bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification and Characterization of Shiga Toxin Type 2 Variants in
Escherichia coli
Isolates from Animals, Food, and Humans
Authors: Zheng Jie , Cui Shenghui , Teel Louise D. , Zhao Shaohua , Singh Ruby , O'Brien Alison D. , Meng Jianghong 
Published: July 2008

ABSTRACT

There is considerable heterogeneity among the Shiga toxin type 2 (Stx2) toxins elaborated by Shiga toxin-producing
Escherichia coli
(STEC). One such Stx2 variant, the Stx2d mucus-activatable toxin (Stx2dact), is rendered more toxic by the action of elastase present in intestinal mucus, which cleaves the last two amino acids of the A2 portion of the toxin A subunit. We screened 153 STEC isolates from food, animals, and humans for the gene encoding Stx2dact by using a novel one-step PCR procedure. This method targeted the region of
stx
2dact
that encodes the elastase recognition site. The presence of
stx
2dact
was confirmed by DNA sequencing of the complete toxin genes. Seven STEC isolates from cows (four isolates), meat (two isolates), and a human (one isolate) that carried the putative
stx
2dact
gene were identified; all were
eae
negative, and none was the O157:H7 serotype. Three of the isolates (CVM9322, CVM9557, and CVM9584) also carried
stx
1
, two (P1332 and P1334) carried
stx
1
and
stx
2c
, and one (CL-15) carried
stx
2c
. One isolate, P1130, harbored only
stx
2dact
. The Vero cell cytotoxicities of supernatants from P1130 and
stx
1
deletion mutants of CVM9322, CVM9557, and CVM9584 were increased 13- to 30-fold after treatment with porcine elastase. Thus, Stx2dact-producing strains, as detected by our one-step PCR method, can be isolated not only from humans, as previously documented, but also from food and animals. The latter finding has important public health implications based on a recent report from Europe of a link between disease severity and infection with STEC isolates that produce Stx2dact.
Morphological Transitions Governed by Density Dependence and Lipoxygenase Activity inAspergillus flavus
Authors: Horowitz Brown S. , Zarnowski R. , Sharpee W. C. , Keller N. P. 
Published: July 2008

ABSTRACTAspergillus flavusdifferentiates to produce asexual dispersing spores (conidia) or overwintering survival structures called sclerotia. Results described here show that these two processes are oppositely regulated by density-dependent mechanisms and that increasing the cell density (from 101to 107cells/plate) results in the lowest numbers of sclerotial and the highest numbers of conidial. Extract from spent medium of low-cell-density cultures induced a high-sclerotium-number phenotype, whereas high-cell-density extract increased conidiation. Density-dependent development is also modified by changes in lipid availability. Exogenous linoleic acid increased sclerotial production at intermediate cell densities (104and 105cells/plate), whereas oleic and linolenic acids inhibited sclerotium formation. Deletion of Afloxencoding a lipoxygenase (LOX) greatly diminished density-dependent development of both sclerotia and conidia, resulting in an overall increase in the number of sclerotia and a decrease in the number of conidia at high cell densities (>105cells/plate). Afloxmutants showed decreased linoleic acid LOX activity. Taken together, these results suggest that there is a quorum-sensing mechanism in which a factor(s) produced in dense cultures, perhaps a LOX-derived metabolite, activates conidium formation, while a factor(s) produced in low-density cultures stimulates sclerotium formation.
Extraction of RNA from Cheese without Prior Separation of Microbial Cells
Authors: Monnet Christophe , Ulvé Vincent , Sarthou Anne-Sophie , Irlinger Françoise 
Published: July 2008

ABSTRACT

In situ gene expression studies are promising approaches for improving our understanding of the cheese microbial flora. This requires efficient RNA extraction methods, but studies of cheeses are scarce. The objective of the present study was to determine whether RNA samples compatible with quantitative mRNA transcript analyses can be obtained without separating the cells from the cheese matrix. In the method that we describe, the cellular processes are stopped at the very beginning of the procedure. When cheeses were produced with
Lactococcus lactis
LD61 as the only starter microorganism, the integrity of the purified RNA was good, even for 2-week-old cheeses that had been incubated at 30°C. In addition, the RNA samples did not contain any traces of RNases, and the amount of genomic DNA was negligible. A good level of reproducibility could also be achieved. When real-time reverse transcription-PCR analyses were normalized to the total RNA concentration, the amounts of 16S and 23S rRNA transcripts were constant during the 2-week incubation period, whereas the amount of
tuf
mRNA transcripts decreased substantially. RNA samples obtained using the method described in this study were compared to samples obtained using the method described by Ulvé et al. (J. Appl. Microbiol., in press), which is based on separation of the cells from the cheese matrix. For most of the 29 genes investigated, the transcript abundance was the same for both types of samples. Differences were observed mainly for genes whose expression has previously been shown to be modified by heat, acid, or osmotic stresses, such as
busAA
and
glnQ
.
Microbial Community Analysis of Field-Grown Soybeans with Different Nodulation Phenotypes
Authors: Ikeda Seishi , Rallos Lynn Esther E. , Okubo Takashi , Eda Shima , Inaba Shoko , Mitsui Hisayuki , Minamisawa Kiwamu 
Published: July 2008

ABSTRACT

Microorganisms associated with the stems and roots of nonnodulated (Nod
−
), wild-type nodulated (Nod
+
), and hypernodulated (Nod
++
) soybeans [
Glycine max
(L.) Merril] were analyzed by ribosomal intergenic transcribed spacer analysis (RISA) and automated RISA (ARISA). RISA of stem samples detected no bands specific to the nodulation phenotype, whereas RISA of root samples revealed differential bands for the nodulation phenotypes.
Pseudomonas fluorescens
was exclusively associated with Nod
+
soybean roots.
Fusarium solani
was stably associated with nodulated (Nod
+
and Nod
++
) roots and less abundant in Nod
−
soybeans, whereas the abundance of basidiomycetes was just the opposite. The phylogenetic analyses suggested that these basidiomycetous fungi might represent a root-associated group in the
Auriculariales
. Principal-component analysis of the ARISA results showed that there was no clear relationship between nodulation phenotype and bacterial community structure in the stem. In contrast, both the bacterial and fungal community structures in the roots were related to nodulation phenotype. The principal-component analysis further suggested that bacterial community structure in roots could be classified into three groups according to the nodulation phenotype (Nod
−
, Nod
+
, or Nod
++
). The analysis of root samples indicated that the microbial community in Nod
−
soybeans was more similar to that in Nod
++
soybeans than to that in Nod
+
soybeans.
Escherichia coliConstructs Expressing Human or Porcine Enterotoxins Induce Identical Diarrheal Diseases in a Piglet Infection Model
Authors: Zhang Weiping , Robertson Donald C. , Zhang Chengxian , Bai Wei , Zhao Mojun , Francis David H. 
Published: July 2008

ABSTRACTTo develop a piglet model for studying diarrheal disease and developing vaccines, we challenged gnotobiotic piglets with isogenicEscherichia colistrains constructed to express porcine 987P(F6) fimbriae and a heat-labile or a heat-stable enterotoxin to examine clinical outcomes. Piglets developed identical diarrheal diseases when inoculated with constructs expressing human or porcine enterotoxins.
Graphite Electrode as a Sole Electron Donor for Reductive Dechlorination of Tetrachlorethene by
Geobacter lovleyi
Authors: Strycharz Sarah M. , Woodard Trevor L. , Johnson Jessica P. , Nevin Kelly P. , Sanford Robert A. , Löffler Frank E. , Lovley Derek R. 
Published: July 2008

ABSTRACT

The possibility that graphite electrodes can serve as the direct electron donor for microbially catalyzed reductive dechlorination was investigated with
Geobacter lovleyi
. In an initial evaluation of whether
G. lovleyi
could interact electronically with graphite electrodes, cells were provided with acetate as the electron donor and an electrode as the sole electron acceptor. Current was produced at levels that were ca. 10-fold lower than those previously reported for
Geobacter sulfurreducens
under similar conditions, and
G. lovleyi
anode biofilms were correspondingly thinner. When an electrode poised at −300 mV (versus a standard hydrogen electrode) was provided as the electron donor,
G. lovleyi
effectively reduced fumarate to succinate. The stoichiometry of electrons consumed to succinate produced was 2:1, the ratio expected if the electrode served as the sole electron donor for fumarate reduction.
G. lovleyi
effectively reduced tetrachloroethene (PCE) to
cis
-dichloroethene with a poised electrode as the sole electron donor at rates comparable to those obtained when acetate serves as the electron donor. Cells were less abundant on the electrodes when the electrodes served as an electron donor than when they served as an electron acceptor. PCE was not reduced in controls without cells or when the current supply to cells was interrupted. These results demonstrate that
G. lovleyi
can use a poised electrode as a direct electron donor for reductive dechlorination of PCE. The ability to colocalize dechlorinating microorganisms with electrodes has several potential advantages for bioremediation of subsurface chlorinated contaminants, especially in source zones where electron donor delivery is challenging and often limits dechlorination.
Effects ofMedicago truncatulaGenetic Diversity, Rhizobial Competition, and Strain Effectiveness on the Diversity of a NaturalSinorhizobiumSpecies Community
Authors: Rangin Cécile , Brunel Brigitte , Cleyet-Marel Jean-Claude , Perrineau Marie-Mathilde , Béna Gilles 
Published: July 2008

ABSTRACTWe investigated the genetic diversity and symbiotic efficiency of 223Sinorhizobiumsp. isolates sampled from a single Mediterranean soil and trapped with fourMedicago truncatulalines. DNA molecular polymorphism was estimated by capillary electrophoresis-single-stranded conformation polymorphism and restriction fragment length polymorphism on five loci (IGSNOD,typA,virB11,avhB11, and the 16S rRNA gene). More than 90% of the rhizobia isolated belonged to theSinorhizobium medicaespecies (others belonged toSinorhizobium meliloti), with different proportions of the two species among the fourM. truncatulalines. TheS. melilotipopulation was more diverse than that ofS. medicae, and significant genetic differentiation among bacterial populations was detected. Single inoculations performed in tubes with each bacterial genotype and each plant line showed significant bacterium-plant line interactions for nodulation and N2fixation levels. Competition experiments within each species highlighted either strong or weak competition among genotypes withinS. medicaeandS. meliloti, respectively. Interspecies competition experiments showedS. melilotito be more competitive thanS. medicaefor nodulation. Although not highly divergent at a nucleotide level, isolates collected from this single soil sample displayed wide polymorphism for both nodulation and N2fixation. EachM. truncatulaline might influenceSinorhizobiumsoil population diversity differently via its symbiotic preferences. Our data suggested that the two species did not evolve similarly, withS. melilotishowing polymorphism and variable selective pressures andS. medicaeshowing traces of a recent demographic expansion. Strain effectiveness might have played a role in the species and genotype proportions, but in conjunction with strain adaptation to environmental factors.
Construction of
arsB
and
tetH
Mutants of the Sulfur-Oxidizing Bacterium
Acidithiobacillus caldus
by Marker Exchange
Authors: van Zyl Leonardo J. , van Munster Jolanda M. , Rawlings Douglas E. 
Published: July 2008

ABSTRACT

Acidithiobacillus caldus
is a moderately thermophilic, acidophilic bacterium that has been reported to be the dominant sulfur oxidizer in stirred-tank processes used to treat gold-bearing arsenopyrite ores. It is also widely distributed in heap reactors used for the extraction of metals from ores. Not only are these bacteria commercially important, they have an interesting physiology, the study of which has been restricted by the nonavailability of defined mutants. A recently reported conjugation system based on the broad-host-range IncW plasmids pSa and R388 was used to transfer mobilizable narrow-host-range suicide plasmid vectors containing inactivated and partially deleted chromosomal genes from
Escherichia coli
to
A. caldus
. Through the dual use of a selectable kanamycin resistance gene and a hybridization probe made from a deleted portion of the target chromosomal gene, single- and double-recombinant mutants of
A. caldus
were isolated. The functionality of the gene inactivation system was shown by the construction of
A. caldus arsB
and
tetH
mutants, and the effects of these mutations on cell growth in the presence of arsenic and by means of tetrathionate oxidation were demonstrated.
Temporal Changes in the Ectomycorrhizal Community in Two Soil Horizons of a Temperate Oak Forest
Authors: Courty Pierre-Emmanuel , Franc Alain , Pierrat Jean-Claude , Garbaye Jean 
Published: July 2008

ABSTRACT

The species structure of an ectomycorrhizal (ECM) community was assessed monthly for 15 months in the two horizons (A1 and A2) of an oak temperate forest in northeastern France. Ectomycorrhizal species were identified each month by internal transcribed spacer sequencing. Seventy-five fungal symbionts were identified. The community was dominated by Tomentellaceae, Russulaceae, Cortinariaceae, and Boletales. Four species are abundant in the study site:
Lactarius quietus
,
Tomentella sublilacina
,
Cenococcum geophilum
, and
Russula
sp1. The relative abundance of each species varied depending on the soil horizon and over time. Some species, such as
L. quietus
, were present in the A1 and A2 horizons.
C. geophilum
was located particularly in the A2 horizon, whereas
T. sublilacina
was more abundant in A1. Some species, such as
Clavulina
sp., were detected in winter, while
T. sublilacina
and
L. quietus
were present all year long. Our results support the hypothesis that a rapid turnover of species composition of the ECM community occurs over the course of a month. The spatial and temporal unequal distribution of ECM species could be explained by their ecological preferences, driven by such factors as root longevity, competition for resources, and resistance to environmental variability.
Heterotrimeric G-Protein Subunit Function in
Candida albicans
: both the α and β Subunits of the Pheromone Response G Protein Are Required for Mating
Authors: Dignard Daniel , André Dominique , Whiteway Malcolm 
Published: July 2008

ABSTRACT

A pheromone-mediated signaling pathway that couples seven-transmembrane-domain (7-TMD) receptors to a mitogen-activated protein kinase module controls
Candida albicans
mating. 7-TMD receptors are typically connected to heterotrimeric G proteins whose activation regulates downstream effectors. Two Gα subunits in
C. albicans
have been identified previously, both of which have been implicated in aspects of pheromone response. Cag1p was found to complement the mating pathway function of the pheromone receptor-coupled Gα subunit in
Saccharomyces cerevisiae
, and Gpa2p was shown to have a role in the regulation of cyclic AMP signaling in
C. albicans
and to repress pheromone-mediated arrest. Here, we show that the disruption of
CAG1
prevented mating, inactivated pheromone-mediated arrest and morphological changes, and blocked pheromone-mediated gene expression changes in opaque cells of
C. albicans
and that the overproduction of
CAG1
suppressed the hyperactive cell cycle arrest exhibited by
sst2
mutant cells. Because the disruption of the
STE4
homolog constituting the only
C. albicans
gene for a heterotrimeric Gβ subunit also blocked mating and pheromone response, it appears that in this fungal pathogen the Gα and Gβ subunits do not act antagonistically but, instead, are both required for the transmission of the mating signal.
Characterization of anEntamoeba histolyticaHigh-Mobility-Group Box Protein Induced during Intestinal Infection
Authors: Abhyankar Mayuresh M. , Hochreiter Amelia E. , Hershey Jessica , Evans Clive , Zhang Yan , Crasta Oswald , Sobral Bruno W. S. , Mann Barbara J. , Petri William A. , Gilchrist Carol A. 
Published: July 2008

ABSTRACTThe unicellular eukaryoteEntamoeba histolyticais a human parasite that causes amebic dysentery and liver abscess. A genome-wide analysis of gene expression modulated by intestinal colonization and invasion identified an upregulated transcript that encoded a putative high-mobility-group box (HMGB) protein, EhHMGB1. We tested if EhHMGB1 encoded a functional HMGB protein and determined its role in control of parasite gene expression. Recombinant EhHMGB1 was able to bend DNA in vitro, a characteristic of HMGB proteins. Core conserved residues required for DNA bending activity in other HMGB proteins were demonstrated by mutational analysis to be essential for EhHMGB1 activity. EhHMGB1 was also able to enhance the binding of human p53 to its cognate DNA sequence in vitro, which is expected for an HMGB1 protein. Confocal microscopy, using antibodies against the recombinant protein, confirmed its nuclear localization. Overexpression of EhHMGB1 in HM1:IMSS trophozoites led to modulation of 33 transcripts involved in a variety of cellular functions. Of these, 20 were also modulated at either day 1 or day 29 in the mouse model of intestinal amebiasis. Notably, four transcripts with known roles in virulence, including two encoding Gal/GalNAc lectin light chains, were modulated in response to EhHMGB1 overexpression. We concluded that EhHMGB1 was a bona fide HMGB protein with the capacity to recapitulate part of the modulation of parasite gene expression seen during adaptation to the host intestine.
The RGS Protein Crg2 Regulates Pheromone and Cyclic AMP Signaling in
Cryptococcus neoformans
Authors: Shen Gui , Wang Yan-Li , Whittington Amy , Li Lie , Wang Ping 
Published: July 2008

ABSTRACT

Crg1 and Crg2 are regulators of G-protein signaling homologs found in the human fungal pathogen
Cryptococcus neoformans
. Crg1 negatively regulates pheromone responses and mating through direct inhibition of Gα subunits Gpa2 and Gpa3. It has also been proposed that Crg2 has a role in mating, as genetic crosses involving
Δcrg2
mutants resulted in formation of hyperfilaments. We found that mutation of Gpa2 and Gpa3 partially suppressed the hyperfilamentation, mutation of Gpa3 alleviated
Δcrg2
-specfic cell swelling, and mutation of the mitogen-activated protein kinase Cpk1 blocked both processes. These findings indicate that Gpa2 and Gpa3 function downstream of Crg2 and that Gpa3 is also epistatic to Crg2 in a Cpk1-dependent morphogenesis process linked to mating. Significantly, we found that
Δcrg2
mutants formed enlarged capsules that mimic cells expressing a constitutively active
GPA1
(
Q284L
) allele and that the levels of intracellular cyclic AMP (cAMP) were also elevated, suggesting that Crg2 also negatively regulates the Gpa1-cAMP signaling pathway. We further showed that Crg2 interacted with Gpa3 and Gpa1, but not Gpa2, in a pulldown assay and that Crg2 maintained a higher in vitro GTPase-activating protein activity toward Gpa3 and Gpa1 than to Gpa2. Finally, we found that dysregulation of cAMP due to the Crg2 mutation attenuated virulence in a murine model of cryptococcosis. Taken together, our study reveals Crg2 as an RGS (regulator of G-protein signaling) protein of multiregulatory function, including one that controls mating distinctly from Crg1 and one that serves as a novel inhibitor of Gpa1-cAMP signaling.
Dispersed Mutations in Histone H3 That Affect Transcriptional Repression and Chromatin Structure of the
CHA1
Promoter in
Saccharomyces cerevisiae
Authors: He Qiye , Yu Cailin , Morse Randall H. 
Published: July 2008

ABSTRACT

The histone H3 amino terminus, but not that of H4, is required to prevent the constitutively bound activator Cha4 from remodeling chromatin and activating transcription at the
CHA1
gene in
Saccharomyces cerevisiae
. Here we show that neither the modifiable lysine residues nor any specific region of the H3 tail is required for repression of
CHA1
. We then screened for histone H3 mutations that cause derepression of the uninduced
CHA1
promoter and identified six mutants, three of which are also temperature-sensitive mutants and four of which exhibit a
sin
−
phenotype. Histone mutant levels were similar to that of wild-type H3, and the mutations did not cause gross alterations in nucleosome structure. One specific and strongly derepressing mutation, H3 A111G, was examined in depth and found to cause a constitutively active chromatin configuration at the uninduced
CHA1
promoter as well as at the
ADH2
promoter. Transcriptional derepression and altered chromatin structure of the
CHA1
promoter depend on the activator Cha4. These results indicate that modest perturbations in distinct regions of the nucleosome can substantially affect the repressive function of chromatin, allowing activation in the absence of a normal inducing signal (at
CHA1
) or of Swi/Snf (resulting in a
sin
−
phenotype).
DNA Supercoiling-Dependent Gene Regulation in
Chlamydia
Authors: Niehus Eike , Cheng Eric , Tan Ming 
Published: July 2008

ABSTRACT

The intracellular pathogen
Chlamydia
has an unusual developmental cycle marked by temporal expression patterns whose mechanisms of regulation are largely unknown. To examine if DNA topology can regulate chlamydial gene expression, we tested the in vitro activity of five chlamydial promoters at different superhelical densities. We demonstrated for the first time that individual chlamydial promoters show a differential response to changes in DNA supercoiling that correlates with the temporal expression pattern. The promoters for two midcycle genes,
ompA
and
pgk
, were responsive to alterations in supercoiling, and promoter activity could be regulated more than eightfold. In contrast, the promoters for three late transcripts,
omcAB
,
hctA
, and
ltuB
, were relatively insensitive to supercoiling, with promoter activity varying by no more than 2.2-fold over a range of superhelicities. To obtain a measure of how DNA supercoiling levels vary during the chlamydial developmental cycle, we recovered the cryptic chlamydial plasmid at different times after infection and assayed its superhelical density. The chlamydial plasmid was most negatively supercoiled at midcycle, with an approximate superhelical density of −0.07. At early and late times, the plasmid was more relaxed, with an approximate superhelicity of −0.03. Thus, we found a correlation between the responsiveness to supercoiling shown by the two midcycle promoters and the increased level of negative supercoiling during mid time points in the developmental cycle. Our results support a model in which the response of individual promoters to alterations in DNA supercoiling can provide a mechanism for global patterns of temporal gene expression in
Chlamydia
.
Regulation of the Intercellular Adhesin Locus Regulator (
icaR
) by SarA, σ
B
, and IcaR in
Staphylococcus aureus
Authors: Cerca Nuno , Brooks Jamie L. , Jefferson Kimberly K. 
Published: July 2008

ABSTRACT

The staphylococcal accessory regulator SarA and the alternative sigma factor σ
B
have been previously identified as positive regulators, and IcaR as a negative regulator, of
icaADBC
expression. Here, we show that in
Staphylococcus aureus
SarA and σ
B
are also required for
icaR
expression and that IcaR does not have a significant effect on its own expression.