bluuebunny/crossref_metadata_2025_split huggingface dataset:
Three Phylogenetic Groups of
nodA
and
nifH
Genes in
Sinorhizobium
and
Mesorhizobium
Isolates from Leguminous Trees Growing in Africa and Latin America
Authors: Haukka Kaisa , Lindström Kristina , Young J. Peter W. 
Published: December 2019

ABSTRACT

The diversity and phylogeny of
nodA
and
nifH
genes were studied by using 52 rhizobial isolates from
Acacia senegal
,
Prosopis chilensis
, and related leguminous trees growing in Africa and Latin America. All of the strains had similar host ranges and belonged to the genera
Sinorhizobium
and
Mesorhizobium
, as previously determined by 16S rRNA gene sequence analysis. The restriction patterns and a sequence analysis of the
nodA
and
nifH
genes divided the strains into the following three distinct groups: sinorhizobia from Africa, sinorhizobia from Latin America, and mesorhizobia from both regions. In a phylogenetic tree also containing previously published sequences, the
nodA
genes of our rhizobia formed a branch of their own, but within the branch no correlation between symbiotic genes and host trees was apparent. Within the large group of African sinorhizobia, similar symbiotic gene types were found in different chromosomal backgrounds, suggesting that transfer of symbiotic genes has occurred across species boundaries. Most strains had plasmids, and the presence of plasmid-borne
nifH
was demonstrated by hybridization for some examples. The
nodA
and
nifH
genes of
Sinorhizobium teranga
ORS1009
T
grouped with the
nodA
and
nifH
genes of the other African sinorhizobia, but
Sinorhizobium saheli
ORS609
T
had a totally different
nodA
sequence, although it was closely related based on the 16S rRNA gene and
nifH
data. This might be because this
S. saheli
strain was originally isolated from
Sesbania
sp., which belongs to a different cross-nodulation group than
Acacia
and
Prosopis
spp. The factors that appear to have influenced the evolution of rhizobial symbiotic genes vary in importance at different taxonomic levels.
Effects of High Pressure on Inactivation Kinetics and Events Related to Proton Efflux in
Lactobacillus plantarum
Authors: Wouters Patrick C. , Glaasker Erwin , Smelt Jan P. P. M. 
Published: December 2019

ABSTRACT

Knowledge of the mechanism of pressure-induced inactivation of microorganisms could be helpful in defining an effective, relatively mild pressure treatment as a means of decontamination, especially in combination with other physical treatments or antimicrobial agents. We have studied the effect of high pressure on
Lactobacillus plantarum
grown at pH 5.0 and 7.0. The classical inactivation kinetics were compared with a number of events related to the acid-base physiology of the cell, i.e., activity of F
0
F
1
ATPase, intracellular pH, acid efflux, and intracellular ATP pool. Cells grown at pH 5.0 were more resistant to pressures of 250 MPa than were cells grown at pH 7.0. This difference in resistance may be explained by a higher F
0
F
1
ATPase activity, better ability to maintain a ΔpH, or a higher acid efflux of the cells grown at pH 5.0. After pressure treatment at 250 MPa, the F
0
F
1
ATPase activity was decreased, the ability to maintain a ΔpH was reduced, and the acid efflux was impaired. The ATP pool increased initially after mild pressure treatment and finally decreased after prolonged treatment. The observations on acid efflux and the ATP pool suggest that the glycolysis is affected by high pressure later than is the F
0
F
1
ATPase activity. Although functions related to the membrane-bound ATPase activity were impaired, no morphological changes of the membrane could be observed.
Reverse Transcription-PCR Analysis of the Regulation of the Manganese Peroxidase Gene Family
Authors: Gettemy Jessica M. , Ma Biao , Alic Margaret , Gold Michael H. 
Published: December 2019

ABSTRACT

Manganese peroxidase (MnP) gene expression in the lignin-degrading fungus
Phanerochaete chrysosporium
is regulated by nutrient nitrogen levels and by Mn(II), the substrate for the enzyme, as well as by heat shock and other factors. Reverse transcription-PCR (RT-PCR) of total RNA can distinguish the mRNAs of each of the three sequenced
P. chrysosporium mnp
genes, i.e.,
mnp1
,
mnp2
, and
mnp3
. Quantitative RT-PCR demonstrates that each of the three transcripts is present at a similar low basal level in nitrogen-sufficient cultures, with or without Mn, and in nitrogen-limited cultures lacking Mn. However, in 5-day-old, nitrogen-limited, stationary cultures supplemented with 180 μM Mn, the levels of the
mnp1
and
mnp2
transcripts increased approximately 100- and 1,700-fold, respectively, over basal levels. In contrast, under these conditions, the level of the
mnp3
transcript did not increase significantly over the basal level. Quantitative RT-PCR of total RNA extracted from nitrogen-deficient, Mn-supplemented cultures on days 2 through 7 demonstrates that whereas the
mnp1
transcript was present at relatively low levels on days 3 through 7, the
mnp2
transcript level peaked on day 5 and the
mnp3
transcript level peaked on day 3. Comparison of total RNA extracted on day 5 from nitrogen-deficient, Mn-supplemented stationary and agitated cultures indicates that in stationary cultures,
mnp2
was the major expressed
mnp
gene, whereas in large agitated cultures,
mnp1
was the major expressed
mnp
gene.
Prevalence of Broad-Host-Range Lytic Bacteriophages of
Sphaerotilus natans
,
Escherichia coli
, and
Pseudomonas aeruginosa
Authors: Jensen Ellen C. , Schrader Holly S. , Rieland Brenda , Thompson Thomas L. , Lee Kit W. , Nickerson Kenneth W. , Kokjohn Tyler A. 
Published: December 2019

ABSTRACT

Two bacteriophage collections were examined with regard to their ability to form plaques on multiple bacterial host species. Nine of 10 phages studied were found to be broad-host-range bacteriophages. These phages fell into two groups. Group 1, the SN series, was isolated from sewage treatment plant samples with
Sphaerotilus natans
ATCC 13338 as a host. The DNAs of these bacteriophages contained modified bases and were insensitive to cleavage by type I and II restriction endonucleases. The efficiency of plating of these bacteriophages was changed only slightly on the alternate host. Group 2, the BHR series, was isolated by a two-host enrichment protocol. These bacteriophages were sensitive to restriction, and their efficiency of plating was dramatically reduced on the alternate host. Our results suggest that a multiple-host enrichment protocol may be more effective for the isolation of broad-host-range bacteriophages by avoiding the selection bias inherent in single-host methods. At least two of the broad-host-range bacteriophages mediated generalized transduction. We suggest that broad-host-range bacteriophages play a key role in phage ecology and gene transfer in nature.
Optimization of Exopolysaccharide Production by
Lactobacillus delbrueckii
subsp.
bulgaricus
RR Grown in a Semidefined Medium
Authors: Kimmel Stacy A. , Roberts Robert F. , Ziegler Gregory R. 
Published: December 2019

ABSTRACT

The optimal fermentation temperature, pH, and Bacto-casitone (Difco Laboratories, Detroit, Mich.) concentration for production of exopolysaccharide by
Lactobacillus delbrueckii
subsp.
bulgaricus
RR in a semidefined medium were determined by using response surface methods. The design consisted of 20 experiments, 15 unique combinations, and five replications. All fermentations were conducted in a fermentor with a 2.5-liter working volume and were terminated when 90% of the glucose in the medium had been consumed. The population of
L. delbrueckii
subsp.
bulgaricus
RR and exopolysaccharide content were measured at the end of each fermentation. The optimum temperature, pH, and Bacto-casitone concentration for exopolysaccharide production were 38°C, 5, and 30 g/liter, respectively, with a predicted yield of 295 mg of exopolysaccharide/liter. The actual yield under these conditions was 354 mg of exopolysaccharide/liter, which was within the 95% confidence interval (217 to 374 mg of exopolysaccharide/liter). An additional experiment conducted under optimum conditions showed that exopolysaccharide production was growth associated, with a specific production at the endpoint of 101.4 mg/g of dry cells. Finally, to obtain material for further characterization, a 100-liter fermentation was conducted under optimum conditions. Twenty-nine grams of exopolysaccharide was isolated from centrifuged, ultrafiltered fermentation broth by ethanol precipitation.
Effects of Ethanol and Other Alkanols on Transport of Acetic Acid in
Saccharomyces cerevisiae
Authors: Casal Margarida , Cardoso Helena , Leão Cecília 
Published: December 2019

ABSTRACT

In glucose-grown cells of
Saccharomyces cerevisiae
IGC 4072, acetic acid enters only by simple diffusion of the undissociated acid. In these cells, ethanol and other alkanols enhanced the passive influx of labelled acetic acid. The influx of the acid followed first-order kinetics with a rate constant that increased exponentially with the alcohol concentration, and an exponential enhancement constant for each alkanol was estimated. The intracellular concentration of labelled acetic acid was also enhanced by alkanols, and the effect increased exponentially with alcohol concentration. Acetic acid is transported across the plasma membrane of acetic acid-, lactic acid-, and ethanol-grown cells by acetate-proton symports. We found that in these cells ethanol and butanol inhibited the transport of labelled acetic acid in a noncompetitive way; the maximum transport velocity decreased with alcohol concentration, while the affinity of the system for acetate was not significantly affected by the alcohol. Semilog plots of
V
max
versus alcohol concentration yielded straight lines with negative slopes from which estimates of the inhibition constant for each alkanol could be obtained. The intracellular concentration of labelled acid was significantly reduced in the presence of ethanol or butanol, and the effect increased with the alcohol concentration. We postulate that the absence of an operational carrier for acetate in glucose-grown cells of
S. cerevisiae
, combined with the relatively high permeability of the plasma membrane for the undissociated acid and the inability of the organism to metabolize acetic acid, could be one of the reasons why this species exhibits low tolerance to acidic environments containing ethanol.
Cloning and Nucleotide Sequence of the
gyrB
Gene of
Vibrio parahaemolyticus
and Its Application in Detection of This Pathogen in Shrimp
Authors: Venkateswaran Kasthuri , Dohmoto Nobuhiko , Harayama Shigeaki 
Published: December 2019

ABSTRACT

Because biochemical testing and 16S rRNA sequence analysis have proven inadequate for the differentiation of
Vibrio parahaemolyticus
from closely related species, we employed the gyrase B gene (
gyrB
) as a molecular diagnostic probe. The
gyrB
genes of
V. parahaemolyticus
and closely related
Vibrio alginolyticus
were cloned and sequenced. Oligonucleotide PCR primers were designed for the amplification of a 285-bp fragment from within
gyrB
specific for
V. parahaemolyticus
. These primers recognized 117 of 117 reference and wild-type
V. parahaemolyticus
strains, whereas amplification did not occur when 90 strains of 37 other
Vibrio
species or 60 strains representing 34 different nonvibrio species were tested. In 100-μl PCR mixtures, the lower detection limits were 5 CFU for live cells and 4 pg for purified DNA. The possible application of
gyrB
primers for the routine identification of
V. parahaemolyticus
in food was examined. We developed and tested a procedure for the specific detection of the target organism in shrimp consisting of an 18-h preenrichment followed by PCR amplification of the 285-bp
V. parahaemolyticus
-specific fragment. This method enabled us to detect an initial inoculum of 1.5 CFU of
V. parahaemolyticus
cells per g of shrimp homogenate. By this approach, we were able to detect
V. parahaemolyticus
in all of 27 shrimp samples artificially inoculated with this bacterium. We present here a rapid, reliable, and sensitive protocol for the detection of
V. parahaemolyticus
in shrimp.
Ambient pH Is a Major Determinant in the Expression of Cuticle-Degrading Enzymes and Hydrophobin by
Metarhizium anisopliae
Authors: St. Leger Raymond J. , Joshi Lokesh , Roberts Donald 
Published: December 2019

ABSTRACT

Secretion of proteolytic and chitinolytic enzymes is a hallmark of infection processes of
Metarhizium anisopliae
in response to host (insect) cuticular signals. The regulation of these enzymes (subtilisin-like proteases [Pr1a and Pr1b], trypsin-like proteases [Pr2], metalloproteases, aspartyl proteases, aminopeptidase, and chitinases) and a hydrophobin was investigated by Northern analysis and/or enzyme assay. The production of each enzyme showed a differential expression pattern in response to ambient pH; enzymes were synthesized only at pHs at which they function effectively, irrespective of whether the medium contained an inductive cuticle substrate. Three aspartyl proteases (pH optimum, 3), and chitinase (pH optimum, 5) showed maximal accumulation at acidic pHs. The highest level of aminopeptidase (pH optimum, 7) was detected at pH 7. The highest levels of five metalloproteases (pH optima, ca. 7) were detected over the pH range 6 to 8. Two trypsins and several subtilisin-like Pr1 isoforms with pH optima of ca. 8 were produced only under alkaline conditions. Northern analysis of RNA species corresponding to seven cDNA sequences encoding proteases and chitinase confirmed that the ambient pH played a major role in gene expression of secreted proteins. Hydrophobin was expressed almost equally at pHs 5 and 8 but was not expressed at pH 3. During fungal penetration, the pH of infected cuticle rises from about 6.3 to 7.7. Consistent with pH regulation of enzyme production, serine and metalloproteases were produced in situ during infection, but no production of aspartyl proteases was found. We propose that the alkalinity of infected cuticle represents a physiological signal that triggers the production of virulence factors.
Cloning and Nucleotide Sequencing of a
Staphylococcus aureus
Gene Encoding a Branched-Chain-Amino-Acid Transporter
Authors: Vijaranakul Uriwan , Xiong Anming , Lockwood Katherine , Jayaswal R. K. 
Published: December 2019

ABSTRACT

We recently characterized a transposon-induced NaCl-sensitive mutant of
Staphylococcus aureus
(U. Vijaranakul, M. J. Nadakavukaren, D. O. Bayles, B. J. Wilkinson, and R. K. Jayaswal, Appl. Environ. Microbiol. 63:1889–1897, 1997). To further characterize this mutant, we determined the nucleotide sequence at the insertion site of the transposon on the
S. aureus
chromosome. Nucleotide sequencing revealed a 1,326-bp open reading frame (ORF442) encoding a hydrophobic 442-amino-acid polypeptide with a calculated molecular mass of 49,058 Da. The hydrophilicity profile of the gene product revealed the existence of 12 hydrophobic domains predicted to form membrane-associated α-helices. Comparison of the amino acid sequence of ORF442 with amino acid sequences in the GenBank database showed extensive homology with the branched-chain-amino-acid transport genes of gram-positive and gram-negative bacteria. This is the first
brnQ
gene in staphylococci to be described.
Development and Testing of a Bacterial Biosensor for Toluene-Based Environmental Contaminants
Authors: Willardson Barry M. , Wilkins Jon F. , Rand Timothy A. , Schupp James M. , Hill Karen K. , Keim Paul , Jackson Paul J. 
Published: December 2019

ABSTRACT

A bacterial biosensor for benzene, toluene, and similar compounds has been constructed, characterized, and field tested on contaminated water and soil. The biosensor is based on a plasmid incorporating the transcriptional activator
xylR
from the TOL plasmid of
Pseudomonas putida
mt-2. The XylR protein binds a subset of toluene-like compounds and activates transcription at its promoter, P
u
. A reporter plasmid was constructed by placing the
luc
gene for firefly luciferase under the control of XylR and P
u
. When
Escherichia coli
cells were transformed with this plasmid vector, luminescence from the cells was induced in the presence of benzene, toluene, xylenes, and similar molecules. Accurate concentration dependencies of luminescence were obtained and exhibited
K
1/2
values ranging from 39.0 ± 3.8 μM for 3-xylene to 2,690 ± 160 μM for 3-methylbenzylalcohol (means ± standard deviations). The luminescence response was specific for only toluene-like molecules that bind to and activate XylR. The biosensor cells were field tested on deep aquifer water, for which contaminant levels were known, and were able to accurately detect toluene derivative contamination in this water. The biosensor cells were also shown to detect BETX (benzene, toluene, and xylene) contamination in soil samples. These results demonstrate the capability of such a bacterial biosensor to accurately measure environmental contaminants and suggest a potential for its inexpensive application in field-ready assays.
Utilization of Oligopeptides by
Listeria monocytogenes
Scott A
Authors: Verheul Annette , Rombouts Frank M. , Abee Tjakko 
Published: December 2019

ABSTRACT

For effective utilization of peptides,
Listeria monocytogenes
possesses two different peptide transport systems. The first one is the previously described proton motive force (PMF)-driven di- and tripeptide transport system (A. Verheul, A. Hagting, M.-R. Amezaga, I. R. Booth, F. M. Rombouts, and T. Abee, Appl. Environ. Microbiol. 61:226–233, 1995). The present results reveal that
L. monocytogenes
possesses an oligopeptide transport system, presumably requiring ATP rather than the PMF as the driving force for translocation. Experiments to determine growth in a defined medium containing peptides of various lengths suggested that the oligopeptide permease transports peptides of up to 8 amino acid residues. Peptidase activities towards several oligopeptides were demonstrated in cell extract from
L. monocytogenes
, which indicates that upon internalization, the oligopeptides are hydrolyzed to serve as sources of amino acids for growth. The peptide transporters of the nonproteolytic
L. monocytogenes
might play an important role in foods that harbor indigenous proteinases and/or proteolytic microorganisms, since
Pseudomonas fragi
as well as
Bacillus cereus
was found to enhance the growth of
L. monocytogenes
to a large extent in a medium in which the milk protein casein was the sole source of nitrogen. In addition, growth stimulation was elicited in this medium when casein was hydrolyzed by using purified protease from
Bacillus licheniformis
. The possible contribution of the oligopeptide transport system in the establishment of high numbers of
L. monocytogenes
cells in fermented milk products is discussed.
Survival of Infectious
Cryptosporidium parvum
Oocysts in Seawater and Eastern Oysters (
Crassostrea virginica
) in the Chesapeake Bay
Authors: Fayer Ronald , Graczyk Thaddeus K. , Lewis Earl J. , Trout James M. , Farley C. Austin 
Published: December 2019

ABSTRACT

Oocysts of
Cryptosporidium parvum
placed in artificial seawater at salinities of 10, 20, and 30 ppt at 10°C and at 10 ppt at 20°C were infectious after 12 weeks. Those placed in seawater at 20 ppt and 30 ppt at 20°C were infectious for 8 and 4 weeks, respectively. These findings suggested that oocysts could survive in estuarine waters long enough to be removed by filter feeders such as oysters. Thereafter, 30 Eastern oysters,
Crassostrea virginica
, were collected with a dredge or with hand tongs at each of six sites within Maryland tributaries of the Chesapeake Bay in May and June and in August and September of 1997. Hemocytes and gill washings from all oysters were examined for the presence of
Cryptosporidium
oocysts and
Giardia
cysts by immunofluorescence microscopy utilizing a commercially available kit containing fluorescein isothiocyanate-conjugated monoclonal antibodies.
Giardia
was not detected by this method from any of the 360 oysters examined. Presumptive identification of
Cryptosporidium
oocysts was made in either hemocytes or gill washings of oysters from all six sites both times that surveys were conducted. In addition, during August and September, for each of the six sites, hemocytes from the 30 oysters were pooled and gill washings from the oysters were pooled. Each pool was delivered by gastric intubation to a litter of neonatal mice to produce a bioassay for oocyst infectivity. Intestinal tissue from two of three mice that received gill washings from oysters collected at a site near a large cattle farm and shoreline homes with septic tanks was positive for developmental stages of
C. parvum
. These findings demonstrate for the first time that oysters in natural waters harbor infectious
C. parvum
oocysts and can serve as mechanical vectors of this pathogen.
Methane and Trichloroethylene Degradation by
Methylosinus trichosporium
OB3b Expressing Particulate Methane Monooxygenase
Authors: Lontoh Sonny , Semrau Jeremy D. 
Published: December 2019

ABSTRACT

Whole-cell assays of methane and trichloroethylene (TCE) consumption have been performed on
Methylosinus trichosporium
OB3b expressing particulate methane monooxygenase (pMMO). From these assays it is apparent that varying the growth concentration of copper causes a change in the kinetics of methane and TCE degradation. For
M. trichosporium
OB3b, increasing the copper growth concentration from 2.5 to 20 μM caused the maximal degradation rate of methane (
V
max
) to decrease from 300 to 82 nmol of methane/min/mg of protein. The methane concentration at half the maximal degradation rate (

K
s

) also decreased from 62 to 8.3 μM. The pseudo-first-order rate constant for methane,
V
max
/

K
s

, doubled from 4.9 × 10
−3
to 9.9 × 10
−3
liters/min/mg of protein, however, as the growth concentration of copper increased from 2.5 to 20 μM. TCE degradation by
M. trichosporium
OB3b was also examined with varying copper and formate concentrations.
M. trichosporium
OB3b grown with 2.5 μM copper was unable to degrade TCE in both the absence and presence of an exogenous source of reducing equivalents in the form of formate. Cells grown with 20 μM copper, however, were able to degrade TCE regardless of whether formate was provided. Without formate the
V
max
for TCE was 2.5 nmol/min/mg of protein, while providing formate increased the
V
max
to 4.1 nmol/min/mg of protein. The affinity for TCE also increased with increasing copper, as seen by a change in

K
s

from 36 to 7.9 μM.
V
max
/

K
s

for TCE degradation by pMMO also increased from 6.9 × 10
−5
to 5.2 × 10
−4
liters/min/mg of protein with the addition of formate. From these whole-cell studies it is apparent that the amount of copper available is critical in determining the oxidation of substrates in methanotrophs that are expressing only pMMO.
Potential of Unicellular Cyanobacteria from Saline Environments as Exopolysaccharide Producers
Authors: De Philippis Roberto , Margheri Maria Cristina , Materassi Riccardo , Vincenzini Massimo 
Published: December 2019

ABSTRACT

Fifteen
Cyanothece
strains isolated from saline environments have been characterized with regard to exopolysaccharide (EPS) production. The polymers contained six to eight monosaccharides, with one or two acidic sugars. In some EPS samples, the additional presence of acetyl, pyruvyl, and/or sulfate groups was also detected.
Expression of Psychrophilic Genes in Mesophilic Hosts: Assessment of the Folding State of a Recombinant α-Amylase
Authors: Feller Georges , Le Bussy Olivier , Gerday Charles 
Published: December 2019

ABSTRACT

α-Amylase from the antarctic psychrophile
Alteromonas haloplanktis
is synthesized at 0 ± 2°C by the wild strain. This heat-labile α-amylase folds correctly when overexpressed in
Escherichia coli
, providing the culture temperature is sufficiently low to avoid irreversible denaturation. In the described expression system, a compromise between enzyme stability and
E. coli
growth rate is reached at 18°C.