bluuebunny/crossref_metadata_2025 huggingface dataset:
Estimating the Barents Sea polar bear subpopulation size
Authors: Aars J. , Marques T. A. , Buckland S. T. , Andersen M. , Belikov S. , Boltunov A. , Wiig Ø.
Published: July 2008

Abstract

A large‐scale survey was conducted in August 2004 to estimate the size of the Barents Sea polar bear subpopulation. We combined helicopter line transect distance sampling (DS) surveys in most of the survey area with total counts in small areas not suitable for DS. Due to weather constraints we failed to survey some of the areas originally planned to be covered by DS. For those, abundance was estimated using a ratio estimator, in which the auxiliary variable was the number of satellite telemetry fixes (in previous years). We estimated that the Barents Sea subpopulation had approximately 2,650 (95% CI approximately 1,900–3,600) bears. Given current intense interest in polar bear management due to the potentially disastrous effects of climate change, it is surprising that many subpopulation sizes are still unknown. We show here that line transect sampling is a promising method for addressing the need for abundance estimates.
Asymmetrical local adaptation of maize landraces along an altitudinal gradient
Authors: Mercer Kristin , Martínez‐Vásquez Ángel , Perales Hugo R.
Published: July 2008

Crop landraces are managed populations that evolve in response to gene flow and selection. Cross‐pollination among fields, seed sharing by farmers, and selection by management and environmental conditions play roles in shaping crop characteristics. We used common gardens to explore the local adaptation of maize (Zea mays ssp. mays) landrace populations from Chiapas, Mexico to altitude. We sowed seeds of 21 populations from three altitudinal ranges in two common gardens and measured two characteristics that estimate fitness: likelihood of producing good quality seed and the total mass of good quality seed per plant. The probability of lowland plants producing good quality seed was invariably high regardless of garden, while highland landraces were especially sensitive to altitude. Their likelihood of producing good seed quadrupled in the highland site. The mass of good quality seed showed a different pattern, with lowland landraces producing 25% less seed mass than the other types at high elevations. Combining these two measures of fitness revealed that the highland landraces were clearly adapted to highland sites, while lowland and midland landraces appear more adapted to the midland site. We discuss this asymmetry in local adaptation in light of climate change and in situ conservation of crop genetic resources.
Oncogenic BRAFV600E inhibits BIM expression to promote melanoma cell survival
Authors: Cartlidge Robert A. , Thomas G. R. , Cagnol Sebastien , Jong Kimberly A. , Molton Sarah A. , Finch Andrew J. , McMahon Martin
Published: July 2008

Somatic activating mutations of BRAF are the earliest and most common genetic abnormality detected in the genesis of human melanoma. However, the mechanism(s) by which activated BRAF promotes melanoma cell cycle progression and/or survival remain unclear. Here we demonstrate that expression of BIM, a pro‐apoptotic member of the BCL‐2 family, is inhibited by BRAF→MEK→ERK signaling in mouse and human melanocytes and in human melanoma cells. Trophic factor deprivation of melanocytes leads to elevated BIM expression. However, re‐addition of trophic factors or activation of a conditional form of BRAFV600E leads to rapid inhibition of BIM expression. In both cases, inhibition of BIM expression was dependent on the activity of MEK1/2 and the proteasome. Consistent with these observations, pharmacological inhibition of BRAFV600E or MEK1/2 in human melanoma cells (using PLX4720 and CI‐1040 respectively) led to a striking elevation of BIM expression. Re‐activation of BRAF→MEK→ERK signaling led to phosphorylation of BIM‐EL on serine 69 and its subsequent degradation. Interestingly, endogenous expression of BIM in melanoma cells was insufficient to induce apoptosis unless combined with serum deprivation. Under these circumstances, inhibition of BIM expression by RNA interference provided partial protection from apoptosis. These data suggest that regulation of BIM expression by BRAF→MEK→ERK signaling is one mechanism by which oncogenic BRAFV600E can influence the aberrant physiology of melanoma cells.
Downregulated melanogenic paracrine cytokine linkages in hypopigmented palmoplantar skin
Authors: Hasegawa Junichi , Goto Yasufumi , Murata Hiroshi , Takata Minoru , Saida Toshiaki , Imokawa Genji
Published: July 2008

The hypo‐pigmentation of human skin on the palms and the soles compared with other areas of the body has recently been reported to be due to mesenchymal–epithelial interactions via a fibroblast‐derived factor, dickkopf 1, an inhibitor of the canonical Wnt signaling pathway. Recently, it has been reported that keratinocytes play a significant role in skin color determination by producing cytokines involved in melanogenesis. Thus, we hypothesized that the downregulated expression of keratinocyte‐ or fibroblast‐derived melanogenic cytokines may also be responsible for the decreased function of palmoplantar (PP) melanocytes in addition to the suppressive effects of dickkopf 1 on melanogenic function in epidermal melanocytes. Immunohistochemistry revealed that the number of tyrosinase, S100α, c‐KIT, endothelin B receptor (ETBR), SOX10, and microphthalmia‐associated transcription factor (MITF) immuno‐positive melanocytes is significantly reduced in PP epidermis. In contrast, dopa‐histochemistry demonstrated no substantial reduction in melanocyte populations in PP epidermis. Real‐time RT‐PCR revealed that the expression of stem cell factor (SCF) and endothelin (ET)‐1 mRNAs in PP skin was significantly downregulated. In parallel, immunohistochemistry revealed that SCF and ET‐1 immuno‐staining was markedly attenuated in PP skin. Western blotting revealed that the expression of SCF, c‐KIT, and MITF‐M proteins was significantly decreased in PP skin. These findings suggest the possibility that downregulation of ET‐1/SCF/receptor linkages is also associated with the decreased function of melanocytes in PP skin.
Effects of low‐frequency whole‐body vibration on motor‐evoked potentials in healthy men
Authors: Mileva Katya N. , Bowtell Joanna L. , Kossev Andon R.
Published: July 2008

The aim of this study was to determine whether low‐frequency whole‐body vibration (WBV) modulates the excitability of the corticospinal and intracortical pathways related to tibialis anterior (TA) muscle activity, thus contributing to the observed changes in neuromuscular function during and after WBV exercise. Motor‐evoked potentials (MEPs) elicited in response to transcranial magnetic stimulation (TMS) of the leg area of the motor cortex were recorded in TA and soleus (SOL) muscles of seven healthy male subjects whilst performing 330 s continuous static squat exercise. Each subject completed two conditions: control (no WBV) and WBV (30 Hz, 1.5 mm vibration applied from 111 to 220 s). Five single suprathreshold and five paired TMS were delivered during each squat period lasting 110 s (pre‐, during and post‐WBV). Two interstimulus intervals (ISIs) between the conditioning and the testing stimuli were employed in order to study the effects of WBV on short‐interval intracortical inhibition (SICI, ISI = 3 ms) and intracortical facilitation (ICF, ISI = 13 ms). During vibration relative to squat exercise alone, single‐pulse TMS provoked significantly higher TA MEP amplitude (56 ± 14%, P= 0.003) and total area (71 ± 19%, P= 0.04), and paired TMS with ISI = 13 ms provoked smaller MEP amplitude (−21 ± 4%, P= 0.01) but not in SOL. Paired‐pulse TMS with ISI = 3 ms elicited significantly lower MEP amplitude (TA, −19 ± 4%, P= 0.009; and SOL, −13 ± 4%, P= 0.03) and total area (SOL, −17 ± 6%, P= 0.02) during vibration relative to squat exercise alone in both muscles. Tibialis anterior MEP facilitation in response to single‐pulse TMS suggests that WBV increased corticospinal pathway excitability. Increased TA and SOL SICI and decreased TA ICF in response to paired‐pulse TMS during WBV indicate vibration‐induced alteration of the intracortical processes as well.
Comparative In Vitro Activities of the Investigational Fluoroquinolone DC-159a and Other Antimicrobial Agents against Human Mycoplasmas and Ureaplasmas
Authors: Waites Ken B. , Crabb Donna M. , Duffy Lynn B.
Published: July 2008

The in vitro susceptibilities of 151 unique clinical isolates of
Mycoplasma pneumoniae
,
Mycoplasma hominis
,
Mycoplasma fermentans
,
Mycoplasma genitalium
, and
Ureaplasma
species to DC-159a, an investigational fluoroquinolone, in comparison with those to other agents were determined. Macrolides were the most active agents against
M. pneumoniae
and
M. genitalium
, whereas clindamycin was most active against
M. hominis.
DC-159a MICs were ≤0.5 μg/ml for all
Mycoplasma
species and ≤4 μg/ml for ureaplasmas. DC-159a was the most active fluoroquinolone tested against
M. pneumoniae
and
M. fermentans
, and it was second to moxifloxacin against the other species. It was bactericidal against 10
M. pneumoniae
isolates and demonstrated killing of ≥99.9% of the inoculum at 24 h for 2 isolates. The excellent in vitro activity of DC-159a demonstrates its potential for use in the treatment of infections due to mycoplasmas and ureaplasmas.
Quantification of
Desulfovibrio vulgaris
Dissimilatory Sulfite Reductase Gene Expression during Electron Donor- and Electron Acceptor-Limited Growth
Authors: Villanueva Laura , Haveman Shelley A. , Summers Zara M. , Lovley Derek R.
Published: July 2008

Previous studies have suggested that levels of transcripts for
dsrA
, a gene encoding a subunit of the dissimilatory sulfite reductase, are not directly related to the rates of sulfate reduction in sediments under all conditions. This phenomenon was further investigated with chemostat-grown
Desulfovibrio vulgaris
. Under sulfate-limiting conditions,
dsrA
transcript levels increased as the bulk rates of sulfate reduction in the chemostat increased, but transcript levels were similar at all sulfate reduction rates under electron donor-limiting conditions. When both electron donor- and electron acceptor-limiting conditions were considered, there was a direct correspondence between
dsrA
transcript levels and the rates of sulfate reduction per cell. These results suggest that
dsrA
transcript levels may provide important information on the metabolic state of sulfate reducers.
Polyphasic Approach to Bacterial Dynamics during the Ripening of Spanish Farmhouse Cheese, Using Culture-Dependent and -Independent Methods
Authors: Martín-Platero Antonio M. , Valdivia Eva , Maqueda Mercedes , Martín-Sánchez Inés , Martínez-Bueno Manuel
Published: July 2008

We studied the dynamics of the microbial population during ripening of Cueva de la Magahá cheese using a combination of classical and molecular techniques. Samples taken during ripening of this Spanish goat's milk cheese in which
Lactococcus lactis
and
Streptococcus thermophilus
were used as starter cultures were analyzed. All bacterial isolates were clustered by using randomly amplified polymorphic DNA (RAPD) and identified by 16S rRNA gene sequencing, species-specific PCR, and multiplex PCR. Our results indicate that the majority of the 225 strains isolated and enumerated on solid media during the ripening period were nonstarter lactic acid bacteria, and
Lactobacillus paracasei
was the most abundant species. Other
Lactobacillus
species, such as
Lactobacillus plantarum
and
Lactobacillus parabuchneri
, were also detected at the beginning and end of ripening, respectively. Non-lactic-acid bacteria, mainly
Kocuria
and
Staphylococcus
strains, were also detected at the end of the ripening period. Microbial community dynamics determined by temporal temperature gradient gel electrophoresis provided a more precise estimate of the distribution of bacteria and enabled us to detect
Lactobacillus curvatus
and the starter bacteria
S. thermophilus
and
L. lactis
, which were not isolated. Surprisingly, the bacterium most frequently found using culture-dependent analysis,
L. paracasei
, was scarcely detected by this molecular approach. Finally, we studied the composition of the lactobacilli and their evolution by using length heterogeneity PCR.
Purification, Molecular Cloning, and Enzymatic Properties of a Family 12 Endoglucanase (EG-II) from
Fomitopsis palustris
: Role of EG-II in Larch Holocellulose Hydrolysis
Authors: Shimokawa Tomoko , Shibuya Hajime , Nojiri Masanobu , Yoshida Shigeki , Ishihara Mitsuro
Published: July 2008

A family 12 endoglucanase with a molecular mass of 23,926 Da (EG-II) from the brown-rot basidiomycete
Fomitopsis palustris
was purified and characterized. One of the roles of EG-II in wood degradation is thought to be to loosen the polysaccharide network in cell walls by disentangling hemicelluloses that are associated with cellulose.
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

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

Aspergillus 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

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

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

To 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

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

We 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

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

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

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

The 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

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

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

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

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.
Escherichia coli
NsrR Regulates a Pathway for the Oxidation of 3-Nitrotyramine to 4-Hydroxy-3-Nitrophenylacetate
Authors: Rankin Linda D. , Bodenmiller Diane M. , Partridge Jonathan D. , Nishino Shirley F. , Spain Jim C. , Spiro Stephen
Published: July 2008

Chromatin immunoprecipitation and microarray (ChIP-chip) analysis showed that the nitric oxide (NO)-sensitive repressor NsrR from
Escherichia coli
binds in vivo to the promoters of the
tynA
and
feaB
genes. These genes encode the first two enzymes of a pathway that is required for the catabolism of phenylethylamine (PEA) and its hydroxylated derivatives tyramine and dopamine. Deletion of
nsrR
caused small increases in the activities of the
tynA
and
feaB
promoters in cultures grown on PEA. Overexpression of
nsrR
severely retarded growth on PEA and caused a marked repression of the
tynA
and
feaB
promoters. Both the growth defect and the promoter repression were reversed in the presence of a source of NO. These results are consistent with NsrR mediating repression of the
tynA
and
feaB
genes by binding (in an NO-sensitive fashion) to the sites identified by ChIP-chip.
E. coli
was shown to use 3-nitrotyramine as a nitrogen source for growth, conditions which partially induce the
tynA
and
feaB
promoters. Mutation of
tynA
(but not
feaB
) prevented growth on 3-nitrotyramine. Growth yields, mutant phenotypes, and analyses of culture supernatants suggested that 3-nitrotyramine is oxidized to 4-hydroxy-3-nitrophenylacetate, with growth occurring at the expense of the amino group of 3-nitrotyramine. Accordingly, enzyme assays showed that 3-nitrotyramine and its oxidation product (4-hydroxy-3-nitrophenylacetaldehyde) could be oxidized by the enzymes encoded by
tynA
and
feaB
, respectively. The results suggest that an additional physiological role of the PEA catabolic pathway is to metabolize nitroaromatic compounds that may accumulate in cells exposed to NO.
The Helix-Loop-Helix Motif at the N Terminus of HalI Is Essential for Its Immunity Function against Halocin C8
Authors: Mei Shuangshuang , Sun Chaomin , Liu Xiaoqing , Lu Qiuhe , Cai Lei , Li Yun , Xiang Hua
Published: July 2008

Halocin C8 (HalC8) is a stable microhalocin exhibiting strong antimicrobial activity against a wide range of haloarchaea. HalI, a 207-amino-acid peptide derived from the N terminus of the HalC8 preproprotein, is the immunity protein of HalC8. In this study, the molecular mechanism of the immunity function of HalI was investigated. Both pull-down and surface plasmon resonance assays revealed that HalI directly interacted with HalC8, and a mixture of purified HalI and HalC8 readily formed a heterocomplex, which was verified by gel filtration. Interestingly, HalC8 tended to form a self-associated complex, and one immunity protein likely sequestered multiple halocins. Significantly, the helix-loop-helix (HLH) motif containing a 4-amino-acid repeat (RELA) at the N terminus of HalI played a key role in its immunity activity. Disruption of the HLH motif or mutagenesis of the key residues of the RELA repeat resulted in loss of both the immunity function and the ability of HalI to bind to HalC8. These results demonstrated that HalI sequestered the activity of HalC8 through specific and direct binding.
Formation ofStreptococcus pneumoniaeNon-Phase-Variable Colony Variants Is Due to Increased Mutation Frequency Present under Biofilm Growth Conditions
Authors: Allegrucci Magee , Sauer Karin
Published: July 2008

In this report, we show that biofilm formation byStreptococcus pneumoniaeserotype 19 gives rise to variants (the small mucoid variant [SMV] and the acapsular small-colony variant [SCV]) differing in capsule production, attachment, and biofilm formation compared to wild-type strains. All biofilm-derived variants harbored SNPs incps19F. SCVs reverted to SMV, but no reversion to the wild-type phenotype was noted, indicating that these variants were distinct from opaque- and transparent-phase variants. The SCV-SMV reversion frequency was dependent on growth conditions and treatment with tetracycline. Increased reversion rates were coincident with antibiotic treatment, implicating oxidative stress as a trigger for the SCV-SMV switch. We, therefore, evaluated the role played by hydrogen peroxide, the oxidizing chemical, in the reversion and emergence of variants. Biofilms ofS. pneumoniaeTIGR4-ΔspxB, defective in hydrogen peroxide production, showed a significant reduction in variant formation. Similarly, supplementing the medium with catalase or sodium thiosulfate yielded a significant reduction in variants formed by wild-type biofilms. Resistance to rifampin, an indicator for mutation frequency, was found to increase approximately 55-fold in biofilms compared to planktonic cells for each of the three wild-type strains examined. In contrast, TIGR4-ΔspxBgrown as a biofilm showed no increase in rifampin resistance compared to the same cells grown planktonically. Furthermore, addition of 2.5 and 10 mM hydrogen peroxide to planktonic cells resulted in a 12- and 160-fold increase in mutation frequency, respectively, and gave rise to variants similar in appearance, biofilm-related phenotypes, and distribution of biofilm-derived variants. The results suggest that hydrogen peroxide and environmental conditions specific to biofilms are responsible for the development of non-phase-variable colony variants.
Identification of Functional Tat Signal Sequences in
Mycobacterium tuberculosis
Proteins
Authors: McDonough Justin A. , McCann Jessica R. , Tekippe Erin McElvania , Silverman Jason S. , Rigel Nathan W. , Braunstein Miriam
Published: July 2008

The twin-arginine translocation (Tat) pathway is a system used by some bacteria to export proteins out from the cytosol to the cell surface or extracellular environment. A functional Tat pathway exists in the important human pathogen
Mycobacterium tuberculosis
. Identification of the substrates exported by the Tat pathway can help define the role that this pathway plays in the physiology and pathogenesis of
M. tuberculosis
. Here we used a reporter of Tat export, a truncated β-lactamase, ′BlaC, to experimentally identify
M. tuberculosis
proteins with functional Tat signal sequences. Of the 13 proteins identified, one lacks the hallmark of a Tat-exported substrate, the twin-arginine dipeptide, and another is not predicted by in silico analysis of the annotated
M. tuberculosis
genome. Full-length versions of a subset of these proteins were tested to determine if the native proteins are Tat exported. For three proteins, expression in a Δ
tat
mutant of
Mycobacterium smegmatis
revealed a defect in precursor processing compared to expression in the wild type, indicating Tat export of the full-length proteins. Conversely, two proteins showed no obvious Tat export in
M. smegmatis
. One of this latter group of proteins was the
M. tuberculosis
virulence factor phospholipase C (PlcB). Importantly, when tested in
M. tuberculosis
a different result was obtained and PlcB was exported in a twin-arginine-dependent manner. This suggests the existence of an
M. tuberculosis
-specific factor(s) for Tat export of a proven virulence protein. It also emphasizes the importance of domains beyond the Tat signal sequence and bacterium-specific factors in determining if a given protein is Tat exported.
Formate-Dependent Autotrophic Growth in
Sinorhizobium meliloti
Authors: Pickering Brad S. , Oresnik Ivan J.
Published: July 2008

It was found that
S. meliloti
strain SmA818, which is cured of pSymA, could not grow on defined medium containing only formate and bicarbonate as carbon sources. Growth experiments showed that Rm1021 was capable of formate/bicarbonate-dependent growth, suggesting that it was capable of autotrophic-type growth. The annotated genome of
S. meliloti
Rm1021 contains three formate dehydrogenase genes. A systematic disruption of each of the three formate dehydrogenase genes, as well as the genes encoding determinants of the Calvin-Benson-Bassham, cycle was carried out to determine which of these determinants played a role in growth on this defined medium. The results showed that
S. meliloti
is capable of formate-dependent autotrophic growth. Formate-dependent autotrophic growth is dependent on the presence of the chromosomally located
fdsABCDG
operon, as well as the
cbb
operon carried by pSymB. Growth was also dependent on the presence of either of the two triose-phosphate isomerase genes (
tpiA
or
tpiB
) that are found in the genome. In addition, it was found that
fdoGHI
carried by pSymA encodes a formate dehydrogenase that allows Rm1021 to carry out formate-dependent respiration. Taken together, the data allow us to present a model of how
S. meliloti
can grow on defined medium containing only formate and bicarbonate as carbon sources.
Identification of Sesquiterpene Synthases fromNostoc punctiformePCC 73102 andNostocsp. Strain PCC 7120
Authors: Agger Sean A. , Lopez-Gallego Fernando , Hoye Thomas R. , Schmidt-Dannert Claudia
Published: July 2008

Cyanobacteria are a rich source of natural products and are known to produce terpenoids. These bacteria are the major source of the musty-smelling terpenes geosmin and 2-methylisoborneol, which are found in many natural water supplies; however, no terpene synthases have been characterized from these organisms to date. Here, we describe the characterization of three sesquiterpene synthases identified inNostocsp. strain PCC 7120 (terpene synthase NS1) andNostoc punctiformePCC 73102 (terpene synthases NP1 and NP2). The second terpene synthase inN. punctiforme(NP2) is homologous to fusion-type sesquiterpene synthases fromStreptomycesspp. shown to produce geosmin via an intermediate germacradienol. The enzymes were functionally expressed inEscherichia coli, and their terpene products were structurally identified as germacrene A (from NS1), the eudesmadiene 8a-epi-α-selinene (from NP1), and germacradienol (from NP2). The product of NP1, 8a-epi-α-selinene, so far has been isolated only from termites, in which it functions as a defense compound. Terpene synthases NP1 and NS1 are part of an apparent minicluster that includes a P450 and a putative hybrid two-component protein located downstream of the terpene synthases. Coexpression of P450 genes with their adjacent located terpene synthase genes inE. colidemonstrates that the P450 fromNostocsp. can be functionally expressed inE. coliwhen coexpressed with a ferredoxin gene and a ferredoxin reductase gene fromNostocand that the enzyme oxygenates the NS1 terpene product germacrene A. This represents to the best of our knowledge the first example of functional expression of a cyanobacterial P450 inE. coli.
Identification and Characterization of the Dicarboxylate Uptake System DccT inCorynebacterium glutamicum
Authors: Youn Jung-Won , Jolkver Elena , Krämer Reinhard , Marin Kay , Wendisch Volker F.
Published: July 2008

Many bacteria can utilize C4-carboxylates as carbon and energy sources. However,Corynebacterium glutamicumATCC 13032 is not able to use tricarboxylic acid cycle intermediates such as succinate, fumarate, andl-malate as sole carbon sources. Upon prolonged incubation, spontaneous mutants which had gained the ability to grow on succinate, fumarate, andl-malate could be isolated. DNA microarray analysis showed higher mRNA levels of cg0277, which subsequently was nameddccT, in the mutants than in the wild type, and transcriptional fusion analysis revealed that a point mutation in the promoter region ofdccTwas responsible for increased expression. The overexpression ofdccTwas sufficient to enable theC. glutamicumwild type to grow on succinate, fumarate, andl-malate as the sole carbon sources. Biochemical analyses revealed that DccT, which is a member of the divalent anion/Na+symporter family, catalyzes the effective uptake of dicarboxylates like succinate, fumarate,l-malate, and likely also oxaloacetate in a sodium-dependent manner.