bluuebunny/crossref_metadata_2025 huggingface dataset:
The magnetic resonance imaging subset of the mngu0 articulatory corpus
Authors: Steiner Ingmar , Richmond Korin , Marshall Ian , Gray Calum D.
Published: January 2012

This paper announces the availability of the magnetic resonance imaging (MRI) subset of the mngu0 corpus, a collection of articulatory speech data from one speaker containing different modalities. This subset comprises volumetric MRI scans of the speaker’s vocal tract during sustained production of vowels and consonants, as well as dynamic mid-sagittal scans of repetitive consonant–vowel (CV) syllable production. For reference, high-quality acoustic recordings of the speech material are also available. The raw data are made freely available for research purposes.
Site-Specific Recombination Strategies for Engineering Actinomycete Genomes
Authors: Herrmann Simone , Siegl Theresa , Luzhetska Marta , Petzke Lutz , Jilg Caroline , Welle Elisabeth , Erb Annette , Leadlay Peter F. , Bechthold Andreas , Luzhetskyy Andriy
Published: January 2012

The feasibility of using technologies based on site-specific recombination in actinomycetes was shown several years ago. Despite their huge potential, these technologies mostly have been used for simple marker removal from a chromosome. In this paper, we present different site-specific recombination strategies for genome engineering in several actinomycetes belonging to the genera
Streptomyces
,
Micromonospora
, and
Saccharothrix
. Two different systems based on Cre/
loxP
and Dre/
rox
have been utilized for numerous applications. The activity of the Cre recombinase on the heterospecific
loxLE
and
loxRE
sites was similar to its activity on wild-type
loxP
sites. Moreover, an apramycin resistance marker flanked by the
loxLERE
sites was eliminated from the
Streptomyces coelicolor
M145 genome at a surprisingly high frequency (80%) compared to other bacteria. A synthetic gene encoding the Dre recombinase was constructed and successfully expressed in actinomycetes. We developed a marker-free expression method based on the combination of phage integration systems and site-specific recombinases. The Cre recombinase has been used in the deletion of huge genomic regions, including the phenalinolactone, monensin, and lipomycin biosynthetic gene clusters from
Streptomyces
sp. strain Tü6071,
Streptomyces cinnamonensis
A519, and
Streptomyces aureofaciens
Tü117, respectively. Finally, we also demonstrated the site-specific integration of plasmid and cosmid DNA into the chromosome of actinomycetes catalyzed by the Cre recombinase. We anticipate that the strategies presented here will be used extensively to study the genetics of actinomycetes.
Biodiversity in Oscypek, a Traditional Polish Cheese, Determined by Culture-Dependent and -Independent Approaches
Authors: Alegría Ángel , Szczesny Pawel , Mayo Baltasar , Bardowski Jacek , Kowalczyk Magdalena
Published: January 2012

Oscypek is a traditional Polish scalded-smoked cheese, with a protected-designation-of-origin (PDO) status, manufactured from raw sheep's milk without starter cultures in the Tatra Mountains region of Poland. This study was undertaken in order to gain insight into the microbiota that develops and evolves during the manufacture and ripening stages of Oscypek. To this end, we made use of both culturing and the culture-independent methods of PCR followed by denaturing gradient gel electrophoresis (PCR-DGGE) and pyrosequencing of 16S rRNA gene amplicons. The culture-dependent technique and PCR-DGGE fingerprinting detected the predominant microorganisms in traditional Oscypek, whereas the next-generation sequencing technique (454 pyrosequencing) revealed greater bacterial diversity. Besides members of the most abundant bacterial genera in dairy products, e.g.,
Lactococcus
,
Lactobacillus
,
Leuconostoc
,
Streptococcus
, and
Enterococcus
, identified by all three methods, other, subdominant bacteria belonging to the families
Bifidobacteriaceae
and
Moraxellaceae
(mostly
Enhydrobacter
), as well as various minor bacteria, were identified by pyrosequencing. The presence of bifidobacterial sequences in a cheese system is reported for the first time. In addition to bacteria, a great diversity of yeast species was demonstrated in Oscypek by the PCR-DGGE method. Culturing methods enabled the determination of a number of viable microorganisms from different microbial groups and their isolation for potential future applications in specific cheese starter cultures.
Orally Administered Thermostable
N
-Acyl Homoserine Lactonase from Bacillus sp. Strain AI96 Attenuates Aeromonas hydrophila Infection in Zebrafish
Authors: Cao Yanan , He Suxu , Zhou Zhigang , Zhang Meichao , Mao Wei , Zhang Huitu , Yao Bin
Published: January 2012

N
-Acylated homoserine lactone (AHL) lactonases are capable of degrading signal molecules involved in bacterial quorum sensing and therefore represent a new approach to control bacterial infection. Here a gene responsible for the AHL lactonase activity of
Bacillus
sp. strain AI96, 753 bp in length, was cloned and then expressed in
Escherichia coli
. The deduced amino acid sequence of
Bacillus
sp. AI96 AiiA (AiiA
AI96
) is most similar to those of other
Bacillus
sp. AHL lactonases (∼80% sequence identity) and was consequently categorized as a member of the metallo-β-lactamase superfamily. AiiA
AI96
maintains ∼100% of its activity at 10°C to 40°C at pH 8.0, and it is very stable at 70°C at pH 8.0 for at least 1 h; no other
Bacillus
AHL lactonase has been found to be stable under these conditions. AiiA
AI96
resists digestion by proteases and carp intestinal juice, and it has broad-spectrum substrate specificity. The supplementation of AiiA
AI96
into fish feed by oral administration significantly attenuated
Aeromonas hydrophila
infection in zebrafish. This is the first report of the oral administration of an AHL lactonase for the efficient control of
A. hydrophila
.
External Ecological Niche for Candida albicans within Reducing, Oxygen-Limited Zones of Wetlands
Authors: Stone Wendy , Jones Barbara-Lee , Wilsenach Jac , Botha Alfred
Published: January 2012

Candida albicans
within the human host is well studied; however, identifying environmental reservoirs of pathogens is epidemiologically valuable for disease management. Oxygen-limited, carbohydrate-rich zones of wetlands, to which sewage-borne
C. albicans
is often exposed, are characteristically similar to the gastrointestinal reservoir. Consequently, using quantitative real-time PCR (qRT-PCR) and gas chromatography-mass spectrometry (GC-MS), we demonstrated that oxygen-limited zones in polluted wetlands may act as potential reservoirs of
C. albicans
.
Fulminant Cryptosporidiosis after Near-Drowning: a Human Cryptosporidium parvum Strain Implicated in Invasive Gastrointestinal Adenocarcinoma and Cholangiocarcinoma in an Experimental Model
Authors: Certad Gabriela , Benamrouz Sadia , Guyot Karine , Mouray Anthony , Chassat Thierry , Flament Nicolas , Delhaes Laurence , Coiteux Valerie , Delaire Baptiste , Praet Marleen , Cuvelier Claude , Gosset Pierre , Dei-Cas Eduardo , Creusy Colette
Published: January 2012

In the present work, we report the characterization of aCryptosporidium parvumstrain isolated from a patient who nearly drowned in the Deule River (Lille, France) after being discharged from the hospital where he had undergone allogeneic stem cell transplantation. After being rescued and readmitted to the hospital, he developed fulminant cryptosporidiosis. The strain isolated from the patient's stools was identified asC. parvumII2A15G2R1 (subtype linked to zoonotic exposure) and inoculated into SCID mice. In this host, this virulentC. parvumisolate induced not only severe infection but also invasive gastrointestinal and biliary adenocarcinoma. The observation of adenocarcinomas that progressed through all layers of the digestive tract to the subserosa and spread via blood vessels confirmed the invasive nature of the neoplastic process. These results indicate for the first time that a human-derivedC. parvumisolate is able to induce digestive cancer. This study is of special interest considering the exposure of a large number of humans and animals to this waterborne protozoan, which is highly tumorigenic when inoculated in a rodent model.
Seasonal and Successional Influences on Bacterial Community Composition Exceed That of Protozoan Grazing in River Biofilms
Authors: Wey Jennifer K. , Jürgens Klaus , Weitere Markus
Published: January 2012

The effects of protozoa (heterotrophic flagellates and ciliates) on the morphology and community composition of bacterial biofilms were tested under natural background conditions by applying size fractionation in a river bypass system. Confocal laser scanning microscopy (CLSM) was used to monitor the morphological structure of the biofilm, and fingerprinting methods (single-stranded conformation polymorphism [SSCP] and denaturing gradient gel electrophoresis [DGGE]) were utilized to assess changes in bacterial community composition. Season and internal population dynamics had a greater influence on the bacterial biofilm than the presence of protozoa. Within this general framework, bacterial area coverage and microcolony abundance were nevertheless enhanced by the presence of ciliates (but not by the presence of flagellates). We also found that the richness of bacterial operational taxonomic units was much higher in planktonic founder communities than in the ones establishing the biofilm. Within the first 2 h of colonization of an empty substrate by bacteria, the presence of flagellates additionally altered their biofilm community composition. As the biofilms matured, the number of bacterial operational taxonomic units increased when flagellates were present in high abundances. The additional presence of ciliates tended to at first reduce (days 2 to 7) and later increase (days 14 to 29) bacterial operational taxonomic unit richness. Altogether, the response of the bacterial community to protozoan grazing pressure was small compared to that reported in planktonic studies, but our findings contradict the assumption of a general grazing resistance of bacterial biofilms toward protozoa.
Criteria for Selection of Surrogates Used To Study the Fate and Control of Pathogens in the Environment
Authors: Sinclair Ryan G. , Rose Joan B. , Hashsham Syed A. , Gerba Charles P. , Haas Charles N.
Published: January 2012

This article defines the term surrogate as an organism, particle, or substance used to study the fate of a pathogen in a specific environment. Pathogenic organisms, nonpathogenic organisms, and innocuous particles have been used as surrogates for a variety of purposes, including studies on survival and transport as well as for method development and as “indicators” of certain conditions. This article develops a qualitative surrogate attribute prioritization process and allows investigators to select a surrogate by systematically detailing the experimental process and prioritizing attributes. The results are described through the use of case studies of various laboratories that have used this process. This article also discusses the history of surrogate and microbial indicator use and outlines the method by which surrogates can be used when conducting a quantitative microbial risk assessment. The ultimate goal of selecting a sufficiently representative surrogate is to improve public health through a health-based risk assessment framework. Under- or overestimating the resistance, inactivation, or movement may negatively impact risk assessments that, in turn, will impact health assessments and estimated safety levels. Reducing uncertainty in a risk assessment is one of the objectives of using surrogates and the ultimate motive for any experiment investigating potential exposure of a pathogen.
The Composition of Camembert Cheese-Ripening Cultures Modulates both Mycelial Growth and Appearance
Authors: Lessard Marie-Hélène , Bélanger Gaétan , St-Gelais Daniel , Labrie Steve
Published: January 2012

The fungal microbiota of bloomy-rind cheeses, such as Camembert, forms a complex ecosystem that has not been well studied, and its monitoring during the ripening period remains a challenge. One limitation of enumerating yeasts and molds on traditional agar media is that hyphae are multicellular structures, and colonies on a petri dish rarely develop from single cells. In addition, fungi tend to rapidly invade agar surfaces, covering small yeast colonies and resulting in an underestimation of their number. In this study, we developed a real-time quantitative PCR (qPCR) method using TaqMan probes to quantify a mixed fungal community containing the most common dairy yeasts and molds:
Penicillium camemberti
,
Geotrichum candidum
,
Debaryomyces hansenii
, and
Kluyveromyces lactis
on soft-cheese model curds (SCMC). The qPCR method was optimized and validated on pure cultures and used to evaluate the growth dynamics of a ripening culture containing
P. camemberti
,
G. candidum
, and
K. lactis
on the surface of the SCMC during a 31-day ripening period. The results showed that
P. camemberti
and
G. candidum
quickly dominated the ecosystem, while
K. lactis
remained less abundant. When added to this ecosystem,
D. hansenii
completely inhibited the growth of
K. lactis
in addition to reducing the growth of the other fungi. This result was confirmed by the decrease in the mycelium biomass on SCMC. This study compares culture-dependent and qPCR methods to successfully quantify complex fungal microbiota on a model curd simulating Camembert-type cheese.
Generation of a Monoclonal Antibody against Mycoplasma spp. following Accidental Contamination during Production of a Monoclonal Antibody against Lawsonia intracellularis
Authors: Hwang Jeong-Min , Lee Ji-Hye , Yeh Jung-Yong
Published: January 2012

This report describes
Mycoplasma
contamination of
Lawsonia intracellularis
cultures that led to the unintended acquisition of a monoclonal antibody against
Mycoplasma
spp. during the attempted generation of a monoclonal antibody against
L. intracellularis
.
Swimming Behavior of Selected Species of Archaea
Authors: Herzog Bastian , Wirth Reinhard
Published: January 2012

The swimming behavior of
Bacteria
has been studied extensively, at least for some species like
Escherichia coli
. In contrast, almost no data have been published for
Archaea
on this topic. In a systematic study we asked how the archaeal model organisms
Halobacterium salinarum
,
Methanococcus voltae
,
Methanococcus maripaludis
,
Methanocaldococcus jannaschii
,
Methanocaldococcus villosus
,
Pyrococcus furiosus
, and
Sulfolobus acidocaldarius
swim and which swimming behavior they exhibit. The two
Euryarchaeota M. jannaschii
and
M. villosus
were found to be, by far, the fastest organisms reported up to now, if speed is measured in bodies per second (bps). Their swimming speeds, at close to 400 and 500 bps, are much higher than the speed of the bacterium
E. coli
or of a very fast animal, like the cheetah, each with a speed of ca. 20 bps. In addition, we observed that two different swimming modes are used by some
Archaea
. They either swim very rapidly, in a more or less straight line, or they exhibit a slower kind of zigzag swimming behavior if cells are in close proximity to the surface of the glass capillary used for observation. We argue that such a “relocate-and-seek” behavior enables the organisms to stay in their natural habitat.
Short-Read Sequencing for Genomic Analysis of the Brown Rot Fungus Fibroporia radiculosa
Authors: Tang Juliet D. , Perkins Andy D. , Sonstegard Tad S. , Schroeder Steven G. , Burgess Shane C. , Diehl Susan V.
Published: January 2012

The feasibility of short-read sequencing for genomic analysis was demonstrated for
Fibroporia radiculosa
, a copper-tolerant fungus that causes brown rot decay of wood. The effect of read quality on genomic assembly was assessed by filtering Illumina GAIIx reads from a single run of a paired-end library (75-nucleotide read length and 300-bp fragment size) at three different stringency levels and then assembling each data set with Velvet. A simple approach was devised to determine which filter stringency was “best.” Venn diagrams identified the regions containing reads that were used in an assembly but were of a low-enough quality to be removed by a filter. By plotting base quality histograms of reads in this region, we judged whether a filter was too stringent or not stringent enough. Our best assembly had a genome size of 33.6 Mb, an N50 of 65.8 kb for a
k
-mer of 51, and a maximum contig length of 347 kb. Using GeneMark, 9,262 genes were predicted. TargetP and SignalP analyses showed that among the 1,213 genes with secreted products, 986 had motifs for signal peptides and 227 had motifs for signal anchors. Blast2GO analysis provided functional annotation for 5,407 genes. We identified 29 genes with putative roles in copper tolerance and 73 genes for lignocellulose degradation. A search for homologs of these 102 genes showed that
F. radiculosa
exhibited more similarity to
Postia placenta
than
Serpula lacrymans
. Notable differences were found, however, and their involvements in copper tolerance and wood decay are discussed.
The 60-Kilodalton Protein Encoded by
orf2
in the
cry19A
Operon of Bacillus thuringiensis subsp. jegathesan Functions Like a C-Terminal Crystallization Domain
Authors: Barboza-Corona J. Eleazar , Park Hyun-Woo , Bideshi Dennis K. , Federici Brian A.
Published: January 2012

The
cry19A
operon of
Bacillus thuringiensis
subsp.
jegathesan
encodes two proteins, mosquitocidal Cry19A (ORF1; 75 kDa) and an ORF2 (60 kDa) of unknown function. Expression of the
cry19A
operon in an acrystalliferous strain of
B. thuringiensis
(4Q7) yielded one small crystal per cell, whereas no crystals were produced when
cry19A
or
orf2
was expressed alone. To determine the function of the ORF2 protein, different combinations of Cry19A, ORF2, and the N- or C-terminal half of Cry1C were synthesized in strain 4Q7. Stable crystalline inclusions of these fusion proteins similar in shape to those in the strain harboring the wild-type operon were observed in sporulating cells. Comparative analysis showed that ORF2 shares considerable amino acid sequence identity with the C-terminal region of large Cry proteins. Together, these results suggest that ORF2 assists in synthesis and crystallization of Cry19A by functioning like the C-terminal domain characteristic of Cry protein in the 130-kDa mass range. In addition, to determine whether overexpression of the
cry19A
operon stabilized its shape and increased Cry19A yield, it was expressed under the control of the strong chimeric
cyt1A
-p/STAB-SD promoter. Interestingly, in contrast to the expression seen with the native promoter, overexpression of the operon yielded uniform bipyramidal crystals that were 4-fold larger on average than the wild-type crystal. In bioassays using the 4th instar larvae of
Culex quinquefasciatus
, the strain producing the larger Cry19A crystal showed moderate larvicidal activity that was 4-fold (95% lethal concentration [LC
95
] = 1.9 μg/ml) more toxic than the activity produced in the strain harboring the wild-type operon (LC
95
= 8.2 μg/ml).
Wide Variation in Antibiotic Resistance Proteins Identified by Functional Metagenomic Screening of a Soil DNA Library
Authors: McGarvey Kelly M. , Queitsch Konstantin , Fields Stanley
Published: January 2012

Most genes for antibiotic resistance present in soil microbes remain unexplored because most environmental microbes cannot be cultured. Only recently has the identification of these genes become feasible through the use of culture-independent methods. We screened a soil metagenomic DNA library in an
Escherichia coli
host for genes that can confer resistance to kanamycin, gentamicin, rifampin, trimethoprim, chloramphenicol, or tetracycline. The screen revealed 41 genes that encode novel protein variants of eight protein families, including aminoglycoside acetyltransferases, rifampin ADP-ribosyltransferases, dihydrofolate reductases, and transporters. Several proteins of the same protein family deviate considerably from each other yet confer comparable resistance. For example, five dihydrofolate reductases sharing at most 44% amino acid sequence identity in pairwise comparisons were equivalent in conferring trimethoprim resistance. We identified variants of aminoglycoside acetyltransferases and transporters that differ in the specificity of the drugs for which they confer resistance. We also found wide variation in protein structure. Two forms of rifampin ADP-ribosyltransferases, one twice the size of the other, were similarly effective at conferring rifampin resistance, although the short form was expressed at a much lower level. Functional metagenomic screening provides insight into the large variability in antibiotic resistance protein sequences, revealing divergent variants that preserve protein function.
Candida zemplinina Can Reduce Acetic Acid Produced by Saccharomyces cerevisiae in Sweet Wine Fermentations
Authors: Rantsiou Kalliopi , Dolci Paola , Giacosa Simone , Torchio Fabrizio , Tofalo Rosanna , Torriani Sandra , Suzzi Giovanna , Rolle Luca , Cocolin Luca
Published: January 2012

In this study we investigated the possibility of usingCandida zemplinina, as a partner ofSaccharomyces cerevisiae, in mixed fermentations of must with a high sugar content, in order to reduce its acetic acid production. Thirty-fiveC. zemplininastrains, which were isolated from different geographic regions, were molecularly characterized, and their fermentation performances were determined. Five genetically different strains were selected for mixed fermentations withS. cerevisiae. Two types of inoculation were carried out: coinoculation and sequential inoculation. A balance between the two species was generally observed for the first 6 days, after which the levels ofC. zemplininastarted to decrease. Relevant differences were observed concerning the consumption of sugars, the ethanol and glycerol content, and acetic acid production, depending on which strain was used and which type of inoculation was performed. Sequential inoculation led to the reduction of about half of the acetic acid content compared to the pureS. cerevisiaefermentation, but the ethanol and glycerol amounts were also low. A coinoculation with selected combinations ofS. cerevisiaeandC. zemplininaresulted in a decrease of ∼0.3 g of acetic acid/liter, while maintaining high ethanol and glycerol levels. This study demonstrates that mixedS. cerevisiaeandC. zemplininafermentation could be applied in sweet wine fermentation to reduce the production of acetic acid, connected to theS. cerevisiaeosmotic stress response.
Phenotypic Switching in Pseudomonas brassicacearum Involves GacS- and GacA-Dependent Rsm Small RNAs
Authors: Lalaouna David , Fochesato Sylvain , Sanchez Lisa , Schmitt-Kopplin Philippe , Haas Dieter , Heulin Thierry , Achouak Wafa
Published: January 2012

The plant-beneficial bacterium
Pseudomonas brassicacearum
forms phenotypic variants
in vitro
as well as
in planta
during root colonization under natural conditions. Transcriptome analysis of typical phenotypic variants using microarrays containing coding as well as noncoding DNA fragments showed differential expression of several genes relevant to secondary metabolism and of the small RNA (sRNA) genes
rsmX
,
rsmY
, and
rsmZ
. Naturally occurring mutations in the
gacS-gacA
system accounted for phenotypic switching, which was characterized by downregulation of antifungal secondary metabolites (2,4-diacetylphloroglucinol and cyanide), indoleacetate, exoenzymes (lipase and protease), and three different
N
-acyl-homoserine lactone molecules. Moreover, in addition to abrogating these biocontrol traits,
gacS
and
gacA
mutations resulted in reduced expression of the type VI secretion machinery, alginate biosynthesis, and biofilm formation. In a
gacA
mutant, the expression of
rsmX
was completely abolished, unlike that of
rsmY
and
rsmZ
. Overexpression of any of the three sRNAs in the
gacA
mutant overruled the pleiotropic changes and restored the wild-type phenotypes, suggesting functional redundancy of these sRNAs. In conclusion, our data show that phenotypic switching in
P. brassicacearum
results from mutations in the
gacS-gacA
system.
Use of High Hydrostatic Pressure To Inactivate Escherichia coli O157:H7 and Salmonella enterica Internalized within and Adhered to Preharvest Contaminated Green Onions
Authors: Neetoo Hudaa , Lu Yingjian , Wu Changqing , Chen Haiqiang
Published: January 2012

Green onions grown in soil and hydroponic medium contaminated with
Escherichia coli
O157:H7 and
Salmonella
were found to take up the pathogens in their roots, bulbs, stems, and leaves. Pressure treatment at 400 to 500 MPa for 2 min at 20 to 40°C eliminated both pathogens that were internalized within green onions during plant growth.
Biochemical and Mutational Analyses of a Multidomain Cellulase/Mannanase from Caldicellulosiruptor bescii
Authors: Su Xiaoyun , Mackie Roderick I. , Cann Isaac K. O.
Published: January 2012

Thermophilic cellulases and hemicellulases are of significant interest to the biofuel industry due to their perceived advantages over their mesophilic counterparts. We describe here biochemical and mutational analyses of
Caldicellulosiruptor bescii
Cel9B/Man5A (CbCel9B/Man5A), a highly thermophilic enzyme. As one of the highly secreted proteins of
C. bescii
, the enzyme is likely to be critical to nutrient acquisition by the bacterium. CbCel9B/Man5A is a modular protein composed of three carbohydrate-binding modules flanked at the N terminus and the C terminus by a glycoside hydrolase family 9 (GH9) module and a GH5 module, respectively. Based on truncational analysis of the polypeptide, the cellulase and mannanase activities within CbCel9B/Man5A were assigned to the N- and C-terminal modules, respectively. CbCel9B/Man5A and its truncational mutants, in general, exhibited a pH optimum of ∼5.5 and a temperature optimum of 85°C. However, at this temperature, thermostability was very low. After 24 h of incubation at 75°C, the wild-type protein maintained 43% activity, whereas a truncated mutant, TM1, maintained 75% activity. The catalytic efficiency with phosphoric acid swollen cellulose as a substrate for the wild-type protein was 7.2 s
−1
ml/mg, and deleting the GH5 module led to a mutant (TM1) with a 2-fold increase in this kinetic parameter. Deletion of the GH9 module also increased the apparent
k
cat
of the truncated mutant TM5 on several mannan-based substrates; however, a concomitant increase in the

K
m

led to a decrease in the catalytic efficiencies on all substrates. These observations lead us to postulate that the two catalytic activities are coupled in the polypeptide.
Identification of the Herboxidiene Biosynthetic Gene Cluster in Streptomyces chromofuscus ATCC 49982
Authors: Shao Lei , Zi Jiachen , Zeng Jia , Zhan Jixun
Published: January 2012

The 53-kb biosynthetic gene cluster for the novel anticholesterol natural product herboxidiene was identified in
Streptomyces chromofuscus
ATCC 49982 by genome sequencing and gene inactivation. In addition to herboxidiene, a biosynthetic intermediate, 18-deoxy-herboxidiene, was also isolated from the fermentation broth of
S. chromofuscus
ATCC 49982 as a minor metabolite.
Association of Pandemic Vibrio parahaemolyticus O3:K6 Present in the Coastal Environment of Northwest Mexico with Cases of Recurrent Diarrhea between 2004 and 2010
Authors: Velazquez-Roman Jorge , León-Sicairos Nidia , Flores-Villaseñor Héctor , Villafaña-Rauda Santiago , Canizalez-Roman Adrian
Published: January 2012

In 2004, more than 1,230 cases of gastroenteritis due to pandemic O3:K6 strains of
Vibrio parahaemolyticus
were reported in southern Sinaloa, a state in Northwestern Mexico. Recurrent sporadic cases arose from 2004 to 2010, spreading from the south to the north. In the present study,
Vibrio parahaemolyticus
was detected in both environmental samples and clinical cases along the Pacific coast of Sinaloa during 2004 to 2010. An evaluation was made of the serotypes, distribution of virulence genes, and presence of pandemic O3:K6 strains. A total of 144 strains were isolated from environmental samples (from sediment, seawater, and shrimp), and 154 clinical strains were isolated. A total of 10 O serogroups and 30 serovars were identified in the strains. Environmental strains (
n
= 144) belonged to 10 O serogroups and 28 serovars, while clinical strains (
n
= 154) belonged to 8 O serogroups and 14 serovars. Ten serovars were shared by both environmental and clinical strains. Among 144 environmental isolates, 4.1% (6/144) belonged to the pandemic clone, with 83.3% containing the
orf8
gene and with O3:K6 accounting for 67%. On the other hand, pathogenic strains (
tdh
and/or
trh
) accounted for 52% (75/144) of the environmental isolates. Interestingly, among 154 clinical isolates, 80.5% (124/154) were pandemic strains, with O3:K6 (
tdh
,
toxRS
new
, and
orf8
) representing the predominant serovar (99.2%, 123/124). Overall, our results indicate that in spite of a high serodiversity and prevalence of pathogenic
Vibrio parahaemolyticus
in the environment, the pandemic strain O3:K6 caused >79% of reported cases between 2004 and 2010 in Sinaloa, Mexico.
Conserved Mechanisms of Mycobacterium marinum Pathogenesis within the Environmental Amoeba Acanthamoeba castellanii
Authors: Kennedy George M. , Morisaki J. Hiroshi , DiGiuseppe Champion Patricia A.
Published: January 2012

Mycobacterium marinum
is a waterborne mycobacterial pathogen. Due to their common niche, protozoa likely represent natural hosts for
M. marinum
. We demonstrate that the ESX-1 secretion system is required for
M. marinum
pathogenesis and that
M. marinum
utilizes actin-based motility in amoebae. Therefore, at least two virulence pathways used by
M. marinum
in macrophages are conserved during
M. marinum
infection of amoebae.
FSL J1-208, a Virulent Uncommon Phylogenetic Lineage IV Listeria monocytogenes Strain with a Small Chromosome Size and a Putative Virulence Plasmid Carrying Internalin-Like Genes
Authors: den Bakker Henk C. , Bowen Barbara M. , Rodriguez-Rivera Lorraine D. , Wiedmann Martin
Published: January 2012

The bacterial genusListeriacontains both saprotrophic and facultative pathogenic species. A small genome size has been suggested to be associated with the loss of pathogenic potential ofL. welshimeriandL. seeligeri. In this paper we present data on the genome ofL. monocytogenesstrain FSL J1-208, a representative of phylogenetic lineage IV. Although this strain was isolated from a clinical case in a caprine host and has no decreased invasiveness in human intestinal epithelial cells, our analyses show that this strain has one of the smallestListeriachromosomes reported to date (2.78 Mb). The chromosome contains 2,772 protein-coding genes, including well-characterized virulence-associated genes, such asinlA,inlB, andinlCand the fullprfAgene cluster. The small genome size is mainly caused by the absence of prophages in the genome ofL. monocytogenesFSL J1-208, and further analyses showed that the total size of prophage-related regions is highly correlated to chromosome size in the genusListeria. L. monocytogenesFSL J1-208 carries a unique type of plasmid of approximately 80 kbp that does not carry genes annotated as being involved in resistance to antibiotics or heavy metals. The accessory genes in this plasmid belong to the internalin family, a family of virulence-associated proteins, and therefore this is the first report of a potential virulence plasmid in the genusListeria.
Contribution of Surface β-Glucan Polysaccharide to Physicochemical and Immunomodulatory Properties of Propionibacterium freudenreichii
Authors: Deutsch Stéphanie-Marie , Parayre Sandrine , Bouchoux Antoine , Guyomarc'h Fanny , Dewulf Joëlle , Dols-Lafargue Marguerite , Baglinière François , Cousin Fabien J. , Falentin Hélène , Jan Gwénaël , Foligné Benoît
Published: January 2012

Propionibacterium freudenreichii
is a bacterial species found in Swiss-type cheeses and is also considered for its health properties. The main claimed effect is the bifidogenic property. Some strains were shown recently to display other interesting probiotic potentialities such as anti-inflammatory properties. About 30% of strains were shown to produce a surface exopolysaccharide (EPS) composed of (1→3,1→2)-β
-d-
glucan due to a single gene named
gtfF
. We hypothesized that functional properties of
P. freudenreichii
strains, including their anti-inflammatory properties, could be linked to the presence of β-glucan. To evaluate this hypothesis,
gtfF
genes of three β-glucan-producing strains were disrupted. These knockout (KO) mutants were complemented with a plasmid harboring
gtfF
(KO-C mutants). The absence of β-glucan in KO mutants was verified by immunological detection and transmission electron microscopy. We observed by atomic force microscopy that the absence of β-glucan in the KO mutant dramatically changed the cell's topography. The capacity to adhere to polystyrene surface was increased for the KO mutants compared to wild-type (WT) strains. Anti-inflammatory properties of WT strains and mutants were analyzed by stimulation of human peripheral blood mononuclear cells (PBMCs). A significant increase of the anti-inflammatory interleukin-10 cytokine production by PBMCs was measured in the KO mutants compared to WT strains. For one strain, the role of β-glucan in mice gut persistence was assessed, and no significant difference was observed between the WT strain and its KO mutant. Thus, β-glucan appears to partly hide the anti-inflammatory properties of
P. freudenreichii
; which is an important result for the selection of probiotic strains.
Hyperthermophilic Thermotoga Species Differ with Respect to Specific Carbohydrate Transporters and Glycoside Hydrolases
Authors: Frock Andrew D. , Gray Steven R. , Kelly Robert M.
Published: January 2012

Four hyperthermophilic members of the bacterial genus
Thermotoga
(
T. maritima
,
T. neapolitana
,
T. petrophila
, and
Thermotoga
sp. strain RQ2) share a core genome of 1,470 open reading frames (ORFs), or about 75% of their genomes. Nonetheless, each species exhibited certain distinguishing features during growth on simple and complex carbohydrates that correlated with genomic inventories of specific ABC sugar transporters and glycoside hydrolases. These differences were consistent with transcriptomic analysis based on a multispecies cDNA microarray. Growth on a mixture of six pentoses and hexoses showed no significant utilization of galactose or mannose by any of the four species.
T. maritima
and
T. neapolitana
exhibited similar monosaccharide utilization profiles, with a strong preference for glucose and xylose over fructose and arabinose.
Thermotoga
sp. strain RQ2 also used glucose and xylose, but was the only species to utilize fructose to any extent, consistent with a phosphotransferase system (PTS) specific for this sugar encoded in its genome.
T. petrophila
used glucose to a significantly lesser extent than the other species. In fact, the XylR regulon was triggered by growth on glucose for
T. petrophila
, which was attributed to the absence of a glucose transporter (XylE2F2K2), otherwise present in the other
Thermotoga
species. This suggested that
T. petrophila
acquires glucose through the XylE1F1K1 transporter, which primarily serves to transport xylose in the other three
Thermotoga
species. The results here show that subtle differences exist among the hyperthermophilic
Thermotogales
with respect to carbohydrate utilization, which supports their designation as separate species.
Response of Fatty Acid Synthesis Genes to the Binding of Human Salivary Amylase by Streptococcus gordonii
Authors: Nikitkova Anna E. , Haase Elaine M. , Vickerman M. Margaret , Gill Steven R. , Scannapieco Frank A.
Published: January 2012

Streptococcus gordonii
, an important primary colonizer of dental plaque biofilm, specifically binds to salivary amylase via the surface-associated amylase-binding protein A (AbpA). We hypothesized that a function of amylase binding to
S. gordonii
may be to modulate the expression of chromosomal genes, which could influence bacterial survival and persistence in the oral cavity. Gene expression profiling by microarray analysis was performed to detect genes in
S. gordonii
strain CH1 that were differentially expressed in response to the binding of purified human salivary amylase versus exposure to purified heat-denatured amylase. Selected genes found to be differentially expressed were validated by quantitative reverse transcription-PCR (qRT-PCR). Five genes from the fatty acid synthesis (FAS) cluster were highly (10- to 35-fold) upregulated in
S. gordonii
CH1 cells treated with native amylase relative to those treated with denatured amylase. An
abpA
-deficient strain of
S. gordonii
exposed to amylase failed to show a response in FAS gene expression similar to that observed in the parental strain. Predicted phenotypic effects of amylase binding to
S. gordonii
strain CH1 (associated with increased expression of FAS genes, leading to changes in fatty acid synthesis) were noted; these included increased bacterial growth, survival at low pH, and resistance to triclosan. These changes were not observed in the amylase-exposed
abpA
-deficient strain, suggesting a role for AbpA in the amylase-induced phenotype. These results provide evidence that the binding of salivary amylase elicits a differential gene response in
S. gordonii
, resulting in a phenotypic adjustment that is potentially advantageous for bacterial survival in the oral environment.
Wide Distribution of Closely Related, Antibiotic-Producing Arthrobacter Strains throughout the Arctic Ocean
Authors: Wietz Matthias , Månsson Maria , Bowman Jeff S. , Blom Nikolaj , Ng Yin , Gram Lone
Published: January 2012

We isolated 16 antibiotic-producing bacterial strains throughout the central Arctic Ocean, including seven
Arthrobacter
spp. with almost identical 16S rRNA gene sequences. These strains were numerically rare, as revealed using 454 pyrosequencing libraries.
Arthrobacter
spp. produced arthrobacilins A to C under different culture conditions, but other, unidentified compounds likely contributed to their antibiotic activity.
Evidence of Two Functionally Distinct Ornithine Decarboxylation Systems in Lactic Acid Bacteria
Authors: Romano Andrea , Trip Hein , Lonvaud-Funel Aline , Lolkema Juke S. , Lucas Patrick M.
Published: January 2012

Biogenic amines are low-molecular-weight organic bases whose presence in food can result in health problems. The biosynthesis of biogenic amines in fermented foods mostly proceeds through amino acid decarboxylation carried out by lactic acid bacteria (LAB), but not all systems leading to biogenic amine production by LAB have been thoroughly characterized. Here, putative ornithine decarboxylation pathways consisting of a putative ornithine decarboxylase and an amino acid transporter were identified in LAB by strain collection screening and database searches. The decarboxylases were produced in heterologous hosts and purified and characterizedin vitro, whereas transporters were heterologously expressed inLactococcus lactisand functionally characterizedin vivo. Amino acid decarboxylation by whole cells of the original hosts was determined as well. We concluded that two distinct types of ornithine decarboxylation systems exist in LAB. One is composed of an ornithine decarboxylase coupled to an ornithine/putrescine transmembrane exchanger. Their combined activities results in the extracellular release of putrescine. This typical amino acid decarboxylation system is present in only a few LAB strains and may contribute to metabolic energy production and/or pH homeostasis. The second system is widespread among LAB. It is composed of a decarboxylase active on ornithine andl-2,4-diaminobutyric acid (DABA) and a transporter that mediates unidirectional transport of ornithine into the cytoplasm. Diamines that result from this second system are retained within the cytosol.
Characterization of the ELPhiS Prophage from Salmonella enterica Serovar Enteritidis Strain LK5
Authors: Hanna L. Farris , Matthews T. David , Dinsdale Elizabeth A. , Hasty David , Edwards Robert A.
Published: January 2012

Phages are a primary driving force behind the evolution of bacterial pathogens by transferring a variety of virulence genes into their hosts. Similar to other bacterial genomes, the
Salmonella enterica
serovar Enteritidis LK5 genome contains several regions that are homologous to phages. Although genomic analysis demonstrated the presence of prophages, it was unable to confirm which phage elements within the genome were viable. Genetic markers were used to tag one of the prophages in the genome to allow monitoring of phage induction. Commonly used laboratory strains of
Salmonella
were resistant to phage infection, and therefore a rapid screen was developed to identify susceptible hosts. This approach showed that a genetically tagged prophage, ELPhiS (Enteritidis lysogenic phage S), was capable of infecting
Salmonella
serovars that are diverse in host range and virulence and has the potential to laterally transfer genes between these serovars via lysogenic conversion. The rapid screen approach is adaptable to any system with a large collection of isolates and may be used to test the viability of prophages found by sequencing the genomes of various bacterial pathogens.
Fine-Tuned Transcriptional Regulation of Malate Operons in Enterococcus faecalis
Authors: Mortera Pablo , Espariz Martín , Suárez Cristian , Repizo Guillermo , Deutscher Josef , Alarcón Sergio , Blancato Víctor , Magni Christian
Published: January 2012

In
Enterococcus faecalis
, the
mae
locus is constituted by two putative divergent operons,
maePE
and
maeKR
. The first operon encodes a putative H
+
/malate symporter (MaeP) and a malic enzyme (MaeE) previously shown to be essential for malate utilization in this bacterium. The
maeKR
operon encodes two putative proteins with significant similarity to two-component systems involved in sensing malate and activating its assimilation in bacteria. Our transcriptional and genetic assays showed that
maePE
and
maeKR
are induced in response to malate by the response regulator MaeR. In addition, we observed that both operons were partially repressed in the presence of glucose. Accordingly, the cometabolism of this sugar and malate was detected. The binding of the complex formed by CcpA and its corepressor P-Ser-HPr to a
cre
site located in the
mae
region was demonstrated
in vitro
and explains the carbon catabolite repression (CCR) observed for the
maePE
operon. However, our results also provide evidence for a CcpA-independent CCR mechanism regulating the expression of both operons. Finally, a biomass increment of 40 or 75% was observed compared to the biomass of cells grown only on glucose or malate, respectively. Cells cometabolizing both carbon sources exhibit a higher rate of glucose consumption and a lower rate of malate utilization. The growth improvement achieved by
E. faecalis
during glucose-malate cometabolism might explain why this microorganism employs different regulatory systems to tightly control the assimilation of both carbon sources.
Development and Validation of a Predictive Model for the Growth of Vibrio vulnificus in Postharvest Shellstock Oysters
Authors: DaSilva Ligia , Parveen Salina , DePaola Angelo , Bowers John , Brohawn Kathy , Tamplin Mark L.
Published: January 2012

Postharvest growth of
Vibrio vulnificus
in oysters can increase risk of human infection. Unfortunately, limited information is available regarding
V. vulnificus
growth and survival patterns over a wide range of storage temperatures in oysters harvested from different estuaries and in different oyster species. In this study, we developed a predictive model for
V. vulnificus
growth in Eastern oysters (
Crassostrea virginica
) harvested from Chesapeake Bay, MD, over a temperature range of 5 to 30°C and then validated the model against
V. vulnificus
growth rates (GRs) in Eastern and Asian oysters (
Crassostrea ariakensis
) harvested from Mobile Bay, AL, and Chesapeake Bay, VA, respectively. In the model development studies,
V. vulnificus
was slowly inactivated at 5 and 10°C with average GRs of −0.0045 and −0.0043 log most probable number (MPN)/h, respectively. Estimated average growth rates at 15, 20, 25, and 30°C were 0.022, 0.042, 0.087, and 0.093 log MPN/h, respectively. With respect to Eastern oysters, bias (
B
f
) and accuracy (
A
f
) factors for model-dependent and -independent data were 1.02 and 1.25 and 1.67 and 1.98, respectively. For Asian oysters,
B
f
and
A
f
were 0.29 and 3.40. Residual variations in growth rate about the fitted model were not explained by season, region, water temperature, or salinity at harvest. Growth rate estimates for Chesapeake Bay and Mobile Bay oysters stored at 25 and 30°C showed relatively high variability and were lower than Food and Agricultural Organization (FAO)/WHO
V. vulnificus
quantitative risk assessment model predictions. The model provides an improved tool for designing and implementing food safety plans that minimize the risk associated with
V. vulnificus
in oysters.
Marked Genomic Diversity of Norovirus Genogroup I Strains in a Waterborne Outbreak
Authors: Nenonen Nancy P. , Hannoun Charles , Larsson Charlotte U. , Bergström Tomas
Published: January 2012

Marked norovirus (NoV) diversity was detected in patient samples from a large community outbreak of gastroenteritis with waterborne epidemiology affecting approximately 2,400 people. NoV was detected in 33 of 50 patient samples examined by group-specific real-time reverse transcription-PCR. NoV genotype I (GI) strains predominated in 31 patients, with mixed GI infections occurring in 5 of these patients. Sequence analysis of RNA-dependent polymerase-N/S capsid-coding regions (∼900 nucleotides in length) confirmed the dominance of the GI strains (
n
= 36). Strains of NoV GI.4 (
n
= 21) and GI.7 (
n
= 9) were identified, but six strains required full capsid amino acid analyses (530 to 550 amino acids) based on control sequencing of cloned amplicons before the virus genotype could be determined. Three strains were assigned to a new NoV GI genotype, proposed as GI.9, based on capsid amino acid analyses showing 26% dissimilarity from the established genotypes GI.1 to GI.8. Three other strains grouped in a sub-branch of GI.3 with 13 to 15% amino acid dissimilarity to GI.3 GenBank reference strains. Phylogenetic analysis (2.1 kb) of 10 representative strains confirmed these genotype clusters. Strains of NoV GII.4 (
n
= 1), NoV GII.6 (
n
= 2), sapovirus GII.2 (
n
= 1), rotavirus (
n
= 3), adenovirus (
n
= 1), and
Campylobacter
spp. (
n
= 2) were detected as single infections or as mixtures with NoV GI. Marked NoV GI diversity detected in patients was consistent with epidemiologic evidence of waterborne NoV infections, suggesting human fecal contamination of the water supply. Recognition of NoV diversity in a cluster of patients provided a useful warning marker of waterborne contamination in the Lilla Edet outbreak.