were found to be identical insertion sequences (ISs), designated IS
1600
, which formed a composite transposon designated Tn
5707
. Although the
tcbR-CDEF
genes were not associated with similar ISs, a DNA fragment homologous to IS
1600
was cloned from the chromosome of strain P51. The sequence of the fragment suggested that it might be a remnant of an IS. The two sequences, together with IS
1326
and
nmoT
, formed a distinct cluster on a phylogenetic tree of the IS
21
family. The diversity of the sources of these IS or IS-like elements suggests the prevalence of ISs of this type.
Alterations in Adhesion, Transport, and Membrane Characteristics in an Adhesion-Deficient Pseudomonad
Authors: DeFlaun M. F. , Oppenheimer S. R. , Streger S. , Condee C. W. , Fletcher M. 
Published: December 2019

ABSTRACT

A stable adhesion-deficient mutant of
Burkholderia cepacia
G4, a soil pseudomonad, was selected in a sand column assay. This mutant (ENV435) was compared to the wild-type strain by examining the adhesion of the organisms to silica sand and their transport through two aquifer sediments that differed in their sand, silt, and clay contents. We compared the longitudinal transport of the wild type and the adhesion mutant to the transport of a conservative chloride tracer in 25-cm-long glass columns. The transport of the wild-type strain was severely retarded compared to the transport of the conservative tracer in a variety of aquifer sediments, while the adhesion mutant and the conservative tracer traveled at similar rates. An intact sediment core study produced similar results; ENV435 was transported at a faster rate and in much greater numbers than G4. The results of hydrophobic interaction chromatography revealed that G4 was significantly more hydrophobic than ENV435, and polyacrylamide gel electrophoresis revealed significant differences in the lipopolysaccharide O-antigens of the adhesion mutant and the wild type. Differences in this cell surface polymer may explain the decreased adhesion of strain ENV435.
Simultaneous Monitoring of Cell Number and Metabolic Activity of Specific Bacterial Populations with a Dual
gfp-luxAB
Marker System
Authors: Unge Annika , Tombolini Riccardo , Mølbak Lars , Jansson Janet K. 
Published: December 2019

ABSTRACT

A dual marker system was developed for simultaneous quantification of bacterial cell numbers and their activity with the
luxAB
and
gfp
genes, encoding bacterial luciferase and green fluorescent protein (GFP), respectively. The bioluminescence phenotype of the
luxAB
biomarker is dependent on cellular energy status. Since cellular metabolism requires energy, bioluminescence output is directly related to the metabolic activity of the cells. By contrast, GFP fluorescence has no energy requirement. Therefore, by combining these two biomarkers, total cell number and metabolic activity of a specific marked cell population could be monitored simultaneously. Two different bacterial strains,
Escherichia coli
DH5α and
Pseudomonas fluorescens
SBW25, were chromosomally tagged with the dual marker cassette, and the cells were monitored under different conditions by flow cytometry, plate counting, and luminometry. During log-phase growth, the luciferase activity was proportional to the number of GFP-fluorescent cells and culturable cells. Upon entrance into stationary phase or during starvation, luciferase activity decreased due to a decrease in cellular metabolic activity of the population, but the number of GFP-fluorescing cells and culturable cells remained relatively stable. In addition, we optimized a procedure for extraction of bacterial cells from soil, allowing GFP-tagged bacteria in soil samples to be quantitated by flow cytometry. After 30 days of incubation of
P. fluorescens
SBW25::gfp/lux in soil, the cells were still maintained at high population densities, as determined by GFP fluorescence, but there was a slow decline in luciferase activity, implicating nutrient limitation. In conclusion, the dual marker system allowed simultaneous monitoring of the metabolic activity and cell number of a specific bacterial population and is a promising tool for monitoring of specific bacteria in situ in environmental samples.
Poly-3-Hydroxybutyrate in
Legionella pneumophila
, an Energy Source for Survival in Low-Nutrient Environments
Authors: James Brian W. , Mauchline W. Stuart , Dennis P. Julian , Keevil C. William , Wait Robin 
Published: December 2019

ABSTRACT

Chloroform-soluble material was extracted from two strains of
L. pneumophila
serogroup 1 following growth in continuous culture. The purified material was identified as poly-3-hydroxybutyrate (PHB) by nuclear magnetic resonance spectroscopy and by gas chromatography-mass spectrometry. PHB yields of up to 16% of cell dry weight were extracted from culture samples. The PHB was located in electron-dense intracellular inclusions, which fluoresced bright yellow when stained with the lipophilic dye Nile red. A Nile red spectrofluorometric assay provided a more accurate and reliable determination of the PHB content. PHB accumulation increased threefold during iron-limited culture and was inversely related to the concentration of iron metabolized. Chemostat-grown cells survived in a culturable state for at least 600 days when incubated at 24°C in a low-nutrient tap water environment. Nile red spectrofluorometry and flow cytometry demonstrated that PHB reserves were utilized during starvation. PHB utilization, as revealed by the decline in mean cellular fluorescence and cell complexity, correlated with loss of culturability. Fluorescence microscopy provided visual evidence of PHB utilization, with a marked reduction in the number of Nile red-stained granules during starvation. Heat shock treatment failed to resuscitate nonculturable cells. This study demonstrates that
L. pneumophila
accumulates significant intracellular reserves of PHB, which promote its long-term survival under conditions of starvation.
New Assay for Rhizobitoxine Based on Inhibition of 1-Aminocyclopropane-1-Carboxylate Synthase
Authors: Yasuta Tsuyoshi , Satoh Shigeru , Minamisawa Kiwamu 
Published: December 2019

ABSTRACT

Rhizobitoxine is synthesized by the legume symbiont
Bradyrhizobium elkanii
and the plant pathogen
Burkholderia andropogonis
. Rhizobitoxine competitively inhibited 1-aminocyclopropane-1-carboxylate (ACC) synthase bLE-ACS2 from the tomato, a key enzyme in the pathway of ethylene biosynthesis. Based on this inhibition of ACC synthase, we have developed a new assay for rhizobitoxine.
A Phosphonate-Induced Gene Which Promotes
Penicillium
-Mediated Bioconversion of
cis
-Propenylphosphonic Acid to Fosfomycin
Authors: Watanabe M. , Sumida N. , Murakami S. , Anzai H. , Thompson C. J. , Tateno Y. , Murakami T. 
Published: December 2019

ABSTRACT

Penicillium decumbens
is able to epoxidize
cis
-propenylphosphonic acid (cPA) to produce the antibiotic fosfomycin [FOM; also referred to as phosphonomycin and (−)-
cis
-1,2-epoxypropylphosphonic acid], a bioconversion of considerable commercial significance. We sought to improve the efficiency of the process by overexpression of the genes involved. A conventional approach of isolating the presumed epoxidase and its corresponding gene was not possible since cPA epoxidation could not be achieved with protein extracts. As an alternative approach, proteins induced by cPA were detected by two-dimensional gel electrophoresis. The observation that a 31-kDa protein (EpoA) was both cPA induced and overaccumulated in a strain which more efficiently converted cPA suggested that it might take part in the bioconversion. EpoA was purified, its amino acid sequence was partially determined, and the corresponding gene was isolated from cosmid and cDNA libraries with oligonucleotide probes. The DNA sequence for this gene (
epoA
) contained two introns and an open reading frame encoding a peptide of 277 amino acids having some similarity to oxygenases. When the gene was subcloned into
P. decumbens
, a fourfold increase in epoxidation activity was achieved.
epoA
-disruption mutants which were obtained by homologous recombination could not convert cPA to FOM. To investigate the regulation of the
epoA
promoter, the bialaphos resistance gene (
bar
, encoding phosphinothricin acetyltransferase) was used to replace the
epoA
-coding region. In
P. decumbens
, expression of the
bar
reporter gene was induced by cPA, FOM, and phosphorous acid but not by phosphoric acid.
Induction of Defense Responses in Cucumber Plants (
Cucumis sativus
L.) by the Biocontrol Agent
Trichoderma harzianum
Authors: Yedidia I. , Benhamou N. , Chet I. 
Published: December 2019

ABSTRACT

The potential of the biocontrol agent
Trichoderma harzianum
T-203 to trigger plant defense responses was investigated by inoculating roots of cucumber seedlings with
Trichoderma
in an aseptic, hydroponic system.
Trichoderma
-treated plants were more developed than nontreated plants throughout the experiment. Electron microscopy of ultrathin sections from
Trichoderma
-treated roots revealed penetration of
Trichoderma
into the roots, restricted mainly to the epidermis and outer cortex. Strengthening of the epidermal and cortical cell walls was observed, as was the deposition of newly formed barriers. These typical host reactions were found beyond the sites of potential fungal penetration. Wall appositions contained large amounts of callose and infiltrations of cellulose. The wall-bound chitin in
Trichoderma
hyphae was preserved, even when the hyphae had undergone substantial disorganization. Biochemical analyses revealed that inoculation with
Trichoderma
initiated increased peroxidase and chitinase activities within 48 and 72 h, respectively. These results were observed for both the roots and the leaves of treated seedlings, providing evidence that
T. harzianum
may induce systemic resistance mechanisms in cucumber plants.
Diversity of Dissimilatory Bisulfite Reductase Genes of Bacteria Associated with the Deep-Sea Hydrothermal Vent Polychaete Annelid
Alvinella pompejana
Authors: Cottrell Matthew T. , Cary S. Craig 
Published: December 2019

ABSTRACT

A unique community of bacteria colonizes the dorsal integument of the polychaete annelid
Alvinella pompejana
, which inhabits the high-temperature environments of active deep-sea hydrothermal vents along the East Pacific Rise. The composition of this bacterial community was characterized in previous studies by using a 16S rRNA gene clone library and in situ hybridization with oligonucleotide probes. In the present study, a pair of PCR primers (P94-F and P93-R) were used to amplify a segment of the dissimilatory bisulfite reductase gene from DNA isolated from the community of bacteria associated with
A. pompejana
. The goal was to assess the presence and diversity of bacteria with the capacity to use sulfate as a terminal electron acceptor. A clone library of bisulfite reductase gene PCR products was constructed and characterized by restriction fragment and sequence analysis. Eleven clone families were identified. Two of the 11 clone families, SR1 and SR6, contained 82% of the clones. DNA sequence analysis of a clone from each family indicated that they are dissimilatory bisulfite reductase genes most similar to the dissimilatory bisulfite reductase genes of
Desulfovibrio vulgaris
,
Desulfovibrio gigas
,
Desulfobacterium autotrophicum
, and
Desulfobacter latus
. Similarities to the dissimilatory bisulfite reductases of
Thermodesulfovibrio yellowstonii
, the sulfide oxidizer
Chromatium vinosum
, the sulfur reducer
Pyrobaculum islandicum
, and the archaeal sulfate reducer
Archaeoglobus fulgidus
were lower. Phylogenetic analysis separated the clone families into groups that probably represent two genera of previously uncharacterized sulfate-reducing bacteria. The presence of dissimilatory bisulfite reductase genes is consistent with recent temperature and chemical measurements that documented a lack of dissolved oxygen in dwelling tubes of the worm. The diversity of dissimilatory bisulfite reductase genes in the bacterial community on the back of the worm suggests a prominent role for anaerobic sulfate-reducing bacteria in the ecology of
A. pompejana
.
The Role of Benzoate in Anaerobic Degradation of Terephthalate
Authors: Kleerebezem Robbert , Pol Look W. Hulshoff , Lettinga Gatze 
Published: December 2019

ABSTRACTThe effects of acetate, benzoate, and periods without substrate on the anaerobic degradation of terephthalate (1,4-benzene-dicarboxylate) by a syntrophic methanogenic culture were studied. The culture had been enriched on terephthalate and was capable of benzoate degradation without a lag phase. When incubated with a mixture of benzoate and terephthalate, subsequent degradation with preference for benzoate was observed. Both benzoate and acetate inhibited the anaerobic degradation of terephthalate. The observed inhibition is partially irreversible, resulting in a decrease (or even a complete loss) of the terephthalate-degrading activity after complete degradation of benzoate or acetate. Irreversible inhibition was characteristic for terephthalate degradation only because the inhibition of benzoate degradation by acetate could well be described by reversible noncompetitive product inhibition. Terephthalate degradation was furthermore irreversibly inhibited by periods without substrate of only a few hours. The inhibition of terephthalate degradation due to periods without substrate could be overcome through incubation of the culture with a mixture of benzoate and terephthalate. In this case no influence of a period without substrate was observed. Based on these observations it is postulated that decarboxylation of terephthalate, resulting in the formation of benzoate, is strictly dependent on the concomitant fermentation of benzoate. In the presence of higher concentrations of benzoate, however, benzoate is the favored substrate over terephthalate, and the culture loses its ability to degrade terephthalate. In order to overcome the inhibition of terephthalate degradation by benzoate and acetate, a two-stage reactor system is suggested for the treatment of wastewater generated during terephthalic acid production.
Thaumatin Production in
Aspergillus awamori
by Use of Expression Cassettes with Strong Fungal Promoters and High Gene Dosage
Authors: Moralejo Francisco-Jose , Cardoza Rosa-Elena , Gutierrez Santiago , Martin Juan F. 
Published: December 2019

ABSTRACT

Four expression cassettes containing strong fungal promoters, a signal sequence for protein translocation, a KEX protease cleavage site, and a synthetic gene (
tha
) encoding the sweet protein thaumatin II were used to overexpress this protein in
Aspergillus awamori lpr66
, a PepA protease-deficient strain. The best expression results were obtained with the
gdhA
promoter of
A. awamori
or with the
gpdA
promoter of
Aspergillus nidulans
. There was good correlation of
tha
gene dosage, transcript levels, and thaumatin secretion. The thaumatin gene was expressed as a transcript of the expected size in each construction (1.9 or 1.4 kb), and the transcript levels and thaumatin production rate decayed at the end of the growth phase, except in the double transformant TB2b1-44-GD5, in which secretion of thaumatin continued until 96 h. The recombinant thaumatin secreted by a high-production transformant was purified to homogeneity, giving one major component and two minor components. In all cases, cleavage of the fused protein occurred at the KEX recognition sequence. This work provides new expression systems in
A. awamori
that result in very high levels of thaumatin production.
Bacteriophage Inactivation at the Air-Water-Solid Interface in Dynamic Batch Systems
Authors: Thompson Shawn S. , Yates Marylynn V. 
Published: December 2019

ABSTRACT
Bacteriophages have been widely used as surrogates for human enteric viruses in many studies on virus transport and fate. In this investigation, the fates of three bacteriophages, MS2, R17, and φX174, were studied in a series of dynamic batch experiments. Both MS2 and R17 readily underwent inactivation in batch experiments where solutions of each phage were percolated through tubes packed with varying ratios of glass and Teflon beads. MS2 and R17 inactivation was the result of exposure to destructive forces at the dynamic air-water-solid interface. φX174, however, did not undergo inactivation in similar studies, suggesting that this phage does not accumulate at air-water interfaces or is not affected by interfacial forces in the same manner. Other batch experiments showed that MS2 and R17 were increasingly inactivated during mixing in polypropylene tubes as the ionic strength of the solution was raised (φX174 was not affected). By the addition of Tween 80 to suspensions of MS2 and R17, phage inactivation was prevented. Our data suggest that viral inactivation in simple dynamic batch experiments is dependent upon (i) the presence of a dynamic air-water-solid interface (where the solid is a hydrophobic surface), (ii) the ionic strength of the solution, (iii) the concentration of surface active compounds in the solution, and (iv) the type of virus used.
A General Method for Selection of α-Acetolactate Decarboxylase-Deficient
Lactococcus lactis
Mutants To Improve Diacetyl Formation
Authors: Curic Mirjana , Stuer-Lauridsen Birgitte , Renault Pierre , Nilsson Dan 
Published: December 2019

ABSTRACT

The enzyme acetolactate decarboxylase (Ald) plays a key role in the regulation of the α-acetolactate pool in both pyruvate catabolism and the biosynthesis of the branched-chain amino acids, isoleucine, leucine, and valine (ILV). This dual role of Ald, due to allosteric activation by leucine, was used as a strategy for the isolation of Ald-deficient mutants of
Lactococcus lactis
subsp.
lactis
biovar diacetylactis. Such mutants can be selected as leucine-resistant mutants in ILV- or IV-prototrophic strains. Most dairy lactococcus strains are auxotrophic for the three amino acids. Therefore, the plasmid pMC004 containing the
ilv
genes (encoding the enzymes involved in the biosynthesis of IV) of
L. lactis
NCDO2118 was constructed. Introduction of pMC004 into ILV-auxotrophic dairy strains resulted in an isoleucine-prototrophic phenotype. By plating the strains on a chemically defined medium supplemented with leucine but not valine and isoleucine, spontaneous leucine-resistant mutants were obtained. These mutants were screened by Western blotting with Ald-specific antibodies for the presence of Ald. Selected mutants lacking Ald were subsequently cured of pMC004. Except for a defect in the expression of Ald, the resulting strain, MC010, was identical to the wild-type strain, as shown by Southern blotting and DNA fingerprinting. The mutation resulting in the lack of Ald in MC010 occurred spontaneously, and the strain does not contain foreign DNA; thus, it can be regarded as food grade. Nevertheless, its application in dairy products depends on the regulation of genetically modified organisms. These results establish a strategy to select spontaneous Ald-deficient mutants from transformable
L. lactis
strains.
Interactions between Nitrogen Fixation and Osmoregulation in the Methanogenic Archaeon
Methanosarcina barkeri
227
Authors: Brabban A. D. , Orcutt E. N. , Zinder S. H. 
Published: December 2019

ABSTRACT

The nitrogenase enzyme complex of
Methanosarcina barkeri
227 was found to be more sensitive to NaCl than previously studied molybdenum nitrogenases are, with total inhibition of activity occurring at 190 mM NaCl, compared with >600 mM NaCl for
Azotobacter vinelandii
and
Clostridium pasteurianum
nitrogenases. Na
+
and K
+
had equivalent effects, whereas Mg
2+
was more inhibitory than either monovalent cation, even on a per-charge basis. The anion Cl
−
was more inhibitory than acetate was. Because
M. barkeri
227 is a facultative halophile, we examined the effects of external salt on growth and diazotrophy and found that inhibition of growth was not greater with N
2
than with NH
4
+
. Cells grown with N
2
and cells grown with NH
4
+
produced equal concentrations of α-glutamate at low salt concentrations and equal concentrations of
N
ɛ
-acetyl-β-lysine at NaCl concentrations greater than 500 mM. Despite the high energetic cost of fixing nitrogen for these osmolytes, we obtained no evidence that there is a shift towards nonnitrogenous osmolytes during diazotrophic growth. In vitro nitrogenase enzyme assays showed that at a low concentration (approximately 100 mM) potassium glutamate enhanced activity but at higher concentrations this compound inhibited activity; 50% inhibition occurred at a potassium glutamate concentration of approximately 400 mM.