bluuebunny/crossref_metadata_2025_split huggingface dataset:
Performance Assessment PCR-Based Assays Targeting
Bacteroidales
Genetic Markers of Bovine Fecal Pollution
Authors: Shanks Orin C. , White Karen , Kelty Catherine A. , Hayes Sam , Sivaganesan Mano , Jenkins Michael , Varma Manju , Haugland Richard A. 
Published: January 2010

ABSTRACT

There are numerous PCR-based assays available to characterize bovine fecal pollution in ambient waters. The determination of which approaches are most suitable for field applications can be difficult because each assay targets a different gene, in many cases from different microorganisms, leading to variation in assay performance. We describe a performance evaluation of seven end-point PCR and real-time quantitative PCR (qPCR) assays reported to be associated with either ruminant or bovine feces. Each assay was tested against a reference collection of DNA extracts from 247 individual bovine fecal samples representing 11 different populations and 175 fecal DNA extracts from 24 different animal species. Bovine-associated genetic markers were broadly distributed among individual bovine samples ranging from 39 to 93%. Specificity levels of the assays spanned 47.4% to 100%. End-point PCR sensitivity also varied between assays and among different bovine populations. For qPCR assays, the abundance of each host-associated genetic marker was measured within each bovine population and compared to results of a qPCR assay targeting 16S rRNA gene sequences from
Bacteroidales
. Experiments indicate large discrepancies in the performance of bovine-associated assays across different bovine populations. Variability in assay performance between host populations suggests that the use of bovine microbial source-tracking applications will require
a priori
characterization at each watershed of interest.
Detection of a Reproducible, Single-Member Shift in Soil Bacterial Communities Exposed to Low Levels of Hydrogen
Authors: Osborne Catherine A. , Peoples Mark B. , Janssen Peter H. 
Published: January 2010

ABSTRACT

Soil is exposed to hydrogen when symbiotic rhizobia in legume root nodules cannot recycle the hydrogen that is generated during nitrogen fixation. The hydrogen emitted is most likely taken up by free-living soil bacteria that use hydrogen as an energy source, though the bacteria that do this
in situ
remain unclear. In this study, we investigated the effect of hydrogen exposure on the bacteria of two different soils in a microcosm setup designed to simulate hydrogen-emitting root nodules. Although the size and overall composition of the soil bacterial community did not significantly alter after hydrogen exposure, one ribotype increased in relative abundance within each soil. This single-ribotype shift was identified by generating multiple terminal restriction fragment length polymorphism (T-RFLP) profiles of 16S rRNA genes from each soil sample, with gene sequence confirmation to identify terminal restriction fragments. The increased abundance of a single ribotype after hydrogen exposure, within an otherwise similar community, was found in replicate samples taken from each microcosm and was reproducible across replicate experiments. Similarly, only one member of the soil bacterial community increased in abundance in response to hydrogen exposure in soil surrounding the root nodules of field-grown soybean (
Glycine max
). The ribotypes that increased after hydrogen exposure in each soil system tested were all from known hydrogen-oxidizing lineages within the order
Actinomycetales
. We suggest that soil actinomycetes are important utilizers of hydrogen at relevant concentrations in soil and could be key contributors to soil's function as a sink in the global hydrogen cycle.
Genome Organization and Characterization of the Virulent Lactococcal Phage 1358 and Its Similarities to
Listeria
Phages
Authors: Dupuis Marie-Ève , Moineau Sylvain 
Published: January 2010

ABSTRACT

Virulent phage 1358 is the reference member of a rare group of phages infecting
Lactococcus lactis
. Electron microscopy revealed a typical icosahedral capsid connected to one of the smallest noncontractile tails found in a lactococcal phage of the
Siphoviridae
family. Microbiological characterization identified a burst size of 72 virions released per infected host cell and a latent period of 90 min. The host range of phage 1358 was limited to 3 out of the 60 lactococcal strains tested. Moreover, this phage was insensitive to four Abi systems (AbiK, AbiQ, AbiT, and AbiV). The genome of phage 1358 consisted of a linear, double-stranded, 36,892-bp DNA molecule containing 43 open reading frames (ORFs). At least 14 ORFs coded for structural proteins, as identified by SDS-PAGE coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses. The genomic organization was similar to those of other siphophages. All genes were on the same coding strand and in the same orientation. This lactococcal phage was unique, however, in its 51.4% GC content, much higher than those of other phages infecting this low-GC Gram-positive host. A bias for GC-rich codons was also observed. Comparative analyses showed that several phage 1358 structural proteins shared similarity with two
Listeria monocytogenes
phages, P35 and P40. The possible origin and evolution of lactococcal phage 1358 is discussed.
Viral Multiplex Quantitative PCR Assays for Tracking Sources of Fecal Contamination
Authors: Wolf Sandro , Hewitt Joanne , Greening Gail E. 
Published: January 2010

ABSTRACT
Human and animal fecal pollution of the environment presents a risk to human health because of the presence of pathogenic viruses and bacteria. To distinguish between human and animal sources of pollution, we designed specific real-time reverse transcription (RT)-PCR assays for human and animal enteric viruses, including norovirus genogroups I, II, and III; porcine adenovirus types 3 and 5; ovine adenovirus; atadenovirus; and human adenovirus species C and F, which are excreted by infected humans, pigs, cattle, sheep, deer, and goats, and for the detection of F+ RNA bacteriophage genogroups I to IV, which are associated with human and animal wastes. The sensitivity of this viral toolbox (VTB) was tested against 10-fold dilution series of DNA plasmids that carry the target sequences of the respective viruses and was shown to detect at least 10 plasmid copies for each assay. A panel of human and animal enteric and respiratory viruses showed these assays to be highly sensitive and specific to their respective targets. The VTB was used to detect viruses in fecal and environmental samples, including raw sewage and biosolids from municipal sewage treatment plants, abattoir sewage, and fecally contaminated shellfish and river water, which were likely to contain animal or human viruses.
Growth of
Arthrobacter
sp. Strain JBH1 on Nitroglycerin as the Sole Source of Carbon and Nitrogen
Authors: Husserl Johana , Spain Jim C. , Hughes Joseph B. 
Published: January 2010

ABSTRACT

Arthrobacter
sp. strain JBH1 was isolated from nitroglycerin-contaminated soil by selective enrichment. Detection of transient intermediates and simultaneous adaptation studies with potential intermediates indicated that the degradation pathway involves the conversion of nitroglycerin to glycerol via 1,2-dinitroglycerin and 1-mononitroglycerin, with concomitant release of nitrite. Glycerol then serves as the source of carbon and energy.
Transcriptome Analysis of
Escherichia coli
O157:H7 Exposed to Lysates of Lettuce Leaves
Authors: Kyle Jennifer L. , Parker Craig T. , Goudeau Danielle , Brandl Maria T. 
Published: January 2010

ABSTRACT

Harvesting and processing of leafy greens inherently cause plant tissue damage, creating niches on leaves that human pathogens can exploit. We previously demonstrated that
Escherichia coli
O157:H7 (EcO157) multiplies more rapidly on shredded leaves than on intact leaves (M. T. Brandl, Appl. Environ. Microbiol.
74:
5285-5289, 2008). To investigate how EcO157 cells adapt to physicochemical conditions in injured lettuce tissue, we used microarray-based whole-genome transcriptional profiling to characterize gene expression patterns in EcO157 after 15- and 30-min exposures to romaine lettuce lysates. Multiple carbohydrate transport systems that have a role in the utilization of substrates known to be prevalent in plant cells were activated in EcO157. This indicates the availability to the human pathogen of a variety of carbohydrates released from injured plant cells that may promote its extensive growth in leaf lysates and, thus, in wounded leaf tissue. In addition, microarray analysis revealed the upregulation of numerous genes associated with EcO157 attachment and virulence, with oxidative stress and antimicrobial resistance (including the OxyR and Mar regulons), with detoxification of noxious compounds, and with DNA repair. Upregulation of oxidative stress and antimicrobial resistance genes in EcO157 was confirmed on shredded lettuce by quantitative reverse transcription-PCR. We further demonstrate that this adaptation to stress conditions imparts the pathogen with increased resistance to hydrogen peroxide and calcium hypochlorite. This enhanced resistance to chlorinated sanitizers combined with increased expression of virulence determinants and multiplication at sites of injury on the leaves may help explain the association of processed leafy greens with outbreaks of EcO157.
Prevalence ofBartonella henselaeandBorrelia burgdorferiSensu Lato DNA inIxodes ricinusTicks in Europe
Authors: Dietrich Florian , Schmidgen Thomas , Maggi Ricardo G. , Richter Dania , Matuschka Franz-Rainer , Vonthein Reinhard , Breitschwerdt Edward B. , Kempf Volkhard A. J. 
Published: January 2010

ABSTRACTBartonellaspp. can cause persistent bloodstream infections in humans and animals. To determine whetherBartonella henselaeis present in questingIxodes ricinusticks, we analyzed the prevalence ofB. henselaeDNA among tick stages compared to the prevalence of DNA fromBorrelia burgdorferisensu lato, the pathogen most frequently transmitted by ticks.B. henselaeDNA was present with a prevalence of up to ∼40% in tick populations sampled in four European sites (Eberdingen, Germany; Klasdorf, Germany; Lembach, France; and Madeira, Portugal). The odds of detectingB. henselaeDNA in nymphal ticks was ∼14-fold higher than in adult ticks. No tick was found to be coinfected withB. henselaeandB. burgdorferisensu lato. Taken together, our data indicate that ticks might serve as a vector for the transmission ofB. henselaeto humans.
Galactoxylomannans from Cryptococcus neoformans Varieties
neoformans
and
grubii
Are Structurally and Antigenically Variable
Authors: De Jesus Magdia , Chow Siu-Kei , Cordero Radames J. B. , Frases Susana , Casadevall Arturo 
Published: January 2010

ABSTRACT

Prior studies have established that the
Cryptococcus neoformans
capsular polysaccharide component galactoxylomannan (GalXM) manifests serotype-related structural differences that translate into antigenic differences. We analyzed GalXM from acapsular serotype A and D strains by carbohydrate analysis and static and dynamic light scattering to determine mass, effective diameter, polydispersity, and diffusion coefficients. Multiangle laser light scattering showed that GalXM from
C. neoformans
var.
grubii
strain cap59 (serotype A) had larger molecular mass (4.21 × 10
6
± 0.95 × 10
6
g/mol) and radius of gyration (207 ± 27 nm) than GalXM from
C. neoformans
var.
neoformans
cap67 (serotype D). cap67 GalXM had corresponding values of 0.70 × 10
6
± 0.05 × 10
6
g/mol and 120 ± 22 nm, respectively. The effective diameter for GalXM and polydispersity from the two strains varied depending on temperature and medium growth conditions, indicating that GalXM structure can vary within a strain, depending on its environment. Zeta potential determinations were negative for GalXM from both strains under all conditions, consistent with the recently reported presence of glucuronic acid. These results imply that
C. neoformans
GalXM, like glucuronoxylomannan, can manifest variety- and growth condition-related variations. Analysis of 16
C. neoformans
and 7
Cryptococcus gattii
strains with polyclonal antibody to a GalXM strain revealed antigenic similarities among the
C. neoformans
variety
neoformans
and
grubii
strains and no reactivity with
C. gattii
. As a result of the deleterious effects of GalXM on immune function, structural and antigenic variability between serotypes may translate into differences in immunomodulatory effects.
Global Change of Gene Expression and Cell Physiology in YidC-DepletedEscherichia coli
Authors: Wang Peng , Kuhn Andreas , Dalbey Ross E. 
Published: January 2010

ABSTRACTYidC depletion affects membrane protein insertion and leads to a defect in the growth of theEscherichia colicell. We analyzed global changes in gene expression upon YidC depletion to determine the importance of YidC for cellular functions using a gene chip method to compare the transcriptomes of JS71 (control) and JS7131 (yidCdepletion strain). Of the more than 4,300 genes identified, 163 were upregulated and 99 were downregulated upon YidC depletion, including genes which are responsible for DNA/RNA repair; energy metabolism; various transporters, proteases and chaperones; stress response; and translation and transcription functions. Real-time PCR was performed on selected genes to confirm the results. Specifically, we found upregulation of the genes encoding the energy transduction proteins F1FoATP synthase and cytochromebo3oxidase due to perturbation in assembly when YidC was depleted. We also determined that the high-level induction of the PspA stress protein under YidC depletion conditions is roughly 10-fold higher than the activation due to the addition of protonophore carbonyl cyanidem-chlorophenylhydrazone (CCCP), which dissipates the proton motive force. In addition, the gene chip data reveal the Cpx stress pathway is activated upon YidC depletion. The data show the broad physiological contribution of YidC to the bacterial cell and the considerable ramification to the cell when it is depleted.
Characterization of a Thioredoxin-Thioredoxin Reductase System from the Hyperthermophilic BacteriumThermotoga maritima
Authors: Yang Xianqin , Ma Kesen 
Published: January 2010

ABSTRACTA thioredoxin reductase and a thioredoxin were purified to homogeneity from a cell extract ofThermotoga maritima. The thioredoxin reductase was a homodimeric flavin adenine dinucleotide (FAD)-containing protein with a subunit of 37 kDa estimated using SDS-PAGE, which was identified to be TM0869. The amino acid sequence of the enzyme showed high identities and similarities to those of typical bacterial thioredoxin reductases. Although the purifiedT. maritimathioredoxin reductase could not use thioredoxin fromSpirulinaas an electron acceptor, it used thioredoxin that was purified fromT. maritimaby monitoring the dithiothreitol-dependent reduction of bovine insulin. This enzyme also catalyzed the reduction of benzyl viologen using NADH or NADPH as an electron donor with apparentVmaxvalues of 1,111 ± 35 μmol NADH oxidized min−1mg−1and 115 ± 2.4 μmol NADPH oxidized min−1mg−1, respectively. The apparentKmvalues were determined to be 89 ± 1.1 μM, 73 ± 1.6 μM, and 780 ± 20 μM for benzyl viologen, NADH, and NADPH, respectively. Optimal pH values were determined to be 9.5 and 6.5 for NADH and NADPH, respectively. The enzyme activity increased along with the rise of temperature up to 95°C, and more than 60% of the activity remained after incubation for 28 h at 80°C. The purifiedT. maritimathioredoxin was a monomer with a molecular mass of 31 kDa estimated using SDS-PAGE and identified as TM0868, which exhibited both thioredoxin and thioltransferase activities.T. maritimathioredoxin and thioredoxin reductase together were able to reduce insulin or 5,5′-dithio-bis(2-nitrobenzoic acid) using NAD(P)H as an electron donor. This is the first thioredoxin-thioredoxin reductase system characterized from hyperthermophilic bacteria.
The Cyclic-di-GMP Phosphodiesterase BinA Negatively Regulates Cellulose-Containing Biofilms inVibrio fischeri
Authors: Bassis Christine M. , Visick Karen L. 
Published: January 2010

ABSTRACTBacteria produce different types of biofilms under distinct environmental conditions.Vibrio fischerihas the capacity to produce at least two distinct types of biofilms, one that relies on the symbiosis polysaccharide Syp and another that depends upon cellulose. A key regulator of biofilm formation in bacteria is the intracellular signaling molecule cyclic diguanylate (c-di-GMP). In this study, we focused on a predicted c-di-GMP phosphodiesterase encoded by the genebinA, located directly downstream ofsyp, a cluster of 18 genes critical for biofilm formation and the initiation of symbiotic colonization of the squidEuprymna scolopes. Disruption or deletion ofbinAincreased biofilm formation in culture and led to increased binding of Congo red and calcofluor, which are indicators of cellulose production. Using random transposon mutagenesis, we determined that the phenotypes of the ΔbinAmutant strain could be disrupted by insertions in genes in the bacterial cellulose biosynthesis cluster (bcs), suggesting that cellulose production is negatively regulated by BinA. Replacement of critical amino acids within the conserved EAL residues of the EAL domain disrupted BinA activity, and deletion ofbinAincreased c-di-GMP levels in the cell. Together, these data support the hypotheses that BinA functions as a phosphodiesterase and that c-di-GMP activates cellulose biosynthesis. Finally, overexpression of thesypregulatorsypGinducedbinAexpression. Thus, this work reveals a mechanism by whichV. fischeriinhibits cellulose-dependent biofilm formation and suggests that the production of two different polysaccharides may be coordinated through the action of the cellulose inhibitor BinA.
PepD Participates in the Mycobacterial Stress Response Mediated through MprAB and SigE
Authors: White Mark J. , He Hongjun , Penoske Renee M. , Twining Sally S. , Zahrt Thomas C. 
Published: January 2010

ABSTRACT

Currently, one-third of the world's population is believed to be latently infected with
Mycobacterium tuberculosis
. The mechanisms by which
M. tuberculosis
establishes latent infection remain largely undefined.
mprAB
encodes a two-component signal transduction system required by
M. tuberculosis
for aspects of persistent infection. MprAB regulates a large and diverse group of genetic determinants in response to membrane stress, including the extracytoplasmic function (ECF) sigma factor
sigE
and the HtrA-like serine protease
pepD
. Recent studies have demonstrated that PepD functions as both a protease and chaperone
in vitro
. In addition, inactivation of
pepD
alters the virulence of
M. tuberculosis
in a mouse model system of infection. Here, we demonstrate that PepD plays an important role in the stress response network of
Mycobacterium
mediated through MprAB and SigE. In particular, we demonstrate that the protease activity of PepD requires the PDZ domain, in addition to the catalytic serine at position 317.
pepD
expression initiates from at least three promoters in
M. tuberculosis
, including one that is regulated by SigE and is located upstream of the
mprA
coding sequence. Deletion of
pepD
or
mprAB
in
Mycobacterium smegmatis
and
M. tuberculosis
alters the stress response phenotypes of these strains, including increasing sensitivity to SDS and cell wall antibiotics and upregulating the expression of stress-responsive determinants, including
sigE
. Taking these data together, we hypothesize that PepD utilizes its PDZ domain to recognize and process misfolded proteins at the cell membrane, leading to activation of the MprAB and SigE signaling pathways and subsequent establishment of a positive feedback loop that facilitates bacterial adaptation.
Modified 3-Oxoadipate Pathway for the Biodegradation of Methylaromatics inPseudomonas reinekeiMT1
Authors: Marín Macarena , Pérez-Pantoja Danilo , Donoso Raul , Wray Victor , González Bernardo , Pieper Dietmar H. 
Published: January 2010

ABSTRACTCatechols are central intermediates in the metabolism of aromatic compounds. Degradation of 4-methylcatechol via intradiol cleavage usually leads to the formation of 4-methylmuconolactone (4-ML) as a dead-end metabolite. Only a few microorganisms are known to mineralize 4-ML. Themmlgene cluster ofPseudomonasreinekeiMT1, which encodes enzymes involved in the metabolism of 4-ML, is shown here to encode 10 genes found in a 9.4-kb chromosomal region. Reverse transcription assays revealed that these genes form a single operon, where their expression is controlled by two promoters. Promoter fusion assays identified 4-methyl-3-oxoadipate as an inducer. Mineralization of 4-ML is initiated by the 4-methylmuconolactone methylisomerase encoded bymmlI. This reaction produces 3-ML and is followed by a rearrangement of the double bond catalyzed by the methylmuconolactone isomerase encoded bymmlJ. Deletion ofmmlL, encoding a protein of the metallo-β-lactamase superfamily, resulted in a loss of the capability of the strain MT1 to open the lactone ring, suggesting its function as a 4-methyl-3-oxoadipate enol-lactone hydrolase. Further metabolism can be assumed to occur by analogy with reactions known from the 3-oxoadipate pathway.mmlFandmmlGprobably encode a 4-methyl-3-oxoadipyl-coenzyme A (CoA) transferase, and themmlCgene product functions as a thiolase, transforming 4-methyl-3-oxoadipyl-CoA into methylsuccinyl-CoA and acetyl-CoA, as indicated by the accumulation of 4-methyl-3-oxoadipate in the respective deletion mutant. Accumulation of methylsuccinate by anmmlKdeletion mutant indicates that the encoded acetyl-CoA hydrolase/transferase is crucial for channeling methylsuccinate into the central metabolism.
FimR and FimS: Biofilm Formation and Gene Expression inPorphyromonas gingivalis
Authors: Lo Alvin , Seers Christine , Dashper Stuart , Butler Catherine , Walker Glenn , Walsh Katrina , Catmull Deanne , Hoffmann Brigitte , Cleal Steven , Lissel Patricia , Boyce John , Reynolds Eric 
Published: January 2010

ABSTRACTPorphyromonas gingivalisis a late-colonizing bacterium of the subgingival dental plaque biofilm associated with periodontitis. TwoP. gingivalisgenes,fimRandfimS, are predicted to encode a two-component signal transduction system comprising a response regulator (FimR) and a sensor histidine kinase (FimS). In this study, we show thatfimSandfimR, although contiguous on the genome, are not part of an operon. We inactivatedfimRandfimSin both the afimbriated strain W50 and the fimbriated strain ATCC 33277 and demonstrated that both mutants formed significantly less biofilm than their respective wild-type strains. Quantitative reverse transcription-real-time PCR showed that expression of fimbriation genes was reduced in both thefimSandfimRmutants of strain ATCC 33277. The mutations had no effect, in either strain, on theP. gingivalisgrowth rate or on the response to hydrogen peroxide or growth at pH 9, at 41°C, or at low hemin availability. Transcriptome analysis using DNA microarrays revealed that inactivation offimSresulted in the differential expression of 10% of theP. gingivalisgenome (>1.5-fold;P< 0.05). Notably genes encoding seven different transcriptional regulators, including thefimRgene and three extracytoplasmic sigma factor genes, were differentially expressed in thefimSmutant.