bluuebunny/crossref_metadata_2025_split huggingface dataset:
Association of Novel and Highly Diverse Acid-Tolerant Denitrifiers with N
2
O Fluxes of an Acidic Fen
Authors: Palmer Katharina , Drake Harold L. , Horn Marcus A. 
Published: December 2009

ABSTRACT

Wetlands are sources of denitrification-derived nitrous oxide (N
2
O). Thus, the denitrifier community of an N
2
O-emitting fen (pH 4.7 to 5.2) was investigated. N
2
O was produced and consumed to subatmospheric concentrations in unsupplemented anoxic soil microcosms. Total cell counts and most probable numbers of denitrifiers approximated 10
11
cells·g
DW
−1
(where DW is dry weight) and 10
8
cells·g
DW
−1
, respectively, in both 0- to 10-cm and 30- to 40-cm depths. Despite this uniformity, depth-related maximum reaction rate (
v
max
) values for denitrification in anoxic microcosms ranged from 1 to 24 and −19 to −105 nmol N
2
O h
−1
· g
DW
−1
, with maximal values occurring in the upper soil layers. Denitrification was enhanced by substrates that might be formed via fermentation in anoxic microzones of soil. N
2
O approximated 40% of total nitrogenous gases produced at
in situ
pH, which was likewise the optimal pH for denitrification. Gene libraries of
narG
and
nosZ
(encoding nitrate reductase and nitrous oxide reductase, respectively) from fen soil DNA yielded 15 and 18 species-level operational taxonomic units, respectively, many of which displayed phylogenetic novelty and were not closely related to cultured organisms. Although statistical analyses of
narG
and
nosZ
sequences indicated that the upper 20 cm of soil contained the highest denitrifier diversity and species richness, terminal restriction fragment length polymorphism analyses of
narG
and
nosZ
revealed only minor differences in denitrifier community composition from a soil depth of 0 to 40 cm. The collective data indicate that the regional fen harbors novel, highly diverse, acid-tolerant denitrifier communities capable of complete denitrification and consumption of atmospheric N
2
O at
in situ
pH.
Functional Role of
Bradyrhizobium japonicum
Trehalose Biosynthesis and Metabolism Genes during Physiological Stress and Nodulation
Authors: Sugawara Masayuki , Cytryn Eddie J. , Sadowsky Michael J. 
Published: December 2009

ABSTRACT

Trehalose, a disaccharide accumulated by many microorganisms, acts as a protectant during periods of physiological stress, such as salinity and desiccation. Previous studies reported that the trehalose biosynthetic genes (
otsA
,
treS
, and
treY
) in
Bradyrhizobium japonicum
were induced by salinity and desiccation stresses. Functional mutational analyses indicated that disruption of
otsA
decreased trehalose accumulation in cells and that an
otsA treY
double mutant accumulated an extremely low level of trehalose. In contrast, trehalose accumulated to a greater extent in a
treS
mutant, and maltose levels decreased relative to that seen with the wild-type strain. Mutant strains lacking the OtsA pathway, including the single, double, and triple Δ
otsA
, Δ
otsA
Δ
treS
and Δ
otsA
Δ
treY
, and Δ
otsA
Δ
treS
Δ
treY
mutants, were inhibited for growth on 60 mM NaCl. While mutants lacking functional OtsAB and TreYZ pathways failed to grow on complex medium containing 60 mM NaCl, there was no difference in the viability of the double mutant strain when cells were grown under conditions of desiccation stress. In contrast, mutants lacking a functional TreS pathway were less tolerant of desiccation stress than the wild-type strain. Soybean plants inoculated with mutants lacking the OtsAB and TreYZ pathways produced fewer mature nodules and a greater number of immature nodules relative to those produced by the wild-type strain. Taken together, results of these studies indicate that stress-induced trehalose biosynthesis in
B. japonicum
is due mainly to the OtsAB pathway and that the TreS pathway is likely involved in the degradation of trehalose to maltose. Trehalose accumulation in
B. japonicum
enhances survival under conditions of salinity stress and plays a role in the development of symbiotic nitrogen-fixing root nodules on soybean plants.
Thermococcus kodakarensis
Genetics: TK1827-Encoded β-Glycosidase, New Positive-Selection Protocol, and Targeted and Repetitive Deletion Technology
Authors: Santangelo Thomas J. , Čuboňová L'ubomíra , Reeve John N. 
Published: December 2009

ABSTRACT

Inactivation of TK1761, the reporter gene established for
Thermococcus kodakarensis
, revealed the presence of a second β-glycosidase that we have identified as the product of TK1827. This enzyme (pTK1827) has been purified and shown to hydrolyze glucopyranoside but not mannopyranoside, have optimal activity at 95°C and from pH 8 to 9.5, and have a functional half-life of ∼7 min at 100°C. To generate a strain with both TK1761 and TK1827 deleted, a new selection/counterselection protocol has been developed, and the levels of β-glycosidase activity in
T. kodakarensis
strains with TK1761 and/or TK1827 deleted and with these genes expressed from heterologous promoters are described. Genetic tools and strains have been developed that extend the use of this selection/counterselection procedure to delete any nonessential gene from the
T. kodakarensis
chromosome. Using this technology, TK0149 was deleted to obtain an agmatine auxotroph that grows on nutrient-rich medium only when agmatine is added. Transformants can therefore be selected rapidly, and replicating plasmids can be maintained in this strain growing in rich medium by complementation of the TK0149 deletion.
Candida albicans
PEP12
Is Required for Biofilm Integrity and
In Vivo
Virulence
Authors: Palanisamy Suresh K. A. , Ramirez Melissa A. , Lorenz Michael , Lee Samuel A. 
Published: December 2009

ABSTRACT

To investigate the role of the prevacuolar secretion pathway in biofilm formation and virulence in
Candida albicans
, we cloned and analyzed the
C. albicans
homolog of the
Saccharomyces cerevisiae
prevacuolar trafficking gene
PEP12
.
C. albicans
PEP12
encodes a deduced t-SNARE that is 28% identical to
S. cerevisiae
Pep12p, and plasmids bearing
C. albicans
PEP12
complemented the abnormal vacuolar morphology and temperature-sensitive growth of an
S. cerevisiae
pep12
null mutant. The
C. albicans
pep12
Δ null mutant was defective in endocytosis and vacuolar acidification and accumulated 40- to 60-nm cytoplasmic vesicles near the plasma membrane. Secretory defects included increased extracellular proteolytic activity and absent lipolytic activity. The
pep12
Δ null mutant was more sensitive to cell wall stresses and antifungal agents than the isogenic complemented strain or the control strain DAY185. Notably, the biofilm formed by the
pep12
Δ mutant was reduced in overall mass and fragmented completely upon the slightest disturbance. The
pep12
Δ mutant was markedly reduced in virulence in an
in vitro
macrophage infection model and an
in vivo
mouse model of disseminated candidiasis. These results suggest that
C. albicans
PEP12
plays a key role in biofilm integrity and
in vivo
virulence.
Glycine Betaine Biosynthesized from Glycine Provides an Osmolyte for Cell Growth and Spore Germination during Osmotic Stress in
Myxococcus xanthus
Authors: Kimura Yoshio , Kawasaki Shinji , Yoshimoto Hinae , Takegawa Kaoru 
Published: December 2009

ABSTRACT

Glycine sarcosine methyltransferase (Gsm) and sarcosine dimethylglycine methyltransferase (Sdm) catalyze glycine betaine synthesis from glycine. Disruption of the
M. xanthus gsmA
(MXAN 7068) or
sdmA
(MXAN 3190) gene, encoding Gsm or Sdm homologue proteins, respectively, generated mutants that exhibited a longer lag period of growth and delayed spore germination under osmostress.
Evidence for Rare Capsular Switching in
Streptococcus agalactiae
Authors: Martins Elisabete Raquel , Melo-Cristino José , Ramirez Mário 
Published: December 2009

ABSTRACT

The polysaccharide capsule is a major antigenic factor in
Streptococcus agalactiae
(Lancefield group B streptococcus [GBS]). Previous observations suggest that exchange of capsular loci is likely to occur rather frequently in GBS, even though GBS is not known to be naturally transformable. We sought to identify and characterize putative capsular switching events, by means of a combination of phenotypic and genotypic methods, including pulsed-field gel electrophoretic profiling, multilocus sequence typing, and surface protein and pilus gene profiling. We show that capsular switching by horizontal gene transfer is not as frequent as previously suggested. Serotyping errors may be the main reason behind the overestimation of capsule switching, since phenotypic techniques are prone to errors of interpretation. The identified putative capsular transformants involved the acquisition of the entire capsular locus and were not restricted to the serotype-specific central genes, the previously suggested main mechanism underlying capsular switching. Our data, while questioning the frequency of capsular switching, provide clear evidence for
in vivo
capsular transformation in
S. agalactiae
, which may be of critical importance in planning future vaccination strategies against this pathogen.
Characterization of a Gene Family of Outer Membrane Proteins (
ropB
) in
Rhizobium leguminosarum
bv.
viciae
VF39SM and the Role of the Sensor Kinase ChvG in Their Regulation
Authors: Foreman Dallas L. , Vanderlinde Elizabeth M. , Bay Denise C. , Yost Christopher K. 
Published: December 2009

ABSTRACT

The outer membrane of Gram-negative bacteria represents the interface between the bacterium and its external environment. It has a critical role as a protective barrier against harmful substances and is also important in host-bacteria interactions representing the initial physical point of contact between the host cell and bacterial cell. RopB is a previously identified outer membrane protein from
Rhizobium leguminosarum
bv.
viciae
that is present in free-living cells but absent in bacteroids (H. P. Roest, I. H. Mulders, C. A. Wijffelman, and B. J. Lugtenberg, Mol. Plant Microbe Interact. 8:576-583, 1995). The functions of RopB and the molecular mechanisms of
ropB
gene regulation have remained unknown. We identified and cloned
ropB
and two homologs (
ropB2
and
ropB3
) from the
R. leguminosarum
VF39SM genome. Reporter gene fusions indicated that the expression of
ropB
was 8-fold higher when cells were grown in complex media than when they were grown in minimal media, while
ropB3
expression was constitutively expressed at low levels in both complex and minimal media. Expression of
ropB2
was negligible under all conditions tested. The use of minimal media supplemented with various sources of peptides resulted in a 5-fold increase in
ropB
expression. An increase in
ropB
expression in the presence of peptides was not observed in a
chvG
mutant background, indicating a role for the sensor kinase in regulating
ropB
expression. Each member of the
ropB
gene family was mutated using insertional mutagenesis, and the mutants were assayed for susceptibility to antimicrobial agents and symbiotic phenotypes. All mutants formed effective nodules on pea plants, and gene expression for each
rop
gene in bacteroids was negligible. The functions of
ropB2
and
ropB3
remain cryptic, while the
ropB
mutant had an increased sensitivity to detergents, hydrophobic antibiotics, and weak organic acids, suggesting a role for RopB in outer membrane stability.
The El Tor Biotype of
Vibrio cholerae
Exhibits a Growth Advantage in the Stationary Phase in Mixed Cultures with the Classical Biotype
Authors: Pradhan Subhra , Baidya Amit K. , Ghosh Amalendu , Paul Kalidas , Chowdhury Rukhsana 
Published: December 2009

ABSTRACT

Vibrio cholerae
strains of the O1 serogroup that typically cause epidemic cholera can be classified into two biotypes, classical and El Tor. The El Tor biotype emerged in 1961 and subsequently displaced the classical biotype as a cause of cholera throughout the world. In this study we demonstrate that when strains of the El Tor and classical biotypes were cocultured in standard LB medium, the El Tor strains clearly had a competitive growth advantage over the classical biotype starting from the late stationary phase and could eventually take over the population. The classical biotype produces extracellular protease(s) in the stationary phase, and the amounts of amino acids and small peptides in the late stationary and death phase culture filtrates of the classical biotype were higher than those in the corresponding culture filtrates of the El Tor biotype. The El Tor biotype cells could utilize the amino acids more efficiently than the classical biotype under the alkaline pH of the stationary phase cultures but not in medium buffered to neutral pH. The growth advantage of the El Tor biotype was also observed
in vivo
using the ligated rabbit ileal loop and infant mouse animal models.
Discovery and Characterization of Cadherin Domains in
Saccharophagus degradans
2-40
Authors: Fraiberg Milana , Borovok Ilya , Weiner Ronald M. , Lamed Raphael 
Published: December 2009

ABSTRACT

Saccharophagus degradans
strain 2-40 is a prominent member of newly discovered group of marine and estuarine bacteria that recycle complex polysaccharides. The
S. degradans
2-40 genome codes for 15 extraordinary long polypeptides, ranging from 274 to 1,600 kDa. Five of these contain at least 52 cadherin (CA) and cadherin-like (CADG) domains, the types of which were reported to bind calcium ions and mediate protein/protein interactions in metazoan systems. In order to evaluate adhesive features of these domains, recombinant CA doublet domains (two neighboring domains) from CabC (Sde_3323) and recombinant CADG doublet domains from CabD (Sde_0798) were examined qualitatively and quantitatively for homophilic and heterophilic interactions. In addition, CA and CADG doublet domains were tested for adhesion to the surface of
S. degradans
2-40. Results showed obvious homophilic and heterophilic, calcium ion-dependent interactions between CA and CADG doublet domains. Likewise, CA and CADG doublet domains adhered to the
S. degradans
2-40 surface of cells that were grown on xylan from birch wood or pectin, respectively, as a sole carbon source. This research shows for the first time that bacterial cadherin homophilic and heterophilic interactions may be similar in their nature to cadherin domains from metazoan lineages. We hypothesize that
S. degradans
2-40 cadherin and cadherin-like multiple domains contribute to protein-protein interactions that may mediate cell-cell contact in the marine environment.
The Nonphosphorylative Entner-Doudoroff Pathway in the Thermoacidophilic Euryarchaeon
Picrophilus torridus
Involves a Novel 2-Keto-3-Deoxygluconate- Specific Aldolase
Authors: Reher Matthias , Fuhrer Tobias , Bott Michael , Schönheit Peter 
Published: December 2009

ABSTRACT

The pathway of glucose degradation in the thermoacidophilic euryarchaeon
Picrophilus torridus
has been studied by
in vivo
labeling experiments and enzyme analyses. After growth of
P. torridus
in the presence of [1-
13
C]- and [3-
13
C]glucose, the label was found only in the C-1 and C-3 positions, respectively, of the proteinogenic amino acid alanine, indicating the exclusive operation of an Entner-Doudoroff (ED)-type pathway
in vivo
. Cell extracts of
P. torridus
contained all enzyme activities of a nonphosphorylative ED pathway, which were not induced by glucose. Two key enzymes, gluconate dehydratase (GAD) and a novel 2-keto-3-deoxygluconate (KDG)-specific aldolase (KDGA), were characterized. GAD is a homooctamer of 44-kDa subunits, encoded by Pto0485. KDG aldolase, KDGA, is a homotetramer of 32-kDa subunits. This enzyme was highly specific for KDG with up to 2,000-fold-higher catalytic efficiency compared to 2-keto-3-deoxy-6-phosphogluconate (KDPG) and thus differs from the bifunctional KDG/KDPG aldolase, KD(P)GA of crenarchaea catalyzing the conversion of both KDG and KDPG with a preference for KDPG. The KDGA-encoding gene,
kdgA
, was identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) as Pto1279, and the correct translation start codon, an ATG 24 bp upstream of the annotated start codon of Pto1279, was determined by N-terminal amino acid analysis. The
kdgA
gene was functionally overexpressed in
Escherichia coli
. Phylogenetic analysis revealed that KDGA is only distantly related to KD(P)GA, both enzymes forming separate families within the dihydrodipicolinate synthase superfamily. From the data we conclude that
P. torridus
degrades glucose via a strictly nonphosphorylative ED pathway with a novel KDG-specific aldolase, thus excluding the operation of the branched ED pathway involving a bifunctional KD(P)GA as a key enzyme.
Escherichia coliRNase R Has Dual Activities, Helicase and RNase
Authors: Awano Naoki , Rajagopal Vaishnavi , Arbing Mark , Patel Smita , Hunt John , Inouye Masayori , Phadtare Sangita 
Published: December 2009

ABSTRACTInEscherichia coli, the cold shock response occurs when there is a temperature downshift from 37°C to 15°C, and this response is characterized by induction of several cold shock proteins, including the DEAD-box helicase CsdA, during the acclimation phase. CsdA is involved in a variety of cellular processes. Our previous studies showed that the helicase activity of CsdA is critical for its function in cold shock acclimation of cells and that the only proteins that were able to complement its function were another helicase, RhlE, an RNA chaperone, CspA, and a cold-inducible exoribonuclease, RNase R. Interestingly, other major 3′-to-5′ processing exoribonucleases ofE. coli, such as polynucleotide phosphorylase and RNase II, cannot complement the cold shock function of CsdA. Here we carried out a domain analysis of RNase R and showed that this protein has two distinct activities, RNase and helicase, which are independent of each other and are due to different domains. Mutant RNase R proteins that lack the RNase activity but exhibit the helicase activity were able to complement the cold shock function of CsdA, suggesting that only the helicase activity of RNase R is essential for complementation of the cold shock function of CsdA. We also observed thatin vivodeletion of the two cold shock domains resulted in a loss of the ability of RNase R to complement the cold shock function of CsdA. We further demonstrated that RNase R exhibits helicase activityin vitroindependent of its RNase activity. Our results shed light on the unique properties of RNase R and how it is distinct from other exoribonucleases inE. coli.
Genome Diversity ofPseudomonas aeruginosaPAO1 Laboratory Strains
Authors: Klockgether Jens , Munder Antje , Neugebauer Jens , Davenport Colin F. , Stanke Frauke , Larbig Karen D. , Heeb Stephan , Schöck Ulrike , Pohl Thomas M. , Wiehlmann Lutz , Tümmler Burkhard 
Published: December 2009

ABSTRACTPseudomonas aeruginosaPAO1 is the most commonly used strain for research on this ubiquitous and metabolically versatile opportunistic pathogen. Strain PAO1, a derivative of the original Australian PAO isolate, has been distributed worldwide to laboratories and strain collections. Over decades discordant phenotypes of PAO1 sublines have emerged. Taking the existing PAO1-UW genome sequence (named after the University of Washington, which led the sequencing project) as a blueprint, the genome sequences of reference strains MPAO1 and PAO1-DSM (stored at the German Collection for Microorganisms and Cell Cultures [DSMZ]) were resolved by physical mapping and deep short read sequencing-by-synthesis. MPAO1 has been the source of near-saturation libraries of transposon insertion mutants, and PAO1-DSM is identical in its SpeI-DpnI restriction map with the original isolate. The major genomic differences of MPAO1 and PAO1-DSM in comparison to PAO1-UW are the lack of a large inversion, a duplication of a mobile 12-kb prophage region carrying a distinct integrase and protein phosphatases or kinases, deletions of 3 to 1,006 bp in size, and at least 39 single-nucleotide substitutions, 17 of which affect protein sequences. The PAO1 sublines differed in their ability to cope with nutrient limitation and their virulence in an acute murine airway infection model. Subline PAO1-DSM outnumbered the two other sublines in late stationary growth phase. In conclusion,P. aeruginosaPAO1 shows an ongoing microevolution of genotype and phenotype that jeopardizes the reproducibility of research. High-throughput genome resequencing will resolve more cases and could become a proper quality control for strain collections.
αB-crystallin regulation of angiogenesis by modulation of VEGF
Authors: Kase Satoru , He Shikun , Sonoda Shozo , Kitamura Mizuki , Spee Christine , Wawrousek Eric , Ryan Stephen J. , Kannan Ram , Hinton David R. 
Published: December 2009

Abstract
αB-crystallin is a chaperone belonging to the small heat shock protein family. Herein we show attenuation of intraocular angiogenesis in αB-crystallin knockout (αB-crystallin−/−) mice in 2 models of intraocular disease: oxygen-induced retinopathy and laser-induced choroidal neovascularization. Vascular endothelial growth factor A (VEGF-A) mRNA and hypoxia inducible factor-1α protein expression were induced during retinal angiogenesis, but VEGF-A protein expression remained low in αB-crystallin−/− retina versus wild-type mice, whereas VEGF-R2 expression was not affected. Both αB-crystallin and its phosphorylated serine59 formwere expressed, and immunoprecipitation revealed αB-crystallin binding to VEGF-A but not transforming growth factor-β in cultured retinal pigment epithelial (RPE) cells. αB-crystallin and VEGF-A are colocalized in the endoplasmic reticulum in RPE cells under chemical hypoxia. αB-crystallin−/− RPE showed low VEGF-A secretion under serum-starved conditions compared with wild-type cells. VEGF-A is polyubiquitinated in control and αB-crystallin siRNA treated RPE; however, mono-tetra ubiquitinated VEGF-A increases with αB-crystallin knockdown. Endothelial cell apoptosis in newly formed vessels was greater in αB-crystallin−/− than wild-type mice. Proteasomal inhibition in αB-crystallin−/− mice partially restores VEGF-A secretion and angiogenic phenotype in choroidal neovascularization. Our studies indicate an important role for αB-crystallin as a chaperone for VEGF-A in angiogenesis and its potential as a therapeutic target.