bluuebunny/crossref_metadata_2025_split huggingface dataset:
Phylogeny of the Defined Murine Microbiota: Altered Schaedler Flora
Authors: Dewhirst Floyd E. , Chien Chih-Ching , Paster Bruce J. , Ericson Rebecca L. , Orcutt Roger P. , Schauer David B. , Fox James G. 
Published: December 2019

ABSTRACT

The “altered Schaedler flora” (ASF) was developed for colonizing germfree rodents with a standardized microbiota. The purpose of this study was to identify each of the eight ASF strains by 16S rRNA sequence analysis. Three strains were previously identified as
Lactobacillus acidophilus
(strain ASF 360),
Lactobacillus salivarius
(strain ASF 361), and
Bacteroides distasonis
(strain ASF 519) based on phenotypic criteria. 16S rRNA analysis indicated that each of the strains differed from its presumptive identity. The 16S rRNA sequence of strain ASF 361 is essentially identical to the 16S rRNA sequences of the type strains of
Lactobacillus murinis
and
Lactobacillus animalis
(both isolated from mice), and all of these strains probably belong to a single species. Strain ASF 360 is a novel lactobacillus that clusters with
L. acidophilus
and
Lactobacillus lactis
. Strain ASF 519 falls into an unnamed genus containing [
Bacteroides
]
distasonis
, [
Bacteroides
]
merdae
, [
Bacteroides
]
forsythus
, and CDC group DF-3. This unnamed genus is in the
Cytophaga-Flavobacterium-Bacteroides
phylum and is most closely related to the genus
Porphyromonas
. The spiral-shaped strain, strain ASF 457, is in the
Flexistipes
phylum and exhibits sequence identity with rodent isolates of Robertson. The remaining four ASF strains, which are extremely oxygen-sensitive fusiform bacteria, group phylogenetically with the low-G+C-content gram-positive bacteria (
Firmicutes
,
Bacillus-Clostridium
group). ASF 356, ASF 492, and ASF 502 fall into
Clostridium
cluster XIV of Collins et al. Morphologically, ASF 492 resembles members of this cluster,
Roseburia cecicola
, and
Eubacterium plexicaudatum
. The 16S rRNA sequence of ASF 492 is identical to that of
E. plexicaudatum
. Since the type strain and other viable original isolates of
E. plexicaudatum
have been lost, strain ASF 492 is a candidate for a neotype strain. Strain ASF 500 branches deeply in the low-G+C-content gram-positive phylogenetic tree but is not closely related to any organisms whose 16S rRNA sequences are currently in the GenBank database. The 16S rRNA sequence information determined in the present study should allow rapid identification of ASF strains and should permit detailed analysis of the interactions of ASF organisms during development of intestinal disease in mice that are coinfected with a variety of pathogenic microorganisms.
Degradation of 3-Phenoxybenzoic Acid in Soil by
Pseudomonas pseudoalcaligenes
POB310(pPOB) and Two Modified
Pseudomonas
Strains
Authors: Halden Rolf U. , Tepp Sandra M. , Halden Barbara G. , Dwyer Daryl F. 
Published: December 2019

ABSTRACT

Pseudomonas pseudoalcaligenes
POB310(pPOB) and
Pseudomonas
sp. strains B13-D5(pD30.9) and B13-ST1(pPOB) were introduced into soil microcosms containing 3-phenoxybenzoic acid (3-POB) in order to evaluate and compare bacterial survival, degradation of 3-POB, and transfer of plasmids to a recipient bacterium. Strain POB310 was isolated for its ability to use 3-POB as a growth substrate; degradation is initiated by POB-dioxygenase, an enzyme encoded on pPOB. Strain B13-D5 contains pD30.9, a cloning vector harboring the genes encoding POB-dioxygenase; strain B13-ST1 contains pPOB. Degradation of 3-POB in soil by strain POB310 was incomplete, and bacterial densities decreased even under the most favorable conditions (100 ppm of 3-POB, supplementation with P and N, and soil water-holding capacity of 90%). Strains B13-D5 and B13-ST1 degraded 3-POB (10 to 100 ppm) to concentrations of <50 ppb with concomitant increases in density from 10
6
to 10
8
CFU/g (dry weight) of soil. Thus, in contrast to strain POB310, the modified strains had the following two features that are important for in situ bioremediation: survival in soil and growth concurrent with removal of an environmental contaminant. Strains B13-D5 and B13-ST1 also completely degraded 3-POB when the inoculum was only 30 CFU/g (dry weight) of soil. This suggests that in situ bioremediation may be effected, in some cases, with low densities of introduced bacteria. In pure culture, transfer of pPOB from strains POB310 and B13-ST1 to
Pseudomonas
sp. strain B13 occurred at frequencies of 5 × 10
−7
and 10
−1
transconjugant per donor, respectively. Transfer of pPOB from strain B13-ST1 to strain B13 was observed in autoclaved soil but not in nonautoclaved soil; formation of transconjugant bacteria was more rapid in soil containing clay and organic matter than in sandy soil. Transfer of pPOB from strain POB310 to strain B13 in soil was never observed.
Phenotypic and Genotypic Characterization of Non-Starter Lactic Acid Bacteria in Mature Cheddar Cheese
Authors: Fitzsimons N. A. , Cogan T. M. , Condon S. , Beresford T. 
Published: December 2019

ABSTRACT

Non-starter lactic acid bacteria were isolated from 14 premium-quality and 3 sensorially defective mature Irish Cheddar cheeses, obtained from six manufacturers. From countable plates of
Lactobacillus
-selective agar, 20 single isolated colonies were randomly picked per cheese. All 331 viable isolates were biochemically characterized as mesophilic (i.e., group II)
Lactobacillus
spp. Phenotypically, the isolates comprised 96.4%
L. paracasei
, 2.1%
L. plantarum
, 0.3%
L. curvatus
, 0.3%
L. brevis
, and 0.9% unidentified species. Randomly amplified polymorphic DNA (RAPD) analysis was used to rapidly identify the dominant strain groups in nine cheeses from three of the factories, and through clustering by the unweighted pair group method with arithmetic averages, an average of seven strains were found per cheese. In general, strains isolated from cheese produced at the same factory clustered together. The majority of isolates associated with premium-quality cheese grouped together and apart from clusters of strains from defective-quality cheese. No correlation was found between the isomer of lactate produced and RAPD profiles, although isolates which did not ferment ribose clustered together. The phenotypic and genotypic methods employed were validated with a selection of 31 type and reference strains of mesophilic
Lactobacillus
spp. commonly found in Cheddar cheese. RAPD analysis was found to be a useful and rapid method for identifying isolates to the species level. The low homology exhibited between RAPD banding profiles for cheese isolates and collection strains demonstrated the heterogeneity of the
L. paracasei
complex.
Detection of Infectious
Cryptosporidium parvum
Oocysts in Surface and Filter Backwash Water Samples by Immunomagnetic Separation and Integrated Cell Culture-PCR
Authors: Di Giovanni George D. , Hashemi F. Helen , Shaw Nancy J. , Abrams Felicia A. , LeChevallier Mark W. , Abbaszadegan Morteza 
Published: December 2019

ABSTRACT

A new strategy for the detection of infectious
Cryptosporidium parvum
oocysts in water samples, which combines immunomagnetic separation (IMS) for recovery of oocysts with in vitro cell culturing and PCR (CC-PCR), was field tested with a total of 122 raw source water samples and 121 filter backwash water grab samples obtained from 25 sites in the United States. In addition, samples were processed by Percoll-sucrose flotation and oocysts were detected by an immunofluorescence assay (IFA) as a baseline method. Samples of different water quality were seeded with viable
C. parvum
to evaluate oocyst recovery efficiencies and the performance of the CC-PCR protocol. Mean method oocyst recoveries, including concentration of seeded 10-liter samples, from raw water were 26.1% for IMS and 16.6% for flotation, while recoveries from seeded filter backwash water were 9.1 and 5.8%, respectively. There was full agreement between IFA oocyst counts of IMS-purified seeded samples and CC-PCR results. In natural samples, CC-PCR detected infectious
C. parvum
in 4.9% (6) of the raw water samples and 7.4% (9) of the filter backwash water samples, while IFA detected oocysts in 13.1% (16) of the raw water samples and 5.8% (7) of the filter backwash water samples. All CC-PCR products were confirmed by cloning and DNA sequence analysis and were greater than 98% homologous to the
C. parvum
KSU-1
hsp70
gene product. DNA sequence analysis also revealed reproducible nucleotide substitutions among the
hsp70
fragments, suggesting that several different strains of infectious
C. parvum
were detected.
Development of a System for Genetic Manipulation of
Bartonella bacilliformis
Authors: Battisti James M. , Minnick Michael F. 
Published: December 2019

ABSTRACT

Lack of a system for site-specific genetic manipulation has severely hindered studies on the molecular biology of all
Bartonella
species. We report the first site-specific mutagenesis and complementation for a
Bartonella
species. A highly transformable strain of
B. bacilliformis
, termed JB584, was isolated and found to exhibit a significant increase in transformation efficiency with the broad-host-range plasmid pBBR1MCS-2, relative to wild-type strains. Restriction analyses of genomic preparations with the methylation-sensitive restriction enzymes
Cla
I and
Stu
I suggest that strain JB584 possesses a
dcm
methylase mutation that contributes to its enhanced transformability. A suicide plasmid, pUB1, which contains a polylinker, a pMB1 replicon, and a
nptI
kanamycin resistance cassette, was constructed. An internal 508-bp fragment of the
B. bacilliformis
flagellin gene (
fla
) was cloned into pUB1 to generate pUB508, a
fla
-targeting suicide vector. Introduction of pUB508 into JB584 by electroporation generated eight Kan
r
clones of
B. bacilliformis
. Characterization of one of these strains, termed JB585, indicated that allelic exchange between pUB508 and
fla
had occurred. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and electron microscopy showed that synthesis of flagellin encoded by
fla
and secretion/assembly of flagella were abolished. Complementation of
fla
in
trans
was accomplished with a pBBR1MCS recombinant containing the entire wild-type
fla
gene (pBBRFLAG). These data conclusively show that inactivation of
fla
results in a bald, nonmotile phenotype and that pMB1 and REP replicons make suitable
B. bacilliformis
suicide and shuttle vectors, respectively. When used in conjunction with the highly transformable strain JB584, this system for site-specific genetic manipulation and complementation provides a new venue for studying the molecular biology of
B. bacilliformis
.
A Predictive Model That Describes the Effect of Prolonged Heating at 70 to 90°C and Subsequent Incubation at Refrigeration Temperatures on Growth from Spores and Toxigenesis by Nonproteolytic
Clostridium botulinum
in the Presence of Lysozyme
Authors: Fernández Pablo S. , Peck Michael W. 
Published: December 2019

ABSTRACT

Refrigerated processed foods of extended durability such as cook-chill and sous-vide foods rely on a minimal heat treatment at 70 to 95°C and then storage at a refrigeration temperature for safety and preservation. These foods are not sterile and are intended to have an extended shelf life, often up to 42 days. The principal microbiological hazard in foods of this type is growth of and toxin production by nonproteolytic
Clostridium botulinum
. Lysozyme has been shown to increase the measured heat resistance of nonproteolytic
C. botulinum
spores. However, the heat treatment guidelines for prevention of risk of botulism in these products have not taken into consideration the effect of lysozyme, which can be present in many foods. In order to assess the botulism hazard, the effect of heat treatments at 70, 75, 80, 85, and 90°C combined with refrigerated storage for up to 90 days on growth from 10
6
spores of nonproteolytic
C. botulinum
(types B, E, and F) in an anaerobic meat medium containing 2,400 U of lysozyme per ml (50 μg per ml) was studied. Provided that the storage temperature was no higher than 8°C, the following heat treatments each prevented growth and toxin production during 90 days; 70°C for ≥2,545 min, 75°C for ≥463 min, 80°C for ≥230 min, 85°C for ≥84 min, and 90°C for ≥33.5 min. A factorial experimental design allowed development of a predictive model that described the incubation time required before the first sample showed growth, as a function of heating temperature (70 to 90°C), period of heat treatment (up to 2,545 min), and incubation temperature (5 to 25°C). Predictions from the model provided a valid description of the data used to generate the model and agreed with observations made previously.
IS
1631
Occurrence in
Bradyrhizobium japonicum
Highly Reiterated Sequence-Possessing Strains with High Copy Numbers of Repeated Sequences RSα and RSβ
Authors: Isawa Tsuyoshi , Sameshima Reiko , Mitsui Hisayuki , Minamisawa Kiwamu 
Published: December 2019

ABSTRACT

From
Bradyrhizobium japonicum
highly reiterated sequence-possessing (HRS) strains indigenous to Niigata and Tokachi in Japan with high copy numbers of the repeated sequences RSα and RSβ (K. Minamisawa, T. Isawa, Y. Nakatsuka, and N. Ichikawa, Appl. Environ. Microbiol. 64:1845–1851, 1998), several insertion sequence (IS)-like elements were isolated by using the formation of DNA duplexes by denaturation and renaturation of total DNA, followed by treatment with S1 nuclease. Most of these sequences showed structural features of bacterial IS elements, terminal inverted repeats, and homology with known IS elements and transposase genes. HRS and non-HRS strains of
B. japonicum
differed markedly in the profiles obtained after hybridization with all the elements tested. In particular, HRS strains of
B. japonicum
contained many copies of IS
1631
, whereas non-HRS strains completely lacked this element. This association remained true even when many field isolates of
B. japonicum
were examined. Consequently, IS
1631
occurrence was well correlated with
B. japonicum
HRS strains possessing high copy numbers of the repeated sequence RSα or RSβ. DNA sequence analysis indicated that IS
1631
is 2,712 bp long. In addition, IS
1631
belongs to the IS
21
family, as evidenced by its two open reading frames, which encode putative proteins homologous to IstA and IstB of IS
21
, and its terminal inverted repeat sequences with multiple short repeats.
Gene Sequence and Properties of an
s
-Triazine Ring-Cleavage Enzyme from
Pseudomonas
sp. Strain NRRLB-12227
Authors: Karns Jeffrey S. 
Published: December 2019

ABSTRACT

Pesticides based on the
s
-triazine ring structure are widely used in cultivation of food crops. Cleavage of the
s
-triazine ring is an important step in the mineralization of
s
-triazine compounds and hence in their complete removal from the environment. Cyanuric acid amidohydrolase cleaves cyanuric acid (2,4,6-trihydroxy-
s
-triazine), which yields carbon dioxide and biuret; the biuret is subject to further metabolism, which yields CO
2
and ammonia. The
trzD
gene encoding cyanuric acid amidohydrolase was cloned into pMMB277 from
Pseudomonas
sp. strain NRRLB-12227, a strain that is capable of utilizing
s
-triazines as nitrogen sources. Hydrolysis of cyanuric acid was detected in crude extracts of
Escherichia coli
containing the cloned gene by monitoring the disappearance of cyanuric acid and the appearance of biuret by high-performance liquid chromatography (HPLC). DEAE and hydrophobic interaction HPLC were used to purify cyanuric acid amidohydrolase to homogeneity, and a spectrophotometric assay for the purified enzyme was developed. The purified enzyme had an apparent

K
m

of 0.05 mM for cyanuric acid at pH 8.0. The enzyme did not cleave any other
s
-triazine or hydroxypyrimidine compound, although barbituric acid (2,4,6-trihydroxypyrimidine) was found to be a strong competitive inhibitor. Neither the nucleotide sequence of
trzD
nor the amino acid sequence of the gene product exhibited a significant level of similarity to any known gene or protein.
The Viable-but-Nonculturable Condition Is Induced by Copper in
Agrobacterium tumefaciens
and
Rhizobium leguminosarum
Authors: Alexander Emily , Pham Dat , Steck Todd R. 
Published: December 2019

ABSTRACT

Many bacteria respond to changes in environmental conditions by entering the viable-but-nonculturable state. We have determined that copper can induce nutrient-starved
Agrobacterium tumefaciens
and
Rhizobium leguminosarum
cells to become viable but nonculturable. This is the first report of a chemical inducer of this condition.
Role of
N
γ-Acetyldiaminobutyrate as an Enzyme Stabilizer and an Intermediate in the Biosynthesis of Hydroxyectoine
Authors: Cánovas David , Borges Nuno , Vargas Carmen , Ventosa Antonio , Nieto Joaquín J. , Santos Helena 
Published: December 2019

ABSTRACT

Strain CHR63 is a salt-sensitive mutant of the moderately halophilic wild-type strain
Halomonas elongata
DSM 3043 that is affected in the ectoine synthase gene (
ectC
). This strain accumulates large amounts of
N
γ-acetyldiaminobutyrate (NADA), the precursor of ectoine (D. Cánovas, C. Vargas, F. Iglesias-Guerra, L. N. Csonka, D. Rhodes, A. Ventosa, and J. J. Nieto, J. Biol. Chem. 272:25794–25801, 1997). Hydroxyectoine, ectoine, and glucosylglycerate were also identified by nuclear magnetic resonance (NMR) as cytoplasmic organic solutes in this mutant. Accumulation of NADA, hydroxyectoine, and ectoine was osmoregulated, whereas the levels of glucosylglycerate decreased at higher salinities. The effect of the growth stage on the accumulation of solutes was also investigated. NADA was purified from strain CHR63 and was shown to protect the thermolabile enzyme rabbit muscle lactate dehydrogenase against thermal inactivation. The stabilizing effect of NADA was greater than the stabilizing effect of ectoine or potassium diaminobutyrate. A
1
H NMR analysis of the solutes accumulated by the wild-type strain and mutants CHR62 (
ectA
::Tn
1732
) and CHR63 (
ectC
::Tn
1732
) indicated that
H. elongata
can synthesize hydroxyectoine by two different pathways—directly from ectoine or via an alternative pathway that converts NADA into hydroxyectoine without the involvement of ectoine.
Amplified Fragment Length Polymorphism Fingerprinting of
Pseudomonas
Strains from a Poultry Processing Plant
Authors: Geornaras Ifigenia , Kunene Nokuthula F. , von Holy Alexander , Hastings John W. 
Published: December 2019

ABSTRACT

Molecular typing has been used previously to identify and trace dissemination of pathogenic and spoilage bacteria associated with food processing. Amplified fragment length polymorphism (AFLP) is a novel DNA fingerprinting technique which is considered highly reproducible and has high discriminatory power. This technique was used to fingerprint 88
Pseudomonas fluorescens
and
Pseudomonas putida
strains that were previously isolated from plate counts of carcasses at six processing stages and various equipment surfaces and environmental sources of a poultry abattoir. Clustering of the AFLP patterns revealed a high level of diversity among the strains. Six clusters (clusters I through VI) were delineated at an arbitrary Dice coefficient level of 0.65; clusters III (31 strains) and IV (28 strains) were the largest clusters. More than one-half (52.3%) of the strains obtained from carcass samples, which may have represented the resident carcass population, grouped together in cluster III. By contrast, 43.2% of the strains from most of the equipment surfaces and environmental sources grouped together in cluster IV. In most cases, the clusters in which carcass strains from processing stages grouped corresponded to the clusters in which strains from the associated equipment surfaces and/or environmental sources were found. This provided evidence that there was cross-contamination between carcasses and the abattoir environment at the DNA level. The AFLP data also showed that strains were being disseminated from the beginning to the end of the poultry processing operation, since many strains associated with carcasses at the packaging stage were members of the same clusters as strains obtained from carcasses after the defeathering stage.
Development and Use of a Reverse Transcription-PCR Assay To Study Expression of
Tri5
by
Fusarium
Species In Vitro and In Planta
Authors: Doohan F. M. , Weston G. , Rezanoor H. N. , Parry D. W. , Nicholson P. 
Published: December 2019

ABSTRACT

The
Tri5
gene encodes trichodiene synthase, which catalyzes the first reaction in the trichothecene biosynthetic pathway. In vitro, a direct relationship was observed between
Tri5
expression and the increase in deoxynivalenol production over time. We developed a reverse transcription (RT)-PCR assay to quantify
Tri5
gene expression in trichothecene-producing strains of
Fusarium
species. We observed an increase in
Tri5
expression following treatment of
Fusarium culmorum
with fungicides, and we also observed an inverse relationship between
Tri5
expression and biomass, as measured by β-
d
-glucuronidase activity, during colonization of wheat (cv. Avalon) seedlings by
F. culmorum
. RT-PCR analysis also showed that for ears of wheat cv. Avalon inoculated with
F. culmorum
, there were different levels of
Tri5
expression in grain and chaff at later growth stages. We used the
Tri5
-specific primers to develop a PCR assay to detect trichothecene-producing
Fusarium
species in infected plant material.
Cloning of the Gene Encoding a Novel Thermostable α-Galactosidase from
Thermus brockianus
ITI360
Authors: Fridjonsson Olafur , Watzlawick Hildegard , Gehweiler Axel , Rohrhirsch Thilo , Mattes Ralf 
Published: December 2019

ABSTRACT

An α-galactosidase gene from
Thermus brockianus
ITI360 was cloned, sequenced, and expressed in
Escherichia coli
, and the recombinant protein was purified. The gene, designated
agaT
, codes for a 476-residue polypeptide with a calculated molecular mass of 53,810 Da. The native structure of the recombinant enzyme (AgaT) was estimated to be a tetramer. AgaT displays amino acid sequence similarity to the α-galactosidases of
Thermotoga neapolitana
and
Thermotoga maritima
and a low-level sequence similarity to α-galactosidases of family 36 in the classification of glycosyl hydrolases. The enzyme is thermostable, with a temperature optimum of activity at 93°C with
para
-nitrophenyl-α-galactopyranoside as a substrate. Half-lives of inactivation at 92 and 80°C are 100 min and 17 h, respectively. The pH optimum is between 5.5 and 6.5. The enzyme displayed high affinity for oligomeric substrates. The

K
m

s for melibiose and raffinose at 80°C were determined as 4.1 and 11.0 mM, respectively. The α-galactosidase gene in
T. brockianus
ITI360 was inactivated by integrational mutagenesis. Consequently, no α-galactosidase activity was detectable in crude extracts of the mutant strain, and it was unable to use melibiose or raffinose as a single carbohydrate source.