bluuebunny/crossref_metadata_2025_split huggingface dataset:
Monitoring Bacterial Communities in Raw Milk and Cheese by Culture-Dependent and -Independent 16S rRNA Gene-Based Analyses
Authors: Delbès Céline , Ali-Mandjee Leila , Montel Marie-Christine 
Published: January 2007

ABSTRACT

The diversity and dynamics of bacterial populations in Saint-Nectaire, a raw-milk, semihard cheese, were investigated using a dual culture-dependent and direct molecular approach combining single-strand conformation polymorphism (SSCP) fingerprinting and sequencing of 16S rRNA genes. The dominant clones, among 125 16S rRNA genes isolated from milk, belonged to members of the
Firmicutes
(58% of the total clones) affiliated mainly with the orders
Clostridiales
and the
Lactobacillales
, followed by the phyla
Proteobacteria
(21.6%),
Actinobacteria
(16.8%), and
Bacteroidetes
(4%). Sequencing the 16S rRNA genes of 126 milk isolates collected from four culture media revealed the presence of 36 different species showing a wider diversity in the
Gammaproteobacteria
phylum and
Staphylococcus
genus than that found among clones. In cheese, a total of 21 species were obtained from 170 isolates, with dominant species belonging to the
Lactobacillales
and subdominant species affiliated with the
Actinobacteria
,
Bacteroidetes
(
Chryseobacterium
sp.), or
Gammaproteobacteria
(
Stenotrophomonas
sp.). Fingerprinting DNA isolated from milk by SSCP analysis yielded complex patterns, whereas analyzing DNA isolated from cheese resulted in patterns composed of a single peak which corresponded to that of lactic acid bacteria. SSCP fingerprinting of mixtures of all colonies harvested from plate count agar supplemented with crystal violet and vancomycin showed good potential for monitoring the subdominant
Proteobacteria
and
Bacteroidetes
(
Flavobacteria
) organisms in milk and cheese. Likewise, analyzing culturable subcommunities from cheese-ripening bacterial medium permitted assessment of the diversity of halotolerant
Actinobacteria
and
Staphylococcus
organisms. Direct and culture-dependent approaches produced complementary information, thus generating a more accurate view of milk and cheese microbial ecology.
Development of a New, Combined Rapid Method Using Phage and PCR for Detection and Identification of Viable
Mycobacterium paratuberculosis
Bacteria within 48 Hours
Authors: Stanley Emma C. , Mole Richard J. , Smith Rebecca J. , Glenn Sarah M. , Barer Michael R. , McGowan Michael , Rees Catherine E. D. 
Published: January 2007

ABSTRACT

The
FASTPlaque
TB assay is an established diagnostic aid for the rapid detection of
Mycobacterium tuberculosis
from human sputum samples. Using the
FASTPlaque
TB assay reagents, viable
Mycobacterium avium
subsp.
paratuberculosis
cells were detected as phage plaques in just 24 h. The bacteriophage used does not infect
M. avium
subsp.
paratuberculosis
alone, so to add specificity to this assay, a PCR-based identification method was introduced to amplify
M. avium
subsp.
paratuberculosis
-specific sequences from the DNA of the mycobacterial cell detected by the phage. To give further diagnostic information, a multiplex PCR method was developed to allow simultaneous amplification of either
M. avium
subsp.
paratuberculosis
or
M. tuberculosis
complex-specific sequences from plaque samples. Combining the plaque PCR technique with the phage-based detection assay allowed the rapid and specific detection of viable
M. avium
subsp.
paratuberculosis
in milk samples in just 48 h.
Piezotolerant Small-Colony Variants with Increased Thermotolerance, Antibiotic Susceptibility, and Low Invasiveness in a Clonal
Staphylococcus aureus
Population
Authors: Karatzas Kimon A. G. , Zervos Angelos , Tassou Chrysoula C. , Mallidis Costas G. , Humphrey Tom J. 
Published: January 2007

ABSTRACT

Following a pressure treatment of a clonal
Staphylococcus aureus
culture with 400 MPa for 30 min, piezotolerant variants were isolated. Among 21 randomly selected survivors, 9 were piezotolerant and all formed small colonies on several agar media. The majority of the isolates showed increased thermotolerance, impaired growth, and reduced antibiotic resistance compared to the wild type. However, several nonpiezotolerant isolates also demonstrated impaired growth and the small-colony phenotype. In agglutination tests for the detection of protein A and fibrinogen, the piezotolerant variants showed weaker agglutination reactions than the wild type and the other isolates. All variants also showed defective production of the typical
S. aureus
golden color, a characteristic which has previously been linked with virulence. They were also less able to invade intestinal epithelial cells than the wild type. These
S. aureus
variants showed phenotypic similarities to previously isolated
Listeria monocytogenes
piezotolerant mutants that contained mutations in
ctsR
. Because of these similarities, possible alterations in the
ctsR
hypermutable regions of the
S. aureus
variants were investigated through amplified fragment length polymorphism analysis. No mutations were identified, and subsequently we sequenced the
ctsR
and
hrcA
genes of three representative variants, finding no mutations. This work demonstrates that
S. aureus
probably possesses a strategy resulting in an abundance of multiple-stress-resistant variants within clonal populations. This strategy, however, seems to involve genes and regulatory mechanisms different from those previously reported for
L. monocytogenes
. We are in the process of identifying these mechanisms.
Campylobacter insulaenigrae
Isolates from Northern Elephant Seals (
Mirounga angustirostris
) in California
Authors: Stoddard Robyn A. , Miller William G. , Foley Janet E. , Lawrence Judy , Gulland Frances M. D. , Conrad Patricia A. , Byrne Barbara A. 
Published: January 2007

ABSTRACT

There are only two reports in the literature demonstrating the presence of
Campylobacter
spp. in marine mammals. One report describes the isolation of a new species,
Campylobacter insulaenigrae
sp. nov., from three harbor seals (
Phoca vitulina
) and a harbor porpoise (
Phocoena phocoena
) in Scotland, and the other describes the isolation of
Campylobacter jejuni
,
Campylobacter lari
, and an unknown
Campylobacter
species from northern elephant seals (
Mirounga angustirostris
) in California. In this study, 72 presumptive
C. lari
and unknown
Campylobacter
species strains were characterized using standard phenotypic methods, 16S rRNA PCR, and multilocus sequence typing (MLST). Phenotypic characterization of these isolates showed them to be variable in their ability to grow either at 42°C or on agar containing 1% glycine and in their sensitivity to nalidixic acid and cephalothin. Based on both 16S rRNA PCR and MLST, all but 1 of the 72 isolates were
C. insulaenigrae
, with one isolate being similar to but distinct from both
Campylobacter upsaliensis
and
Campylobacter helveticus
. Phylogenetic analysis identified two
C. insulaenigrae
clades: the primary clade, containing exclusively California strains, and a secondary clade, containing some California strains and all of the original Scottish strains. This study demonstrates the inability of phenotypic characterization to correctly identify all
Campylobacter
species and emphasizes the importance of molecular characterization via 16S rRNA sequence analysis or MLST for the identification of
Campylobacter
isolates from marine mammals.
Factors Affecting Survival of Bacteriophage on Tomato Leaf Surfaces
Authors: Iriarte F. B. , Balogh B. , Momol M. T. , Smith L. M. , Wilson M. , Jones J. B. 
Published: January 2007

ABSTRACT
The ability of bacteriophage to persist in the phyllosphere for extended periods is limited by many factors, including sunlight irradiation, especially in the UV zone, temperature, desiccation, and exposure to copper bactericides. The effects of these factors on persistence of phage and formulated phage (phage mixed with skim milk) were evaluated. In field studies, copper caused significant phage reduction if applied on the day of phage application but not if applied 4 or 7 days in advance. Sunlight UV was evaluated for detrimental effects on phage survival on tomato foliage in the field. Phage was applied in the early morning, midmorning, early afternoon, and late evening, while UVA plus UVB irradiation and phage populations were monitored. The intensity of UV irradiation positively correlated with phage population decline. The protective formulation reduced the UV effect. In order to demonstrate direct effects of UV, phage suspensions were exposed to UV irradiation and assayed for effectiveness against bacterial spot of tomato. UV significantly reduced phage ability to control bacterial spot. Ambient temperature had a pronounced effect on nonformulated phage but not on formulated phages. The effects of desiccation and fluorescent light illumination on phage were investigated. Desiccation caused a significant but only slight reduction in phage populations after 60 days, whereas fluorescent light eliminated phages within 2 weeks. The protective formulation eliminated the reduction caused by both of these factors. Phage persistence was dramatically affected by UV, while the other factors had less pronounced effects. Formulated phage reduced deleterious effects of the studied environmental factors.
Cloning and Characterization of a Novel β-Transaminase fromMesorhizobiumsp. Strain LUK: a New Biocatalyst for the Synthesis of Enantiomerically Pure β-Amino Acids
Authors: Kim Juhan , Kyung Dohyun , Yun Hyungdon , Cho Byung-Kwan , Seo Joo-Hyun , Cha Minho , Kim Byung-Gee 
Published: January 2007

ABSTRACTA novel β-transaminase gene was cloned fromMesorhizobiumsp. strain LUK. By using N-terminal sequence and an internal protein sequence, a digoxigenin-labeled probe was made for nonradioactive hybridization, and a 2.5-kb gene fragment was obtained by colony hybridization of a cosmid library. Through Southern blotting and sequence analysis of the selected cosmid clone, the structural gene of the enzyme (1,335 bp) was identified, which encodes a protein of 47,244 Da with a theoretical pI of 6.2. The deduced amino acid sequence of the β-transaminase showed the highest sequence similarity with glutamate-1-semialdehyde aminomutase of transaminase subgroup II. The β-transaminase showed higher activities towardd-β-aminocarboxylic acids such as 3-aminobutyric acid, 3-amino-5-methylhexanoic acid, and 3-amino-3-phenylpropionic acid. The β-transaminase has an unusually broad specificity for amino acceptors such as pyruvate and α-ketoglutarate/oxaloacetate. The enantioselectivity of the enzyme suggested that the recognition mode of β-aminocarboxylic acids in the active site is reversed relative to that of α-amino acids. After comparison of its primary structure with transaminase subgroup II enzymes, it was proposed that R43 interacts with the carboxylate group of the β-aminocarboxylic acids and the carboxylate group on the side chain of dicarboxylic α-keto acids such as α-ketoglutarate and oxaloacetate. R404 is another conserved residue, which interacts with the α-carboxylate group of the α-amino acids and α-keto acids. The β-transaminase was used for the asymmetric synthesis of enantiomerically pure β-aminocarboxylic acids. (3S)-Amino-3-phenylpropionic acid was produced from the ketocarboxylic acid ester substrate by coupled reaction with a lipase using 3-aminobutyric acid as amino donor.
Diagnostic Real-Time PCR Assays for the Detection of Emetic
Bacillus cereus
Strains in Foods and Recent Food-Borne Outbreaks
Authors: Fricker Martina , Messelhäußer Ute , Busch Ulrich , Scherer Siegfried , Ehling-Schulz Monika 
Published: January 2007

ABSTRACT

Cereulide-producing
Bacillus cereus
can cause an emetic type of food-borne disease that mimics the symptoms provoked by
Staphylococcus aureus
. Based on the recently discovered genetic background for cereulide formation, a novel 5′ nuclease (TaqMan) real-time PCR assay was developed to provide a rapid and sensitive method for the specific detection of emetic
B. cereus
in food. The TaqMan assay includes an internal amplification control and primers and a probe designed to target a highly specific part of the cereulide synthetase genes. Additionally, a specific SYBR green I assay was developed and extended to create a duplex SYBR green I assay for the one-step identification and discrimination of the two emesis-causing food pathogens
B. cereus
and
S. aureus
. The inclusivity and exclusivity of the assay were assessed using a panel of 100 strains, including 23 emetic
B. cereus
and 14
S. aureus
strains. Different methods for DNA isolation from artificially contaminated foods were evaluated, and established real-time assays were used to analyze two recent emetic food poisonings in southern Germany. One of the food-borne outbreaks included 17 children visiting a day care center who vomited after consuming a reheated rice dish, collapsed, and were hospitalized; the other case concerned a single food-poisoning incident occurring after consumption of cauliflower. Within 2 h, the etiological agent of these food poisonings was identified as emetic
B. cereus
by using the real-time PCR assay.
Zooxanthellae Harvested by Ciliates Associated with Brown Band Syndrome of Corals Remain Photosynthetically Competent
Authors: Ulstrup Karin E. , Kühl Michael , Bourne David G. 
Published: January 2007

ABSTRACT

Brown band syndrome is a new coral affliction characterized by a local accumulation of yet-unidentified ciliates migrating as a band along the branches of coral colonies. In the current study, morphologically intact zooxanthellae (=
Symbiodinium
) were observed in great numbers inside the ciliates (>50 dinoflagellates per ciliate). Microscale oxygen measurements and variable chlorophyll
a
fluorescence analysis along with microscopic observations demonstrated that zooxanthellae within the ciliates are photosynthetically competent and do not become compromised during the progression of the brown band zone. Zooxanthellae showed similar trends in light acclimation in a comparison of rapid light curve and steady-state light curve measures of variable chlorophyll
a
fluorescence. Extended light exposure of steady-state light curves resulted in higher quantum yields of photosystem II. The brown band tissue exhibited higher photosynthetically active radiation absorptivity, indicating more efficient light absorption due to a higher density of zooxanthellae in the ciliate-dominated zone. This caused relatively higher gross photosynthesis rates in the zone with zooxanthella-containing ciliates compared to healthy coral tissue. The observation of photosynthetically active intracellular zooxanthellae in the ciliates suggests that the latter can benefit from photosynthates produced by ingested zooxanthellae and from photosynthetic oxygen production that alleviates diffusion limitation of oxic respiration in the densely populated brown band tissue. It remains to be shown whether the zooxanthellae form a stable symbiotic association with the ciliate or are engulfed incidentally during grazing on coral tissue and then maintained as active inside the ciliate for a period before being digested and replaced by new zooxanthellae.
Weak Activity of Haloalkane Dehalogenase LinB with 1,2,3-Trichloropropane Revealed by X-Ray Crystallography and Microcalorimetry
Authors: Monincová Marta , Prokop Zbyněk , Vévodová Jitka , Nagata Yuji , Damborský Jiří 
Published: January 2007

ABSTRACT

1,2,3-Trichloropropane (TCP) is a highly toxic and recalcitrant compound. Haloalkane dehalogenases are bacterial enzymes that catalyze the cleavage of a carbon-halogen bond in a wide range of organic halogenated compounds. Haloalkane dehalogenase LinB from
Sphingobium japonicum
UT26 has, for a long time, been considered inactive with TCP, since the reaction cannot be easily detected by conventional analytical methods. Here we demonstrate detection of the weak activity (
k
cat
= 0.005 s
−1
) of LinB with TCP using X-ray crystallography and microcalorimetry. This observation makes LinB a useful starting material for the development of a new biocatalyst toward TCP by protein engineering. Microcalorimetry is proposed to be a universal method for the detection of weak enzymatic activities. Detection of these activities is becoming increasingly important for engineering novel biocatalysts using the scaffolds of proteins with promiscuous activities.
Construction of an
Escherichia coli
K-12 Mutant for Homoethanologenic Fermentation of Glucose or Xylose without Foreign Genes
Authors: Kim Youngnyun , Ingram L. O. , Shanmugam K. T. 
Published: January 2007

ABSTRACT

Conversion of lignocellulosic feedstocks to ethanol requires microorganisms that effectively ferment both hexose and pentose sugars. Towards this goal, recombinant organisms have been developed in which heterologous genes were added to platform organisms such as
Saccharomyces cerevisiae
,
Zymomonas mobilis
, and
Escherichia coli
. Using a novel approach that relies only on native enzymes, we have developed a homoethanologenic alternative,
Escherichia coli
strain SE2378. This mutant ferments glucose or xylose to ethanol with a yield of 82% under anaerobic conditions. An essential mutation in this mutant was mapped within the
pdh
operon (
pdhR aceEF lpd
), which encodes components of the pyruvate dehydrogenase complex. Anaerobic ethanol production by this mutant is apparently the result of a novel pathway that combines the activities of pyruvate dehydrogenase (typically active during aerobic, oxidative metabolism) with the fermentative alcohol dehydrogenase.
Antisense-RNA-Mediated Decreased Synthesis of Small, Acid-Soluble Spore Proteins Leads to Decreased Resistance of
Clostridium perfringens
Spores to Moist Heat and UV Radiation
Authors: Raju Deepa , Setlow Peter , Sarker Mahfuzur R. 
Published: January 2007

ABSTRACT

Previous work has suggested that a group of α/β-type small, acid-soluble spore proteins (SASP) is involved in the resistance of
Clostridium perfringens
spores to moist heat. However, this suggestion is based on the analysis of
C. perfringens
spores lacking only one of the three genes encoding α/β-type SASP in this organism. We have now used antisense RNA to decrease levels of α/β-type SASP in
C. perfringens
spores by ∼90%. These spores had significantly reduced resistance to both moist heat and UV radiation but not to dry heat. These results clearly demonstrate the important role of α/β-type SASP in the resistance of
C. perfringens
spores.
The Iron-Sulfur Cluster Proteins Isa1 and Isa2 Are Required for the Function but Not for the De Novo Synthesis of the Fe/S Clusters of Biotin Synthase in
Saccharomyces cerevisiae
Authors: Mühlenhoff Ulrich , Gerl Mathias J. , Flauger Birgit , Pirner Heike M. , Balser Sandra , Richhardt Nadine , Lill Roland , Stolz Jürgen 
Published: January 2007

ABSTRACT

The yeast
Saccharomyces cerevisiae
is able to use some biotin precursors for biotin biosynthesis. Insertion of a sulfur atom into desthiobiotin, the final step in the biosynthetic pathway, is catalyzed by biotin synthase (Bio2). This mitochondrial protein contains two iron-sulfur (Fe/S) clusters that catalyze the reaction and are thought to act as a sulfur donor. To identify new components of biotin metabolism, we performed a genetic screen and found that Isa2, a mitochondrial protein involved in the formation of Fe/S proteins, is necessary for the conversion of desthiobiotin to biotin. Depletion of Isa2 or the related Isa1, however, did not prevent the de novo synthesis of any of the two Fe/S centers of Bio2. In contrast, Fe/S cluster assembly on Bio2 strongly depended on the Isu1 and Isu2 proteins. Both
isa
mutants contained low levels of Bio2. This phenotype was also found in other mutants impaired in mitochondrial Fe/S protein assembly and in wild-type cells grown under iron limitation. Low Bio2 levels, however, did not cause the inability of
isa
mutants to utilize desthiobiotin, since this defect was not cured by overexpression of
BIO2
. Thus, the Isa proteins are crucial for the in vivo function of biotin synthase but not for the de novo synthesis of its Fe/S clusters. Our data demonstrate that the Isa proteins are essential for the catalytic activity of Bio2 in vivo.
Novel Method for Preparing Spheroplasts from Cells with an Internal Cellulosic Cell Wall
Authors: Kwok Alvin C. M. , Mak Carmen C. M. , Wong Francis T. W. , Wong Joseph T. Y. 
Published: January 2007

ABSTRACTProtoplast and spheroplast preparations allow the transfer of macromolecules into cells and provide the basis for the generation of engineered organisms.Crypthecodinium cohniicells harvested from polyethylene glycol-containing agar plates possessed significantly lower levels of cellulose in their cortical layers, which facilitated the delivery of fluorescence-labeled oligonucleotides into these cells.
In Vivo and In Vitro Anaerobic Mating in
Candida albicans
Authors: Dumitru Raluca , Navarathna Dhammika H. M. L. P. , Semighini Camile P. , Elowsky Christian G. , Dumitru Razvan V. , Dignard Daniel , Whiteway Malcolm , Atkin Audrey L. , Nickerson Kenneth W. 
Published: January 2007

ABSTRACT

Candida albicans
cells of opposite mating types are thought to conjugate during infection in mammalian hosts, but paradoxically, the mating-competent opaque state is not stable at mammalian body temperatures. We found that anaerobic conditions stabilize the opaque state at 37°C, block production of farnesol, and permit in vitro mating at 37°C at efficiencies of up to 84%. Aerobically, farnesol prevents mating because it kills the opaque cells necessary for mating, and as a corollary, farnesol production is turned off in opaque cells. These in vitro observations suggest that naturally anaerobic sites, such as the efficiently colonized gastrointestinal (GI) tract, could serve as niches for
C. albicans
mating. In a direct test of mating in the mouse GI tract, prototrophic cells were obtained from auxotrophic parent cells, confirming that mating will occur in this organ. These cells were true mating products because they were tetraploid, mononuclear, and prototrophic, and they contained the heterologous
hisG
marker from one of the parental strains.
Following Temperature Stress, Export of Heat Shock mRNA Occurs Efficiently in Cells with Mutations in Genes Normally Important for mRNA Export
Authors: Rollenhagen Christiane , Hodge Christine A. , Cole Charles N. 
Published: January 2007

ABSTRACT

Heat shock leads to accumulation of polyadenylated RNA in nuclei of
Saccharomyces cerevisiae
cells, transcriptional induction of heat shock genes, and efficient export of polyadenylated heat shock mRNAs. These studies were conducted to examine the requirements for export of mRNA following heat shock. We used in situ hybridization to detect
SSA4
mRNA (encoding Hsp70) and flow cytometry to measure the amount of Ssa4p-green fluorescent protein (GFP) produced following heat shock. Npl3p and Yra1p are mRNA-binding proteins recruited to nascent mRNAs and are essential for proper mRNA biogenesis and export. Heat shock mRNA was exported efficiently in temperature-sensitive
npl3
,
yra1
, and
npl3 yra1
mutant strains. Nevertheless, Yra1p was recruited to heat shock mRNA, as were Nab2p and Npl3p. Interestingly, Yra1p was not recruited to heat shock mRNA in
yra1
-
1
cells, suggesting that Npl3p is required for recruitment of Yra1p. The THO complex, which functions in transcription elongation and in recruitment of Yra1p, was not required for heat shock mRNA export, although normal mRNA export is impaired in growing cells lacking THO complex proteins. Taken together, these studies indicate that export following heat shock depends upon fewer factors than does mRNA export in growing cells. Furthermore, even though some mRNA-binding proteins are dispensable for efficient export of heat shock mRNA, those that are present in nuclei of heat shocked cells were recruited to heat shock mRNA.