bluuebunny/crossref_metadata_2025_split huggingface dataset:
Development of a Double-Crossover Markerless Gene Deletion System in Bifidobacterium longum: Functional Analysis of the α-Galactosidase Gene for Raffinose Assimilation
Authors: Hirayama Yosuke , Sakanaka Mikiyasu , Fukuma Hidenori , Murayama Hiroki , Kano Yasunobu , Fukiya Satoru , Yokota Atsushi 
Published: May 2012

ABSTRACT

Functional analysis of
Bifidobacterium
genes is essential for understanding host-
Bifidobacterium
interactions with beneficial effects on human health; however, the lack of an effective targeted gene inactivation system in bifidobacteria has prevented the development of functional genomics in this bacterium. Here, we report the development of a markerless gene deletion system involving a double crossover in
Bifidobacterium longum
. Incompatible plasmid vectors were used to facilitate a second crossover step. The conditional replication vector pBS423-
ΔrepA
, which lacks the plasmid replication gene
repA
, was integrated into the target gene by a first crossover event. Subsequently, the replicative plasmid pTBR101-CM, which harbors
repA
, was introduced into this integrant to facilitate the second crossover step and subsequent elimination of the excised conditional replication vector from the cells by plasmid incompatibility. The proposed system was confirmed to work as expected in
B. longum
105-A using the chromosomal full-length β-galactosidase gene as a target. Markerless gene deletion was tested using the
aga
gene, which encodes α-galactosidase, whose substrates include raffinose. Almost all the pTBR101-CM-transformed strains became double-crossover recombinants after subculture, and 4 out of the 270 double-crossover recombinants had lost the ability to assimilate raffinose. Genotype analysis of these strains revealed markerless gene deletion of
aga
. Carbohydrate assimilation analysis and α-galactosidase activity measurement were conducted using both the representative mutant and a plasmid-based
aga
-complemented strain. These functional analyses revealed that
aga
is the only gene encoding a functional α-galactosidase enzyme in
B. longum
105-A.
Lactobacillus reuteri Protects Epidermal Keratinocytes from Staphylococcus aureus-Induced Cell Death by Competitive Exclusion
Authors: Prince Tessa , McBain Andrew J. , O'Neill Catherine A. 
Published: May 2012

ABSTRACT

Recent studies have suggested that the topical application of probiotic bacteria can improve skin health or combat disease. We have utilized a primary human keratinocyte culture model to investigate whether probiotic bacteria can inhibit
Staphylococcus aureus
infection. Evaluation of the candidate probiotics
Lactobacillus reuteri
ATCC 55730,
Lactobacillus rhamnosus
AC413, and
Lactobacillus salivarius
UCC118 demonstrated that both
L. reuteri
and
L. rhamnosus
, but not
L. salivarius
, reduced
S. aureus
-induced keratinocyte cell death in both undifferentiated and differentiated keratinocytes. Keratinocyte survival was significantly higher if the probiotic was applied prior to (
P
< 0.01) or simultaneously with (
P
< 0.01) infection with
S. aureus
but not when added after infection had commenced (
P
> 0.05). The protective effect of
L. reuteri
was not dependent on the elaboration of inhibitory substances such as lactic acid.
L. reuteri
inhibited adherence of
S. aureus
to keratinocytes by competitive exclusion (
P
= 0.026).
L. salivarius
UCC118, however, did not inhibit
S. aureus
from adhering to keratinocytes (
P
> 0.05) and did not protect keratinocyte viability.
S. aureus
utilizes the α5β1 integrin to adhere to keratinocytes, and blocking of this integrin resulted in a protective effect similar to that observed with probiotics (
P
= 0.03). This suggests that the protective mechanism for
L. reuteri-
mediated protection of keratinocytes was by competitive exclusion of the pathogen from its binding sites on the cells. Our results suggest that use of a topical probiotic prophylactically could inhibit the colonization of skin by
S. aureus
and thus aid in the prevention of infection.
Involvement of PatE, a Prophage-Encoded AraC-Like Regulator, in the Transcriptional Activation of Acid Resistance Pathways of Enterohemorrhagic Escherichia coli Strain EDL933
Authors: Bender Jennifer K. , Praszkier Judyta , Wakefield Matthew J. , Holt Kathryn , Tauschek Marija , Robins-Browne Roy M. , Yang Ji 
Published: May 2012

ABSTRACTEnterohemorrhagicEscherichia coli(EHEC) O157:H7 is a lethal human intestinal pathogen that causes hemorrhagic colitis and the hemolytic-uremic syndrome. EHEC is transmitted by the fecal-oral route and has a lower infectious dose than most other enteric bacterial pathogens in that fewer than 100 CFU are able to cause disease. This low infectious dose has been attributed to the ability of EHEC to survive in the acidic environment of the human stomach.In silicoanalysis of the genome of EHEC O157:H7 strain EDL933 revealed a gene,patE, for a putative AraC-like regulatory protein within the prophage island, CP-933H. Transcriptional analysis inE. colishowed that the expression ofpatEis induced during stationary phase. Data from microarray assays demonstrated that PatE activates the transcription of genes encoding proteins of acid resistance pathways. In addition, PatE downregulated the expression of a number of genes encoding heat shock proteins and the type III secretion pathway of EDL933. Transcriptional analysis and electrophoretic mobility shift assays suggested that PatE also activates the transcription of the gene for the acid stress chaperonehdeAby binding to its promoter region. Finally, assays of acid tolerance showed that increasing the expression of PatE in EHEC greatly enhanced the ability of the bacteria to survive in different acidic environments. Together, these findings indicate that EHEC strain EDL933 carries a prophage-encoded regulatory system that contributes to acid resistance.
Enhanced Tolerance to Naphthalene and Enhanced Rhizoremediation Performance for Pseudomonas putida KT2440 via the NAH7 Catabolic Plasmid
Authors: Fernández Matilde , Niqui-Arroyo José Luis , Conde Susana , Ramos Juan Luis , Duque Estrella 
Published: May 2012

ABSTRACTIn this work, we explore the potential use of thePseudomonas putidaKT2440 strain for bioremediation of naphthalene-polluted soils.Pseudomonas putidastrain KT2440 thrives in naphthalene-saturated medium, establishing a complex response that activates genes coding for extrusion pumps and cellular damage repair enzymes, as well as genes involved in the oxidative stress response. The transfer of the NAH7 plasmid enables naphthalene degradation byP. putidaKT2440 while alleviating the cellular stress brought about by this toxic compound, without affecting key functions necessary for survival and colonization of the rhizosphere.Pseudomonas putidaKT2440(NAH7) efficiently expresses the Nah catabolic pathwayin vitroandin situ, leading to the complete mineralization of [14C]naphthalene, measured as the evolution of14CO2, while the rate of mineralization was at least 2-fold higher in the rhizosphere than in bulk soil.
Ammonia Inactivation of Ascaris Ova in Ecological Compost by Using Urine and Ash
Authors: McKinley James W. , Parzen Rebecca E. , Mercado Guzmán Álvaro 
Published: May 2012

ABSTRACT

Viable ova of
Ascaris lumbricoides
, an indicator organism for pathogens, are frequently found in feces-derived compost produced from ecological toilets, demonstrating that threshold levels of time, temperature, pH, and moisture content for pathogen inactivation are not routinely met. Previous studies have determined that NH
3
has ovicidal properties for pathogens, including
Ascaris
ova. This research attempted to achieve
Ascaris
inactivation via NH
3
under environmental conditions commonly found in ecological toilets and using materials universally available in an ecological sanitation setting, including compost (feces and sawdust), urine, and ash. Compost mixed with stored urine and ash produced the most rapid inactivation, with significant inactivation observed after 2 weeks and with a time to 99% ovum inactivation (
T
99
) of 8 weeks. Compost mixed with fresh urine and ash achieved a
T
99
of 15 weeks, after a 4-week lag phase. Both matrices had relatively high total-ammonia concentrations and pH values of >9.24 (pK
a
of ammonia). In compost mixed with ash only, and in compost mixed with fresh urine only, inactivation was observed after an 11-week lag phase. These matrices contained NH
3
concentrations of 164 to 173 and 102 to 277 mg/liter, respectively, when inactivation occurred, which was below the previously hypothesized threshold for inactivation (280 mg/liter), suggesting that a lower threshold NH
3
concentration may be possible with a longer contact time. Other significant results include the hydrolysis of urea to ammonia between pH values of 10.4 and 11.6, above the literature threshold pH of 10.
Epoxy Coenzyme A Thioester Pathways for Degradation of Aromatic Compounds
Authors: Ismail Wael , Gescher Johannes 
Published: May 2012

ABSTRACT
Aromatic compounds (biogenic and anthropogenic) are abundant in the biosphere. Some of them are well-known environmental pollutants. Although the aromatic nucleus is relatively recalcitrant, microorganisms have developed various catabolic routes that enable complete biodegradation of aromatic compounds. The adopted degradation pathways depend on the availability of oxygen. Under oxic conditions, microorganisms utilize oxygen as a cosubstrate to activate and cleave the aromatic ring. In contrast, under anoxic conditions, the aromatic compounds are transformed to coenzyme A (CoA) thioesters followed by energy-consuming reduction of the ring. Eventually, the dearomatized ring is opened via a hydrolytic mechanism. Recently, novel catabolic pathways for the aerobic degradation of aromatic compounds were elucidated that differ significantly from the established catabolic routes. The new pathways were investigated in detail for the aerobic bacterial degradation of benzoate and phenylacetate. In both cases, the pathway is initiated by transforming the substrate to a CoA thioester and all the intermediates are bound by CoA. The subsequent reactions involve epoxidation of the aromatic ring followed by hydrolytic ring cleavage. Here we discuss the novel pathways, with a particular focus on their unique features and occurrence as well as ecological significance.
Detection of Coxiella burnetii DNA in Inhalable Airborne Dust Samples from Goat Farms after Mandatory Culling
Authors: Hogerwerf Lenny , Borlée Floor , Still Kelly , Heederik Dick , van Rotterdam Bart , de Bruin Arnout , Nielen Mirjam , Wouters Inge M. 
Published: May 2012

ABSTRACT

Coxiella burnetii
is thought to infect humans primarily via airborne transmission. However, air measurements of
C. burnetii
are sparse. We detected
C. burnetii
DNA in inhalable and PM10 (particulate matter with an aerodynamic size of 10 μm or less) dust samples collected at three affected goat farms, demonstrating that low levels of
C. burnetii
DNA are present in inhalable size fractions.
Syntrophic Oxidation of Propionate in Rice Field Soil at 15 and 30°C under Methanogenic Conditions
Authors: Gan Yanlu , Qiu Qiongfen , Liu Pengfei , Rui Junpeng , Lu Yahai 
Published: May 2012

ABSTRACTPropionate is one of the major intermediary products in the anaerobic decomposition of organic matter in wetlands and paddy fields. Under methanogenic conditions, propionate is decomposed through syntrophic interaction between proton-reducing and propionate-oxidizing bacteria and H2-consuming methanogens. Temperature is an important environmental regulator; yet its effect on syntrophic propionate oxidation has been poorly understood. In the present study, we investigated the syntrophic oxidation of propionate in a rice field soil at 15°C and 30°C. [U-13C]propionate (99 atom%) was applied to anoxic soil slurries, and the bacteria and archaea assimilating13C were traced by DNA-based stable isotope probing.Syntrophobacterspp.,Pelotomaculumspp., andSmithellaspp. were found significantly incorporating13C into their nucleic acids after [13C]propionate incubation at 30°C. The activity ofSmithellaspp. increased in the later stage, and concurrently that ofSyntrophomonasspp. increased. AceticlasticMethanosaetaceaeand hydrogenotrophicMethanomicrobialesandMethanocellalesacted as methanogenic partners at 30°C. Syntrophic oxidation of propionate also occurred actively at 15°C.Syntrophobacterspp. were significantly labeled with13C, whereasPelotomaculumspp. were less active at this temperature. In addition,Methanomicrobiales,Methanocellales, andMethanosarcinaceaedominated the methanogenic community, whileMethanosaetaceaedecreased. Collectively, temperature markedly influenced the activity and community structure of syntrophic guilds degrading propionate in the rice field soil. Interestingly,Geobacterspp. and some other anaerobic organisms likeRhodocyclaceae,Acidobacteria,Actinobacteria, andThermomicrobiaprobably also assimilated propionate-derived13C. The mechanisms for the involvement of these organisms remain unclear.
Novel Phytases from Bifidobacterium pseudocatenulatum ATCC 27919 and Bifidobacterium longum subsp.
infantis
ATCC 15697
Authors: Tamayo-Ramos Juan Antonio , Sanz-Penella Juan Mario , Yebra María J. , Monedero Vicente , Haros Monika 
Published: May 2012

ABSTRACT

Two novel phytases have been characterized from
Bifidobacterium pseudocatenulatum
and
Bifidobacterium longum
subsp.
infantis
. The enzymes belong to a new subclass within the histidine acid phytases, are highly specific for the hydrolysis of phytate, and render
myo
-inositol triphosphate as the final hydrolysis product. They represent the first phytases characterized from this group of probiotic microorganisms, opening the possibilities for their use in the processing of high-phytate-content foods.
Associations of Yeasts with Spotted-Wing Drosophila (Drosophila suzukii; Diptera: Drosophilidae) in Cherries and Raspberries
Authors: Hamby Kelly A. , Hernández Alejandro , Boundy-Mills Kyria , Zalom Frank G. 
Published: May 2012

ABSTRACTA rich history of investigation documents variousDrosophila-yeast mutualisms, suggesting thatDrosophila suzukiisimilarly has an association with a specific yeast species or community. To discover candidate yeast species, yeasts were isolated from larval frass, adult midguts, and fruit hosts ofD. suzukii. Terminal restriction fragment length polymorphism (TRFLP) technology and decimal dilution plating were used to identify and determine the relative abundance of yeast species present in fruit juice samples that were either infested withD. suzukiior not infested. Yeasts were less abundant in uninfested than infested samples. A total of 126 independent yeast isolates were cultivated from frass, midguts, and fruit hosts ofD. suzukii, representing 28 species of yeasts, withHanseniaspora uvarumpredominating. This suggests an association betweenD. suzukiiandH. uvarumthat could be utilized for pest management of the highly pestiferousD. suzukii.
Phylomark, a Tool To Identify Conserved Phylogenetic Markers from Whole-Genome Alignments
Authors: Sahl Jason W. , Matalka Malcolm N. , Rasko David A. 
Published: May 2012

ABSTRACT

The sequencing and analysis of multiple housekeeping genes has been routinely used to phylogenetically compare closely related bacterial isolates. Recent studies using whole-genome alignment (WGA) and phylogenetics from >100
Escherichia coli
genomes has demonstrated that tree topologies from WGA and multilocus sequence typing (MLST) markers differ significantly. A nonrepresentative phylogeny can lead to incorrect conclusions regarding important evolutionary relationships. In this study, the Phylomark algorithm was developed to identify a minimal number of useful phylogenetic markers that recapitulate the WGA phylogeny. To test the algorithm, we used a set of diverse draft and complete
E. coli
genomes. The algorithm identified more than 100,000 potential markers of different fragment lengths (500 to 900 nucleotides). Three molecular markers were ultimately chosen to determine the phylogeny based on a low Robinson-Foulds (RF) distance compared to the WGA phylogeny. A phylogenetic analysis demonstrated that a more representative phylogeny was inferred for a concatenation of these markers compared to all other MLST schemes for
E. coli
. As a functional test of the algorithm, the three markers (genomic guided
E. coli
markers, or GIG-EM) were amplified and sequenced from a set of environmental
E. coli
strains (ECOR collection) and informatically extracted from a set of 78 diarrheagenic
E. coli
strains (DECA collection). In the instances of the 40-genome test set and the DECA collection, the GIG-EM system outperformed other
E. coli
MLST systems in terms of recapitulating the WGA phylogeny. This algorithm can be employed to determine the minimal marker set for any organism that has sufficient genome sequencing.
A Straightforward DOPE (Double Labeling of Oligonucleotide Probes)-FISH (FluorescenceIn SituHybridization) Method for Simultaneous Multicolor Detection of Six Microbial Populations
Authors: Behnam Faris , Vilcinskas Andreas , Wagner Michael , Stoecker Kilian 
Published: May 2012

ABSTRACTFluorescencein situhybridization (FISH) with rRNA-targeted oligonucleotide probes is an essential tool for the cultivation-independent identification of microbes within environmental and clinical samples. However, one of the major constraints of conventional FISH is the very limited number of different target organisms that can be detected simultaneously with standard epifluorescence or confocal laser scanning microscopy. Recently, this limitation has been overcome via an elegant approach termed combinatorial labeling and spectral imaging FISH (CLASI-FISH) (23). This technique, however, suffers compared to conventional FISH from an inherent loss in sensitivity and potential probe binding biases caused by the competition of two differentially labeled oligonucleotide probes for the same target site. Here we demonstrate that the application of multicolored, double-labeled oligonucleotide probes enables the simultaneous detection of up to six microbial target populations in a straightforward and robust manner with higher sensitivity and less bias. Thus, this newly developed technique should be an attractive option for all researchers interested in applying conventional FISH methods for the study of microbial communities.
Sensitive Quantification of Clostridium difficile Cells by Reverse Transcription-Quantitative PCR Targeting rRNA Molecules
Authors: Matsuda Kazunori , Tsuji Hirokazu , Asahara Takashi , Takahashi Takuya , Kubota Hiroyuki , Nagata Satoru , Yamashiro Yuichiro , Nomoto Koji 
Published: May 2012

ABSTRACT

We established a sensitive and accurate quantification system for
Clostridium difficile
in human intestines, based on rRNA-targeted reverse transcription-quantitative PCR (RT-qPCR). We newly developed a species-specific primer set for
C. difficile
targeting 23S rRNA gene sequences. Both the vegetative cells and the spores of
C. difficile
in human feces were quantified by RT-qPCR, with a lower detection limit of 10
2.4
cells/g of feces. In an analysis of the feces of residents (
n
= 83; age, 85 ± 8 years) and staff (
n
= 19; age, 36 ± 10 years) at a care facility for the elderly,
C. difficile
was detected by RT-qPCR in 43% of the residents (average count, log
10
4.0 ± 2.0 cells/g of feces) and 16% of the staff (average count, log
10
2.2 ± 0.1 cells/g of feces); these rates were far higher than those detected by qPCR (residents, 19%; staff, 0%) or selective cultivation (residents, 18%; staff, 5%). Another analysis of healthy adults (
n
= 63; age, 41 ± 11 years) also revealed the significant carriage rate of
C. difficile
in the intestines (detection rate, 13%; average count, log
10
4.9 ± 1.2 cells/g of feces). From these results, it was suggested that rRNA-targeted RT-qPCR should be an effective tool for analyzing population levels of
C. difficile
in the human intestine.
Topological and Mutational Analysis of Saccharomyces cerevisiae Fks1
Authors: Johnson Michael E. , Edlind Thomas D. 
Published: May 2012

ABSTRACT

Fks1, with orthologs in nearly all fungi as well as plants and many protists, plays a central role in fungal cell wall formation as the putative catalytic component of β-1,3-glucan synthase. It is also the target for an important new antifungal group, the echinocandins, as evidenced by the localization of resistance-conferring mutations to Fks1 hot spots 1, 2, and 3 (residues 635 to 649, 1354 to 1361, and 690 to 700, respectively). Since Fks1 is an integral membrane protein and echinocandins are cyclic peptides with lipid tails, Fks1 topology is key to understanding its function and interaction with echinocandins. We used hemagglutinin (HA)-Suc2-His4C fusions to C-terminally truncated
Saccharomyces cerevisiae
Fks1 to experimentally define its topology and site-directed mutagenesis to test function of selected residues. Of the 15 to 18 transmembrane helices predicted
in silico
for Fks1 from evolutionarily diverse fungi, 13 were experimentally confirmed. The N terminus (residues 1 to 445) is cytosolic and the C terminus (residues 1823 to 1876) external; both are essential to Fks1 function. The cytosolic central domain (residues 715 to 1294) includes newly recognized homology to glycosyltransferases, and residues potentially involved in substrate UDP-glucose binding and catalysis are essential. All three hot spots are external, with hot spot 1 adjacent to and hot spot 3 largely embedded within the outer leaflet of the membrane. This topology suggests a model in which echinocandins interact through their lipid tails with hot spot 3 and through their cyclic peptides with hot spots 1 and 2.
ENVOY Is a Major Determinant in Regulation of Sexual Development in Hypocrea jecorina (
Trichoderma reesei
)
Authors: Seibel Christian , Tisch Doris , Kubicek Christian P. , Schmoll Monika 
Published: May 2012

ABSTRACT

Light is one crucial environmental signal which can determine whether a fungus reproduces asexually or initiates sexual development. Mating in the ascomycete
Hypocrea jecorina
(anamorph
Trichoderma reesei
) occurs preferentially in light. We therefore investigated the relevance of the light response machinery for sexual development in
H. jecorina
. We found that the photoreceptors BLR1 and BLR2 and the light-regulatory protein ENV1 have no effect on male fertility, while ENV1 is essential for female fertility. BLR1 and BLR2 were found to impact fruiting body formation although they are not essential for mating. Quantitative reverse transcription-PCR (qRT-PCR) analyses revealed that BLR1, BLR2, and ENV1 negatively regulate transcript levels of both pheromone receptors as well as peptide pheromone precursors in light but not in darkness and in a mating type-dependent manner. The effect of BLR1 and BLR2 on regulation of pheromone precursor and receptor genes is less severe than that of ENV1 as strains lacking
env1
show 100-fold (for
ppg1
) to more than 100,000-fold (for
hpp1
) increased transcript levels of pheromone precursor genes as well as more than 20-fold increased levels of
hpr1
, the pheromone receptor receiving the HPP1 signal in a MAT1-1 strain. ENV1 likely integrates additional signals besides light, and our results indicate that its function is partially mediated via regulation of
mat1-2-1
. We conclude that ENV1 is essential for balancing the levels of genes regulated in a mating-type-dependent manner, which contributes to determination of sexual identity and fruiting body formation.