bluuebunny/crossref_metadata_2025 huggingface dataset:
Use of Inorganic and Organic Nitrogen by
Synechococcus
spp. and Diatoms on the West Florida Shelf as Measured Using Stable Isotope Probing
Authors: Wawrik Boris , Callaghan Amy V. , Bronk Deborah A.
Published: September 2009

The marine nitrogen (N) cycle is a complex network of biological transformations in different N pools. The linkages among these different reservoirs are often poorly understood. Traditional methods for measuring N uptake rely on bulk community properties and cannot provide taxonomic information.
15
N-based stable isotope probing (SIP), however, is a technique that allows detection of uptake of individual N sources by specific microorganisms. In this study we used
15
N SIP methodology to assess the use of different nitrogen substrates by
Synechococcus
spp. and diatoms on the west Florida shelf. Seawater was incubated in the presence of
15
N-labeled ammonium, nitrate, urea, glutamic acid, and a mixture of 16 amino acids. DNA was extracted and fractionated using CsCl density gradient centrifugation. Quantitative PCR was used to quantify the amounts of
Synechococcus
and diatom DNA as a function of density, and
15
N tracer techniques were used to measure rates of N uptake by the microbial community. The ammonium, nitrate, urea, and dissolved primary amine uptake rates were 0.077, 0.065, 0.013, and 0.055 μmol N liter
−1
h
−1
, respectively. SIP data indicated that diatoms and
Synechococcus
spp. actively incorporated N from [
15
N]nitrate, [
15
N]ammonium, and [
15
N]urea.
Synechococcus
also incorporated nitrogen from [
15
N]glutamate and
15
N-amino acids, but no evidence indicating uptake of labeled amino acids by diatoms was detected. These data suggest that N flow in communities containing
Synechococcus
spp. and diatoms has more plasticity than the new-versus-recycled production paradigm suggests and that these phytoplankters should not be viewed strictly as recycled and new producers, respectively.
High-Level Resistance of
Nosema ceranae
, a Parasite of the Honeybee, to Temperature and Desiccation
Authors: Fenoy S. , Rueda C. , Higes M. , Martín-Hernández R. , del Aguila C.
Published: September 2009

Resistance of
Nosema ceranae
to different exposure conditions has been evaluated by using Sytox green and DAPI (4′,6-diamidino-2-phenylindole) to test spore viability. High thermotolerance at 60 and 35°C and resistance to desiccation were observed. However, a significant decrease in viability after freezing and a rapid degeneration of spores maintained at 4°C were also detected.
Diversity of
Frankia
Populations in Root Nodules of Geographically Isolated Arizona Alder Trees in Central Arizona (United States)
Authors: Welsh Allana K. , Dawson Jeffrey O. , Gottfried Gerald J. , Hahn Dittmar
Published: September 2009

The diversity of uncultured
Frankia
populations in root nodules of
Alnus oblongifolia
trees geographically isolated on mountaintops of central Arizona was analyzed by comparative sequence analyses of
nifH
gene fragments. Sequences were retrieved from
Frankia
populations in nodules of four trees from each of three mountaintops (
n
= 162) and their levels of diversity compared using spatial genetic clustering methods and single-nucleotide or 1, 3, or 5% sequence divergence thresholds. With the single-nucleotide threshold level, 45 different sequences with significant differences between the mountaintops were retrieved, with the southern site partitioning in a separate population from the two other sites. Some of these sequences were identical in nodules from different mountaintops and to those of strains isolated from around the world. A high level of diversity that resulted in the assignment of 14 clusters of sequences was also found on the 1% divergence level. Single-nucleotide and 1% divergence levels thus demonstrate microdiversity of frankiae in root nodules of
A. oblongifolia
trees and suggest a partitioning of diversity by site. At the 3 and 5% divergence levels, however, diversity was reduced to three clusters or one cluster, respectively, with no differentiation by mountaintop. Only at the 5% threshold level do all
Frankia
strains previously assigned to one genomic group cluster together.
Regulation of Mannose Phosphotransferase System Permease and Virulence Gene Expression in
Listeria monocytogenes
by the EII
t
Man
Transporter
Authors: Vu-Khac Hung , Miller Kurt W.
Published: September 2009

The EII
t
Man
phosphotransferase system (PTS) permease encoded by the
mpt
operon is the principal glucose transporter in
Listeria monocytogenes.
EII
t
Man
participates in glucose-mediated carbon catabolite repression (CCR) and downregulation of virulence gene expression, and it is the receptor for class IIa bacteriocins. The regulation of this important protein and its roles in gene control were examined using derivatives of strain EGD-e in which the
mpt
operon or its regulatory genes,
manR
and lmo0095, were deleted. Real-time reverse transcription-PCR analysis showed that the
mpt
mRNA level was 10- and 100-fold lower in the lmo0095 and
manR
deletion strains, respectively. The
manR
mRNA level was higher in the
mpt
deletion mutant in medium lacking glucose, possibly due to disruption of a regulatory process that normally downregulates
manR
transcription in the absence of this sugar. Analysis of the
mpt
deletion mutant also showed that EII
t
Man
participates to various degrees in glucose-mediated CCR of PTS operons. CCR of the lmo0027 gene, which encodes a β-glucoside PTS transporter, required expression of EII
t
Man
. In contrast, genes in two mannose PTS operons (lmo0024, lmo1997, and lmo2002) were repressed by glucose even when EII
t
Man
was not synthesized. A third mannose PTS operon,
mpo
, was not regulated by glucose or by the level of EII
t
Man
. Finally, the mRNA levels for five genes in the
prfA
virulence gene cluster were two- to fourfold higher in the
mpt
deletion mutant. The results show that EII
t
Man
participates to various extents in glucose-mediated CCR of PTS operons and makes a small, albeit significant, contribution to downregulation of virulence gene transcription by glucose in strain EGD-e.
Comparative Analyses of Prophage-Like Elements Present in Bifidobacterial Genomes
Authors: Ventura Marco , Turroni Francesca , Lima-Mendez Gipsi , Foroni Elena , Zomer Aldert , Duranti Sabrina , Giubellini Vanessa , Bottacini Francesca , Horvath Philippe , Barrangou Rodolphe , Sela David A. , Mills David A. , van Sinderen Douwe
Published: September 2009

So far, only scarce and rather diffuse information is available on bacteriophages infecting members of the genus
Bifidobacterium
. In the current study, we investigated the genetic organization, phylogenetic relationships, and, in some cases, transcription profiles and inducibility of 19 prophage-like elements present on the individual chromosomes of nine bifidobacterial strains, which represent six different species.
Processivity, Synergism, and Substrate Specificity of
Thermobifida fusca
Cel6B
Authors: Vuong Thu V. , Wilson David B.
Published: September 2009

A relationship between processivity and synergism has not been reported for cellulases, although both characteristics are very important for hydrolysis of insoluble substrates. Mutation of two residues located in the active site tunnel of
Thermobifida fusca
exocellulase Cel6B increased processivity on filter paper. Surprisingly, mixtures of the Cel6B mutant enzymes and
T. fusca
endocellulase Cel5A did not show increased synergism or processivity, and the mutant enzyme which had the highest processivity gave the poorest synergism. This study suggests that improving exocellulase processivity might be not an effective strategy for producing improved cellulase mixtures for biomass conversion. The inverse relationship between the activities of many of the mutant enzymes with bacterial microcrystalline cellulose and their activities with carboxymethyl cellulose indicated that there are differences in the mechanisms of hydrolysis for these substrates, supporting the possibility of engineering Cel6B to target selected substrates.
Seasonal Distribution of Cyprinid Herpesvirus 3 in Lake Biwa, Japan
Authors: Minamoto Toshifumi , Honjo Mie N. , Kawabata Zen'ichiro
Published: September 2009

The seasonal distribution of the cyprinid herpesvirus 3 (CyHV-3) in Lake Biwa, Japan, was investigated. CyHV-3 was distributed all over the lake 5 years after the first outbreak. The mean concentration of CyHV-3 in water showed annual oscillation, with a peak in the summer and a trough in winter. Our results suggested that CyHV-3 is present at high density in reductive environments, such as reed zones and turbid or eutrophic water.
Identity of the Growth-Limiting Nutrient Strongly Affects Storage Carbohydrate Accumulation in Anaerobic Chemostat Cultures of
Saccharomyces cerevisiae
Authors: Hazelwood Lucie A. , Walsh Michael C. , Luttik Marijke A. H. , Daran-Lapujade Pascale , Pronk Jack T. , Daran Jean-Marc
Published: September 2009

Accumulation of glycogen and trehalose in nutrient-limited cultures of
Saccharomyces cerevisiae
is negatively correlated with the specific growth rate. Additionally, glucose-excess conditions (i.e., growth limitation by nutrients other than glucose) are often implicated in high-level accumulation of these storage carbohydrates. The present study investigates how the identity of the growth-limiting nutrient affects accumulation of storage carbohydrates in cultures grown at a fixed specific growth rate. In anaerobic chemostat cultures (dilution rate, 0.10 h
−1
) of
S. cerevisiae
, the identity of the growth-limiting nutrient (glucose, ammonia, sulfate, phosphate, or zinc) strongly affected storage carbohydrate accumulation. The glycogen contents of the biomass from glucose- and ammonia-limited cultures were 10- to 14-fold higher than those of the biomass from cultures grown under the other three glucose-excess regimens. Trehalose levels were specifically higher under nitrogen-limited conditions. These results demonstrate that storage carbohydrate accumulation in nutrient-limited cultures of
S. cerevisiae
is not a generic response to excess glucose but instead is strongly dependent on the identity of the growth-limiting nutrient. While transcriptome analysis of wild-type and
msn2
Δ
msn4
Δ strains confirmed that transcriptional upregulation of glycogen and trehalose biosynthesis genes is mediated by Msn2p/Msn4p, transcriptional regulation could not quantitatively account for the drastic changes in storage carbohydrate accumulation. The results of assays of glycogen synthase and glycogen phosphorylase activities supported involvement of posttranscriptional regulation. Consistent with the high glycogen levels in ammonia-limited cultures, the ratio of glycogen synthase to glycogen phosphorylase in these cultures was up to eightfold higher than the ratio in the other glucose-excess cultures.
Labeled
Trichoderma reesei
Cellulase as a Marker for
Acanthamoeba
Cyst Wall Cellulose in Infected Tissues
Authors: Derda Monika , Winiecka-Krusnell Jadwiga , Linder Markus B. , Linder Ewert
Published: September 2009

Some protozoans are able to encyst as a protective response to a harmful environment. The cyst wall usually contains chitin as its main structural constituent.
Acanthamoeba
is an exception since its cyst wall contains cellulose. Specific cytochemical differentiation between cellulose and chitin by microscopy has not been possible due to the similarity of the constituent β-1,4-linked hexose backbones of these molecules. Thus, various fluorescent brightening agents and lectins bind to both cellulose and chitin. The identification of
Acanthamoeba
spp., which is based primarily on morphological and biochemical features, is labor-intensive and requires cloning and axenization. We describe a novel immunocytochemical method for identification of
Acanthamoeba
spp. based on selective binding of
Trichoderma reesei
cellulase to protozoan cyst wall cellulose. A recombinant cellulose-binding protein consisting of two cellulose-binding domains (CBDs) from
T. reesei
cellulases was coupled to the fluorescent dyes Alexa Fluor 350 and Alexa Fluor 568 or was labeled with biotin using EZ-Link sulfo-NHS-biotin. No staining reaction was observed with chitin-containing preparations of fungi. Thus, the recombinant CBDs can be used as a marker to distinguish between cellulose and chitin. This allows rapid identification of
Acanthamoeba
cyst wall cellulose in paraffin or frozen sections of infected tissues.
Cloning and Characterization of an Intracellular Esterase from the Wine-Associated Lactic Acid BacteriumOenococcus oeni
Authors: Sumby Krista M. , Matthews Angela H. , Grbin Paul R. , Jiranek Vladimir
Published: September 2009

We report the cloning and characterization of EstB28, the first esterase to be so characterized from the wine-associated lactic acid bacterium,Oenococcus oeni. The published sequence forO. oenistrain PSU-1 was used to identify putative esterase genes and design PCR primers in order to amplify the corresponding region from strain Ooeni28, an isolate intended for inoculation of wines. In this way a 912-bp open reading frame (ORF) encoding a putative esterase of 34.5 kDa was obtained. The amino acid sequence indicated that EstB28 is a member of family IV of lipolytic enzymes and contains the GDSAG motif common to other lactic acid bacteria. This ORF was cloned intoEscherichia coliusing an appropriate expression system, and the recombinant esterase was purified. Characterization of EstB28 revealed that the optimum temperature, pH, and ethanol concentration were 40°C, pH 5.0, and 28% (vol/vol), respectively. EstB28 also retained marked activity under conditions relevant to winemaking (10 to 20°C, pH 3.5, 14% [vol/vol] ethanol). Kinetic constants were determined for EstB28 withp-nitrophenyl (pNP)-linked substrates ranging in chain length from C2to C18. EstB28 exhibited greatest specificity for C2to C4pNP-linked substrates.
Prevalence of
Cardinium
Bacteria in Planthoppers and Spider Mites and Taxonomic Revision of “
Candidatus
Cardinium hertigii” Based on Detection of a New
Cardinium
Group from Biting Midges
Authors: Nakamura Yuki , Kawai Sawako , Yukuhiro Fumiko , Ito Saiko , Gotoh Tetsuo , Kisimoto Ryoiti , Yanase Tohru , Matsumoto Yukiko , Kageyama Daisuke , Noda Hiroaki
Published: September 2009

Cardinium
bacteria, members of the phylum
Cytophaga
-
Flavobacterium
-
Bacteroides
(CFB), are intracellular bacteria in arthropods that are capable of inducing reproductive abnormalities in their hosts, which include parasitic wasps, mites, and spiders. A high frequency of
Cardinium
infection was detected in planthoppers (27 out of 57 species were infected). A high frequency of
Cardinium
infection was also found in spider mites (9 out of 22 species were infected). Frequencies of double infection by
Cardinium
and
Wolbachia
bacteria (
Alphaproteobacteria
capable of manipulating reproduction of their hosts) were disproportionately high in planthoppers but not in spider mites. A new group of bacteria, phylogenetically closely related to but distinct from previously described
Cardinium
bacteria (based on 16S rRNA and
gyrB
genes) was found in 4 out of 25 species of
Culicoides
biting midges. These bacteria possessed a microfilament-like structure that is a morphological feature previously found in
Cardinium
and
Paenicardinium
. The bacteria close to the genus
Cardinium
consist of at least three groups, A, B, and C. Group A is present in various species of arthropods and was previously referred to as “
Candidatus
Cardinium hertigii,” group B is present in plant parasitic nematodes and was previously referred to as “
Candidatus
Paenicardinium endonii,” and group C is present in
Culicoides
biting midges. On the basis of morphological and molecular data, we propose that the nomenclature of these three groups be integrated into a single species, “
Candidatus
Cardinium hertigii.”
Isolation and Identification of Novel Microcystin-Degrading Bacteria
Authors: Manage Pathmalal M. , Edwards Christine , Singh Brajesh K. , Lawton Linda A.
Published: September 2009

Of 31 freshwater bacterial isolates screened using the Biolog MT2 assay to determine their metabolism of the microcystin LR, 10 were positive. Phylogenetic analysis (16S rRNA) identified them as
Arthrobacter
spp.,
Brevibacterium
sp., and
Rhodococcus
sp. This is the first report of microcystin degraders that do not belong to the
Proteobacteria
.
Mss11, a Transcriptional Activator, Is Required for Hyphal Development in
Candida albicans
Authors: Su Chang , Li Yandong , Lu Yang , Chen Jiangye
Published: September 2009

Candida albicans
undergoes a morphological transition from yeast to hyphae in response to a variety of stimuli and growth conditions. We previously isolated a LisH domain containing transcription factor Flo8, which is essential for hyphal development in
C. albicans
. To search the putative binding partner of Flo8 in
C. albicans
, we identified
C. albicans
Mss11, a functional homolog of
Saccharomyces cerevisiae
Mss11, which also contains a LisH motif at its N terminus.
C. albicans
Mss11 can interact with Flo8 via the LisH motif by in vivo coimmunoprecipitation. The results of a chromatin immunoprecipitation (ChIP) assay showed that more Mss11 and Flo8 proteins bound to the upstream activating sequence region of
HWP1
promoter in hyphal cells than in yeast cells, and the increased binding of each of these two proteins responding to hyphal induction was dependent on the other. Overexpression of
MSS11
enhanced filamentous growth. Deletion of
MSS11
caused a profound defect in hyphal development and the induction of hypha-specific genes. Our data suggest that Mss11 functions as an activator in hyphal development of
C. albicans
. Furthermore, overexpression of
FLO8
can bypass the requirement of Mss11 in filamentous formation, whereas overexpression of
MSS11
failed to promote hyphae growth in
flo8
mutants. In summary, we show that the expression level of
MSS11
increases during hyphal induction, and the enhanced expression of
MSS11
may contribute to cooperative binding of Mss11 and Flo8 to the
HWP1
promoter.
Chitinases Are Essential for Sexual Development but Not Vegetative Growth in
Cryptococcus neoformans
Authors: Baker Lorina G. , Specht Charles A. , Lodge Jennifer K.
Published: September 2009

Cryptococcus neoformans
is an opportunistic pathogen that mainly infects immunocompromised individuals. The fungal cell wall of
C. neoformans
is an excellent target for antifungal therapies since it is an essential organelle that provides cell structure and integrity. Importantly, it is needed for localization or attachment of known virulence factors, including melanin, phospholipase, and the polysaccharide capsule. The polysaccharide fraction of the cryptococcal cell wall is a complex structure composed of chitin, chitosan, and glucans. Chitin is an indispensable component of many fungal cell walls that contributes significantly to cell wall strength and integrity. Fungal cell walls are very dynamic, constantly changing during cell division and morphogenesis. Hydrolytic enzymes, such as chitinases, have been implicated in the maintenance of cell wall plasticity and separation of the mother and daughter cells at the bud neck during vegetative growth in yeast. In
C. neoformans
we identified four predicted endochitinases,
CHI2
,
CHI21
,
CHI22
, and
CHI4
, and a predicted exochitinase, hexosaminidase,
HEX1
. Enzymatic analysis indicated that Chi2, Chi22, and Hex1 actively degraded chitinoligomeric substrates. Chi2 and Hex1 activity was associated mostly with the cellular fraction, and Chi22 activity was more prominent in the supernatant. The enzymatic activity of Hex1 increased when grown in media containing only
N
-acetylglucosamine as a carbon source, suggesting that its activity may be inducible by chitin degradation products. Using a quadruple endochitinase deletion strain, we determined that the endochitinases do not affect the growth or morphology of
C. neoformans
during asexual reproduction. However, mating assays indicated that Chi2, Chi21, and Chi4 are each involved in sexual reproduction. In summary, the endochitinases were found to be dispensable for routine vegetative growth but not sexual reproduction.
Two Novel Techniques for Determination of Polysaccharide Cross-Links Show that Crh1p and Crh2p Attach Chitin to both β(1-6)- and β(1-3)Glucan in the
Saccharomyces cerevisiae
Cell Wall
Authors: Cabib Enrico
Published: September 2009

Previous work, using solubilization of yeast cell walls by carboxymethylation, before or after digestion with β(1-3)- or β(1-6)glucanase, followed by size chromatography, showed that the transglycosylases Crh1p and Crh2p/Utr2p were redundantly required for the attachment of chitin to β(1-6)glucan. With this technique,
crh1
Δ
crh2
Δ mutants still appeared to contain a substantial percentage of chitin linked to β(1-3)glucan. Two novel procedures have now been developed for the analysis of polysaccharide cross-links in the cell wall. One is based on the affinity of curdlan, a β(1-3)glucan, for β(1-3)glucan chains in carboxymethylated cell walls. The other consists of in situ deacetylation of cell wall chitin, generating chitosan, which can be extracted with acetic acid, either directly (free chitosan) or after digestion with different glucanases (bound chitosan). Both methodologies indicated that all of the chitin in
crh1
Δ
crh2
Δ strains is free. Reexamination of the previously used procedure revealed that the β(1-3)glucanase preparation used (zymolyase) is contaminated with a small amount of endochitinase, which caused erroneous results with the double mutant. After removing the chitinase from the zymolyase, all three procedures gave coincident results. Therefore, Crh1p and Crh2p catalyze the transfer of chitin to both β(1-3)- and β(1-6)glucan, and the biosynthetic mechanism for all chitin cross-links in the cell wall has been established.
The
rkp-1
Cluster Is Required for Secretion of Kdo Homopolymeric Capsular Polysaccharide in
Sinorhizobium meliloti
Strain Rm1021
Authors: Müller Maike G. , Forsberg Lennart S. , Keating David H.
Published: September 2009

Under conditions of nitrogen stress, leguminous plants form symbioses with soil bacteria called rhizobia. This partnership results in the development of structures called root nodules, in which differentiated endosymbiotic bacteria reduce molecular dinitrogen for the host. The establishment of rhizobium-legume symbioses requires the bacterial synthesis of oligosaccharides, exopolysaccharides, and capsular polysaccharides. Previous studies suggested that the 3-deoxy-
d
-manno-oct-2-ulopyranosonic acid (Kdo) homopolymeric capsular polysaccharide produced by strain
Sinorhizobium meliloti
Rm1021 contributes to symbiosis with
Medicago sativa
under some conditions. However, a conclusive symbiotic role for this polysaccharide could not be determined due to a lack of mutants affecting its synthesis. In this study, we have further characterized the synthesis, secretion, and symbiotic function of the Kdo homopolymeric capsule. We showed that mutants lacking the enigmatic
rkp-1
gene cluster fail to display the Kdo capsule on the cell surface but accumulate an intracellular polysaccharide of unusually high
M
r
. In addition, we have demonstrated that mutations in
kdsB2
, smb20804, and smb20805 affect the polymerization of the Kdo homopolymeric capsule. Our studies also suggest a role for the capsular polysaccharide in symbiosis. Previous reports have shown that the overexpression of
rkpZ
from strain Rm41 allows for the symbiosis of
exoY
mutants of Rm1021 that are unable to produce the exopolysaccharide succinoglycan. Our results demonstrate that mutations in the
rkp-1
cluster prevent this phenotypic suppression of
exoY
mutants, although mutations in
kdsB2
, smb20804, and smb20805 have no effect.
Catabolism of Raffinose, Sucrose, and Melibiose in
Erwinia chrysanthemi
3937
Authors: Hugouvieux-Cotte-Pattat Nicole , Charaoui-Boukerzaza Sana
Published: September 2009

Erwinia chrysanthemi
(
Dickeya dadantii
) is a plant pathogenic bacterium that has a large capacity to degrade the plant cell wall polysaccharides. The present study reports the metabolic pathways used by
E. chrysanthemi
to assimilate the oligosaccharides sucrose and raffinose, which are particularly abundant plant sugars.
E. chrysanthemi
is able to use sucrose, raffinose, or melibiose as a sole carbon source for growth. The two gene clusters
scrKYABR
and
rafRBA
are necessary for their catabolism. The phenotypic analysis of
scr
and
raf
mutants revealed cross-links between the assimilation pathways of these oligosaccharides. Sucrose catabolism is mediated by the genes
scrKYAB
. While the
raf
cluster is sufficient to catabolize melibiose, it is incomplete for raffinose catabolism, which needs two additional steps that are provided by
scrY
and
scrB
. The
scr
and
raf
clusters are controlled by specific repressors, ScrR and RafR, respectively. Both clusters are controlled by the global activator of carbohydrate catabolism, the cyclic AMP receptor protein (CRP).
E. chrysanthemi
growth with lactose is possible only for mutants with a derepressed nonspecific lactose transport system, which was identified as RafB. RafR inactivation allows the bacteria to the assimilate the novel substrates lactose, lactulose, stachyose, and melibionic acid. The
raf
genes also are involved in the assimilation of α- and β-methyl-
d
-galactosides. Mutations in the
raf
or
scr
genes did not significantly affect
E. chrysanthemi
virulence. This could be explained by the large variety of carbon sources available in the plant tissue macerated by
E. chrysanthemi
.
PhoB Regulates Motility, Biofilms, and Cyclic di-GMP inVibrio cholerae
Authors: Pratt Jason T. , McDonough EmilyKate , Camilli Andrew
Published: September 2009

Signaling through the second messenger cyclic di-GMP (c-di-GMP) is central to the life cycle ofVibrio cholerae. However, relatively little is known about the signaling mechanism, including the specific external stimuli that regulate c-di-GMP concentration. Here, we show that the phosphate responsive regulator PhoB regulates an operon,acgAB, which encodes c-di-GMP metabolic enzymes. We show that induction ofacgABby PhoB positively regulatesV. choleraemotility in vitro and that PhoB regulates expression ofacgABat late stages duringV. choleraeinfection in the infant mouse small intestine. These data support a model whereby PhoB becomes activated at a late stage of infection in preparation for dissemination ofV. choleraeto the aquatic environment and suggest that the concentration of exogenous phosphate may become limited at late stages of infection.
Levels of the Secreted
Vibrio cholerae
Attachment Factor GbpA Are Modulated by Quorum-Sensing-Induced Proteolysis
Authors: Jude Brooke A. , Martinez Raquel M. , Skorupski Karen , Taylor Ronald K.
Published: September 2009

Vibrio cholerae
is the etiologic agent of cholera in humans. Intestinal colonization occurs in a stepwise fashion, initiating with attachment to the small intestinal epithelium. This attachment is followed by expression of the toxin-coregulated pilus, microcolony formation, and cholera toxin (CT) production. We have recently characterized a secreted attachment factor, GlcNAc binding protein A (GbpA), which functions in attachment to environmental chitin sources as well as to intestinal substrates. Studies have been initiated to define the regulatory network involved in GbpA induction. At low cell density, GbpA was detected in the culture supernatant of all wild-type (WT) strains examined. In contrast, at high cell density, GbpA was undetectable in strains that produce HapR, the central regulator of the cell density-dependent quorum-sensing system of
V. cholerae
. HapR represses the expression of genes encoding regulators involved in
V. cholerae
virulence and activates the expression of genes encoding the secreted proteases HapA and PrtV. We show here that GbpA is degraded by HapA and PrtV in a time-dependent fashion. Consistent with this, Δ
hapA
Δ
prtV
strains attach to chitin beads more efficiently than either the WT or a Δ
hapA
Δ
prtV
Δ
gbpA
strain. These results suggest a model in which GbpA levels fluctuate in concert with the bacterial production of proteases in response to quorum-sensing signals. This could provide a mechanism for GbpA-mediated attachment to, and detachment from, surfaces in response to environmental cues.
The Glycerol-3-Phosphate Permease GlpT Is the Only Fosfomycin Transporter in
Pseudomonas aeruginosa
Authors: Castañeda-García Alfredo , Rodríguez-Rojas Alexandro , Guelfo Javier R. , Blázquez Jesús
Published: September 2009

Fosfomycin is transported into
Escherichia coli
via both glycerol-3-phosphate (GlpT) and a hexose phosphate transporter (UhpT). Consequently, the inactivation of either
glpT
or
uhpT
confers increased fosfomycin resistance in this species. The inactivation of other genes, including
ptsI
and
cyaA
, also confers significant fosfomycin resistance. It has been assumed that identical mechanisms are responsible for fosfomycin transport into
Pseudomonas aeruginosa
cells. The study of an ordered library of insertion mutants in
P. aeruginosa
PA14 demonstrated that only insertions in
glpT
confer significant resistance. To explore the uniqueness of this resistance target in
P. aeruginosa
, the linkage between fosfomycin resistance and the use of glycerol-3-phosphate was tested. Fosfomycin-resistant (Fos-R) mutants were obtained in LB and minimal medium containing glycerol as the sole carbon source at a frequency of 10
−6
. However, no Fos-R mutants grew on plates containing fosfomycin and glycerol-3-phosphate instead of glycerol (mutant frequency, ≤5 × 10
−11
). In addition, 10 out of 10 independent spontaneous Fos-R mutants, obtained on LB-fosfomycin, harbored mutations in
glpT
, and in all cases the sensitivity to fosfomycin was recovered upon complementation with the wild-type
glpT
gene. The analysis of these mutants provides additional insights into the structure-function relationship of glycerol-3-phosphate the transporter in
P. aeruginosa
. Studies with glucose-6-phosphate and different mutant derivatives strongly suggest that
P. aeruginosa
lacks a specific transport system for this sugar. Thus,
glpT
seems to be the only fosfomycin resistance mutational target in
P. aeruginosa
. The high frequency of Fos-R mutations and their apparent lack of fitness cost suggest that Fos-R variants will be obtained easily in vivo upon the fosfomycin treatment of
P. aeruginosa
infections.
Roles of Periplasmic Chaperone Proteins in the Biogenesis of Serine Protease Autotransporters of
Enterobacteriaceae
Authors: Ruiz-Perez Fernando , Henderson Ian R. , Leyton Denisse L. , Rossiter Amanda E. , Zhang Yinghua , Nataro James P.
Published: September 2009

The serine protease autotransporters of
Enterobacteriaceae
(SPATEs) represent a large family of virulence factors. The prevailing model for autotransporter secretion comprises entry to the periplasm via the Sec apparatus, followed by an obscure series of steps in which the C terminus of the periplasmic species inserts into the outer membrane as a β-barrel protein, accompanied by translocation of the passenger domain to the bacterial cell surface. Little is known about the fate of the autotransporter proteins in the periplasm, including whether accessory periplasmic proteins are involved in translocation to the external milieu. Here we studied the role of the major periplasmic chaperones in the biogenesis of EspP, a prototype SPATE protein produced by
Escherichia coli
O157:H7. The yeast two-hybrid approach, secretion analysis of chaperone mutant strains, and surface plasmon resonance analysis (SPR) revealed direct protein-protein interactions between the periplasmic SurA and DegP chaperones and either the EspP-β or EspP passenger domains. The secretion of EspP was moderately reduced in the
surA
and
skp
mutant strains but severely impaired in the
degP
background. Site-directed mutagenesis of highly conserved aromatic amino acid residues in the SPATE family resulted in ∼80% reduction of EspP secretion. Synthetic peptides containing aromatic residues derived from the EspP passenger domain blocked DegP and SurA binding to the passenger domain. SPR suggested direct protein-protein interaction between periplasmic chaperones and the unfolded EspP passenger domain. Our data suggest that translocation of AT proteins may require accessory factors, calling into question the moniker “autotransporter.”
Comparative Structure-Function Analysis of Mannose-Specific FimH Adhesins from
Klebsiella pneumoniae
and
Escherichia coli
Authors: Stahlhut Steen G. , Tchesnokova Veronika , Struve Carsten , Weissman Scott J. , Chattopadhyay Sujay , Yakovenko Olga , Aprikian Pavel , Sokurenko Evgeni V. , Krogfelt Karen Angeliki
Published: September 2009

FimH, the adhesive subunit of type 1 fimbriae expressed by many enterobacteria, mediates mannose-sensitive binding to target host cells. At the same time, fine receptor-structural specificities of FimH from different species can be substantially different, affecting bacterial tissue tropism and, as a result, the role of the particular fimbriae in pathogenesis. In this study, we compared functional properties of the FimH proteins from
Escherichia coli
and
Klebsiella pneumoniae
, which are both 279 amino acids in length but differ by some ∼15% of residues. We show that
K. pneumoniae
FimH is unable to mediate adhesion in a monomannose-specific manner via terminally exposed Manα(1-2) residues in N-linked oligosaccharides, which are the structural basis of the tropism of
E. coli
FimH for uroepithelial cells. However,
K. pneumoniae
FimH can bind to the terminally exposed Manα(1-3)Manβ(1-4)GlcNAcβ1 trisaccharide, though only in a shear-dependent manner, wherein the binding is marginal at low shear force but enhanced sevenfold under increased shear. A single mutation in the
K. pneumoniae
FimH, S62A, converts the mode of binding from shear dependent to shear independent. This mutation has occurred naturally in the course of endemic circulation of a nosocomial uropathogenic clone and is identical to a pathogenicity-adaptive mutation found in highly virulent uropathogenic strains of
E. coli
, in which it also eliminates the dependence of
E. coli
binding on shear. The shear-dependent binding properties of the
K. pneumoniae
and
E. coli
FimH proteins are mediated via an allosteric catch bond mechanism. Thus, despite differences in FimH structure and fine receptor specificity, the shear-dependent nature of FimH-mediated adhesion is highly conserved between bacterial species, supporting its remarkable physiological significance.
Polar Positioning of a Conjugation Protein from the Integrative and Conjugative Element ICEBs1ofBacillus subtilis
Authors: Berkmen Melanie B. , Lee Catherine A. , Loveday Emma-Kate , Grossman Alan D.
Published: September 2009

ICEBs1is an integrative and conjugative element found in the chromosome ofBacillus subtilis. ICEBs1encodes functions needed for its excision and transfer to recipient cells. We found that the ICEBs1geneconE(formerlyyddE) is required for conjugation and that conjugative transfer of ICEBs1requires a conserved ATPase motif of ConE. ConE belongs to the HerA/FtsK superfamily of ATPases, which includes the well-characterized proteins FtsK, SpoIIIE, VirB4, and VirD4. We found that a ConE-GFP (green fluorescent protein) fusion associated with the membrane predominantly at the cell poles in ICEBs1donor cells. At least one ICEBs1product likely interacts with ConE to target it to the membrane and cell poles, as ConE-GFP was dispersed throughout the cytoplasm in a strain lacking ICEBs1. We also visualized the subcellular location of ICEBs1. When integrated in the chromosome, ICEBs1was located near midcell along the length of the cell, a position characteristic of that chromosomal region. Following excision, ICEBs1was more frequently found near a cell pole. Excision of ICEBs1also caused altered positioning of at least one component of the replisome. Taken together, our findings indicate that ConE is a critical component of the ICEBs1conjugation machinery, that conjugative transfer of ICEBs1fromB. subtilislikely initiates at a donor cell pole, and that ICEBs1affects the subcellular position of the replisome.
Small Stress Response Proteins in
Escherichia coli
: Proteins Missed by Classical Proteomic Studies
Authors: Hemm Matthew R. , Paul Brian J. , Miranda-Ríos Juan , Zhang Aixia , Soltanzad Nima , Storz Gisela
Published: September 2009

Proteins of 50 or fewer amino acids are poorly characterized in all organisms. The corresponding genes are challenging to reliably annotate, and it is difficult to purify and characterize the small protein products. Due to these technical limitations, little is known about the abundance of small proteins, not to mention their biological functions. To begin to characterize these small proteins in
Escherichia coli
, we assayed their accumulation under a variety of growth conditions and after exposure to stress. We found that many small proteins accumulate under specific growth conditions or are stress induced. For some genes, the observed changes in protein levels were consistent with known transcriptional regulation, such as ArcA activation of the operons encoding
yccB
and
ybgT
. However, we also identified novel regulation, such as Zur repression of
ykgMO
, cyclic AMP response protein (CRP) repression of
azuC
, and CRP activation of
ykgR
. The levels of 11 small proteins increase after heat shock, and induction of at least 1 of these, YobF, occurs at a posttranscriptional level. These results show that small proteins are an overlooked subset of stress response proteins in
E. coli
and provide information that will be valuable for determining the functions of these proteins.
Small RNAs and Small Proteins Involved in Resistance to Cell Envelope Stress and Acid Shock in
Escherichia coli
: Analysis of a Bar-Coded Mutant Collection
Authors: Hobbs Errett C. , Astarita Jillian L. , Storz Gisela
Published: September 2009

More than 80 small regulatory RNAs (sRNAs) and 60 proteins of 16 to 50 amino acids (small proteins) are encoded in the
E
scherichia
coli
genome. The vast majority of the corresponding genes have no known function. We screened 125 DNA bar-coded mutants to identify novel cell envelope stress and acute acid shock phenotypes associated with deletions of genes coding for sRNAs and small proteins. Nine deletion mutants (
ssrA
,
micA
,
ybaM
,
ryeF
,
yqcG
,
sroH
,
ybhT
,
yobF
, and
glmY
) were sensitive to cell envelope stress and two were resistant (
rybB
and
blr
). Deletion mutants of genes coding for four small proteins (
yqgB
,
mgrB
,
yobF
, and
yceO
) were sensitive to acute acid stress. We confirmed each of these phenotypes in one-on-one competition assays against otherwise-wild-type
lacZ
mutant cells. A more detailed investigation of the SsrA phenotype suggests that ribosome release is critical for resistance to cell envelope stress. The bar-coded deletion collection we generated can be screened for sensitivity or resistance to virtually any stress condition.
Reciprocal Regulation between SigK and Differentiation Programs inStreptomyces coelicolor
Authors: Mao Xu-Ming , Zhou Zhan , Hou Xiao-Ping , Guan Wen-Jun , Li Yong-Quan
Published: September 2009

Here we reported that deletion of SigK (SCO6520), a sigma factor inStreptomyces coelicolor, caused an earlier switch from vegetative mycelia to aerial mycelia and higher expression ofchpEandchpHthan that in the wild type. Loss of SigK also resulted in accelerated and enhanced production of antibiotics, actinorhodin, and undecylprodigiosin and increased expression ofactII-orf4andredD. These results suggested that SigK had a negative role in morphological transition and secondary metabolism. Furthermore, thesigKpromoter (sigKp) activity gradually increased andsigKexpression was partially dependent on SigK, but this dependence decreased during the developmental course of substrate mycelia. Meanwhile, two potentially nonspecific cleavages occurred between SigK and green fluorescent protein, and the SigK fusion proteins expressed under the constitutive promoterermEp* sharply decreased and disappeared when aerial mycelia emerged. If expressed undersigKp, 3FLAG-SigK showed similar dynamic patterns but did not decrease as sharply as SigK expressed underermEp*. These data suggested that the climbing expression ofsigKmight reduce the prompt degradation of SigK during vegetative hypha development for the proper timing of morphogenesis and that SigK vanished to remove the block for the emergence of aerial mycelia. Thus, we proposed that SigK had inhibitory roles on developmental events and that these inhibitory effects may be released by SigK degradation.
An Interactive Regulatory Network Controls Stress Response inBifidobacterium breveUCC2003
Authors: Zomer Aldert , Fernandez Matilde , Kearney Breda , Fitzgerald Gerald F. , Ventura Marco , van Sinderen Douwe
Published: September 2009

Members of the genusBifidobacteriumare gram-positive bacteria that commonly are found in the gastrointestinal tract (GIT) of mammals, including humans. Because of their perceived probiotic properties, they frequently are incorporated as functional ingredients in food products. From probiotic production to storage and GIT delivery, bifidobacteria encounter a plethora of stresses. To cope with these environmental challenges, they need to protect themselves through stress-induced adaptive responses. We have determined the response ofB. breveUCC2003 to various stresses (heat, osmotic, and solvent) using transcriptome analysis, DNA-protein interactions, and GusA reporter fusions, and we combined these with results from an in silico analysis. The integration of these results allowed the formulation of a model for an interacting regulatory network for stress response inB. breveUCC2003 where HspR controls the SOS response and the ClgR regulon, which in turn regulates and is regulated by HrcA. This model of an interacting regulatory network is believed to represent the paradigm for stress adaptation in bifidobacteria.
QUASINORMAL MODES OF RN BLACK HOLE SPACETIME WITH COSMIC STRING IN A DIRAC FIELD
Authors: SINI R. , KURIAKOSE V. C.
Published: September 2009

We evaluate quasinormal mode frequencies for RN black hole spacetimes with cosmic string perturbed by a massless Dirac field, using Pöschl–Teller potential method. We find that only in the case of RN black hole having small charge, the effect due to cosmic string will dominate when perturbed by a negatively charged Dirac field, but if we are perturbing with positively charged Dirac field decay will be less in the case of black hole having cosmic string compared to the RN black hole without string.
INTERACTING GENERALIZED CHAPLYGIN GAS MODEL WITH HOLOGRAPHIC DARK ENERGY
Authors: WU YA BO , FU HUAN HUAN , CHENG FANG YUAN , WANG JUN , YANG WEI QIANG , ZHANG SHU HONG , WANG LEI , QI FANG
Published: September 2009

In this paper we consider the interacting generalized Chaplygin gas model as a unified scheme, in which the holographic dark energy as the dark energy component interacts with the cold dark matter with wdm= 0. Concretely, we mainly discuss the two kinds of interactions between the dark energy and dark matter, and give the evolution trajectories of density parameter and equation of state of dark energy as well as the decelerate parameter. By analysis, we show that in both cases of Q = 3bHρdeand Q = 3bHρGCG, the evolution laws and the present values of Ωde, wdeand q are compatible with the astronomical observations, but the effects of the coupling constant and holographic constant on them are completely different.
COHERENT STATES AND SCHWINGER MODELS FOR PSEUDO GENERALIZATION OF THE HEISENBERG ALGEBRA
Authors: FAKHRI H. , MOJAVERI B. , DEHGHANI A.
Published: September 2009

We show that the non-Hermitian Hamiltonians of the simple harmonic oscillator with [Formula: see text] and [Formula: see text] symmetries involve a pseudo generalization of the Heisenberg algebra via two pairs of creation and annihilation operators which are [Formula: see text]-pseudo-Hermiticity and [Formula: see text]-anti-pseudo-Hermiticity of each other. The non-unitary Heisenberg algebra is represented by each of the pair of the operators in two different ways. Consequently, the coherent and the squeezed coherent states are calculated in two different approaches. Moreover, it is shown that the approach of Schwinger to construct the su(2), su(1, 1) and sp(4, ℝ) unitary algebras is promoted so that unitary algebras with more linearly dependent number of generators are made.
SUTHERLAND-TYPE TRIGONOMETRIC MODELS, TRIGONOMETRIC INVARIANTS AND MULTIVARIABLE POLYNOMIALS II: E7 CASE
Authors: LÓPEZ VIEYRA J. C. , GARCÍA M. A. G. , TURBINER A. V.
Published: September 2009

It is shown that the E7 trigonometric Olshanetsky–Perelomov Hamiltonian, when written in terms of the Fundamental Trigonometric Invariants (FTI), is in algebraic form, i.e. has polynomial coefficients, and preserves the infinite flag of polynomial spaces with the characteristic vector α = (1, 2, 2, 2, 3, 3, 4). Its flag coincides with one of the minimal characteristic vectors for the E7 rational model, which in turn coincides with the E7 highest root.
MASSIVE METASTABLE CHARGED (S)PARTICLES AT THE LHC
Authors: RAKLEV ARE R.
Published: September 2009

This brief review deals with recent interest in the prospects of observing a Massive Metastable Charged Particle (MMCP) at the Large Hadron Collider (LHC), and measuring its properties there. We discuss the motivation for scenarios with MMCPs in a phenomenological context, focusing on supersymmetric models that allow us to explore the expected experimental signatures of MMCPs at the LHC. We review current bounds and give estimates of the LHC reach in terms of MMCP masses.
VEGF-C regulates lymphangiogenesis and capillary stability by regulation of PDGF-B
Authors: Onimaru Mitsuho , Yonemitsu Yoshikazu , Fujii Takaaki , Tanii Mitsugu , Nakano Toshiaki , Nakagawa Kazunori , Kohno Ri-ichiro , Hasegawa Mamoru , Nishikawa Shin-ichi , Sueishi Katsuo
Published: September 2009

Emerging evidence indicates that the tight communication between vascular endothelial cells and mural cells using platelet-derived growth factor (PDGF)-BB is essential for capillary stabilization during the angiogenic process. However, little is known about the related regulator that determines PDGF-BB expression. Using murine models of therapeutic neovascularization, we here show that a typical lymphangiogenic factor, vascular endothelial growth factor (VEGF)-C, is an essential regulator determining PDGF-BB expression for vascular stabilization via a paracrine mode of action. The blockade of VEGF type 3 receptor (VEGFR3) using neutralizing antibody AFL-4 abrogated FGF-2-mediated limb salvage and blood flow recovery in severely ischemic hindlimb. Interestingly, inhibition of VEGFR3 activity not only diminished lymphangiogenesis, but induced marked dilatation of capillary vessels, showing mural cell dissociation. In these mice, VEGF-C and PDGF-B were upregulated in the later phase after induced ischemia, on day 7, when exogenous FGF-2 expression had already declined, and blockade of VEGFR3 or PDGF-BB activities diminished PDGF-B or VEGF-C expression, respectively. These results clearly indicate that VEGF-C is a critical mediator, not only for lymphangiogenesis, but also for capillary stabilization, the essential molecular mechanism of communication between endothelial cells and mural cells during neovascularization.