bluuebunny/crossref_metadata_2025 huggingface dataset:
A Study of Deep-Sea Natural Microbial Populations and Barophilic Pure Cultures Using a High-Pressure Chemostat
Authors: Wirsen Carl O. , Molyneaux Stephen J.
Published: December 2019

Continuous cultures in which a high-pressure chemostat was used were employed to study the growth responses of (i) deep-sea microbial populations with the naturally occurring carbon available in seawater and with limiting concentrations of supplemental organic substrates and (ii) pure cultures of copiotrophic barophilic and barotolerant deep-sea isolates in the presence of limiting carbon concentrations at various pressures, dilution rates, and temperatures. We found that the growth rates of natural populations could not be measured or were extremely low (e.g., a doubling time of 629 h), as determined from the difference between the dilution rate and the washout rate. A low concentration of supplemental carbon (0.33 mg/liter) resulted in positive growth responses in the natural population, which resulted in an increase in the number of cells and eventually a steady population of cells. We found that the growth responses to imposed growth pressure by barophilic and barotolerant pure-culture isolates that were previously isolated and characterized under high-nutrient-concentration conditions were maintained under the low-nutrient-concentration limiting conditions (0.33 to 3.33 mg of C per liter) characteristic of the deep-sea environment. Our results indicate that deep-sea microbes can respond to small changes in substrate availability. Also, barophilic microbes that are copiotrophic as determined by their isolation in the presence of high carbon concentrations and their preference for high carbon concentrations are versatile and are able to compete and grow as barophiles in the low-carbon-concentration oligotrophic deep-sea environment in which they normally exist.
Molecular Analysis of the 18S rRNA Gene of
Cryptosporidium serpentis
in a Wild-Caught Corn Snake (
Elaphe guttata guttata
) and a Five-Species Restriction Fragment Length Polymorphism- Based Assay That Can Additionally Discern
C. parvum
from
C. wrairi
Authors: Kimbell L. M. , Miller D. L. , Chavez W. , Altman N.
Published: December 2019

An adult wild-caught corn snake (
Elaphe guttata guttata
) was presented for humane euthanasia and necropsy because of severe cryptosporidiosis. The animal was lethargic and >5% dehydrated but in good flesh. Gastric lavage was performed prior to euthanasia. Histopathologic findings included gastric mucosal hypertrophy and a hemorrhagic erosive gastritis. Numerous 5- to 7-μm-diameter round extracellular organisms were associated with the mucosal hypertrophy. A PCR, acid-fast stains, Giemsa stains, and an enzyme immunoassay were all positive for
Cryptosporidium
spp. PCR and restriction fragment length polymorphism (RFLP) analysis on gastric lavage and gastric mucosal specimens, and subsequent sequencing of the 18S rRNA gene, enabled a distinct molecular characterization of the infecting organism as
Cryptosporidium serpentis
. Until recently, studies on snake
Cryptosporidium
have relied on host specificity and gross and histopathologic observations to identify the infecting species. A multiple alignment of our sequence against recently published sequences of the 18S rRNA gene of
C. serpentis
(GenBank accession no.
AF093499
,
AF093500
, and
AF093501
[L. Xiao et al., unpublished data, 1998]) revealed 100% homology with the
C. serpentis
(
Snake
) sequence (
AF093499
) previously described by Xiao et al. An RFLP method to differentiate the five presently sequenced strains of
Cryptosporidium
at this locus was developed. This assay, which uses
Spe
I and
Ssp
I, complements a previously reported assay by additionally distinguishing the bovine strain of
Cryptosporidium
from
Cryptosporidium wrairi
.
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

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

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

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

Feruloyl 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

The 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

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

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

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

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

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

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

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

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

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

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

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.
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

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

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

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

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

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

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

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

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

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

The 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

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

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.