bluuebunny/crossref_metadata_2025 huggingface dataset:
Microbial and Physicochemical Characteristics of Compact Anaerobic Ammonium-Oxidizing Granules in an Upflow Anaerobic Sludge Blanket Reactor
Authors: Ni Bing-Jie , Hu Bao-Lan , Fang Fang , Xie Wen-Ming , Kartal Boran , Liu Xian-Wei , Sheng Guo-Ping , Jetten Mike , Zheng Ping , Yu Han-Qing
Published: February 2010

Anaerobic ammonium oxidation (anammox) is a promising new process to treat high-strength nitrogenous wastewater. Due to the low growth rate of anaerobic ammonium-oxidizing bacteria, efficient biomass retention is essential for reactor operation. Therefore, we studied the settling ability and community composition of the anaerobic ammonium-oxidizing granules, which were cultivated in an upflow anaerobic sludge blanket (UASB) reactor seeded with aerobic granules. With this seed, the start-up period was less than 160 days at a NH
4
+
-N removal efficiency of 94% and a loading rate of 0.064 kg N per kg volatile suspended solids per day. The formed granules were bright red and had a high settling velocity (41 to 79 m h
−1
). Cells and extracellular polymeric substances were evenly distributed over the anaerobic ammonium-oxidizing granules. The high percentage of anaerobic ammonium-oxidizing bacteria in the granules could be visualized by fluorescent
in situ
hybridization and electron microscopy. The copy numbers of 16S rRNA genes of anaerobic ammonium-oxidizing bacteria in the granules were determined to be 4.6 × 10
8
copies ml
−1
. The results of this study could be used for a better design, shorter start-up time, and more stable operation of anammox systems for the treatment of nitrogen-rich wastewaters.
Identification of
Bacillus cereus
Genes Specifically Expressed during Growth at Low Temperatures
Authors: Brillard Julien , Jéhanno Isabelle , Dargaignaratz Claire , Barbosa Isabelle , Ginies Christian , Carlin Frédéric , Fedhila Sinda , Nguyen-the Christophe , Broussolle Véronique , Sanchis Vincent
Published: February 2010

The mechanisms involved in the ability of
Bacillus cereus
to multiply at low temperatures were investigated. It was assumed that many genes involved in cold acclimation would be upregulated at low temperatures. Recombinase-based
in vivo
expression technology (IVET) was adapted to the detection of the transient activation of
B. cereus
promoters during growth at 10°C. Four independent screenings of a promoter library from type strain ATCC 14579 were performed, and 17 clones were isolated. They corresponded to 17 promoter regions that displayed reproducibly elevated expression at 10°C relative to expression at 30°C. This analysis revealed several genes that may be important for
B. cereus
to grow successfully under the restrictive conditions of cold habitats. Among them, a locus corresponding to open reading frames BC5402 to BC5398, harboring a lipase-encoding gene and a putative transcriptional regulator, was identified three times. While a mutation in the putative regulator-encoding gene did not cause any particular phenotype, a mutant deficient in the lipase-encoding gene showed reduced growth abilities at low temperatures compared with the parental strain. The mutant did not change its fatty acid profiles in the same way as the wild type when grown at 12°C instead of 37°C. This study demonstrates the feasibility of a promoter trap strategy for identifying cold-induced genes. It outlines a first picture of the different processes involved in
B. cereus
cold acclimation.
Putative TetR Family Transcriptional Regulator SCO1712 Encodes an Antibiotic Downregulator in
Streptomyces coelicolor
Authors: Lee Han-Na , Huang Jianqiang , Im Jong-Hyuk , Kim Seon-Hye , Noh Jun-Hee , Cohen Stanley N. , Kim Eung-Soo
Published: February 2010

A
tetR
family transcriptional regulatory gene (SCO1712) was identified as a global antibiotic regulatory gene from a
Streptomyces
interspecies DNA microarray analysis. SCO1712 disruption in
S
treptomyces coelicolor
not only upregulated antibiotic biosynthesis through pathway-specific regulators when a previously identified pleiotropic downregulatory
wblA
was expressed but also further stimulated antibiotic production in a
wblA
deletion mutant, implying that SCO1712 might encode a novel antibiotic downregulator.
Responses of Ammonia-Oxidizing Bacterial and Archaeal Populations to Organic Nitrogen Amendments in Low-Nutrient Groundwater
Authors: Reed David W. , Smith Jason M. , Francis Christopher A. , Fujita Yoshiko
Published: February 2010

To evaluate the potential for organic nitrogen addition to stimulate the
in situ
growth of ammonia oxidizers during a field scale bioremediation trial, samples collected from the Eastern Snake River Plain Aquifer in Idaho before, during, and after the addition of molasses and urea were subjected to PCR analysis of ammonia monooxygenase subunit A (
amoA
) genes. Ammonia-oxidizing bacteria (AOB) and archaea (AOA) were present in all of the samples tested, with AOA
amoA
genes outnumbering AOB
amoA
genes in all of the samples. Following urea addition, nitrate levels rose and bacterial
amoA
copy numbers increased dramatically, suggesting that urea hydrolysis stimulated nitrification. Bacterial
amoA
diversity was limited to two
Nitrosomonas
phylotypes, whereas archaeal
amoA
analyses revealed 20 distinct operational taxonomic units, including several that were markedly different from all previously reported sequences. Results from this study demonstrate the likelihood of stimulating ammonia-oxidizing communities during field scale manipulation of groundwater conditions to promote urea hydrolysis.
Bacterial and Viral Pathogens in Live Oysters: 2007 United States Market Survey
Authors: DePaola Angelo , Jones Jessica L. , Woods Jacquelina , Burkhardt William , Calci Kevin R. , Krantz Jeffrey A. , Bowers John C. , Kasturi Kuppuswamy , Byars Robin H. , Jacobs Emily , Williams-Hill Donna , Nabe Khamphet
Published: February 2010

Two samples of market oysters, primarily from retail establishments, were collected twice each month in each of nine states during 2007. Samples were shipped refrigerated overnight to five U.S. Food and Drug Administration laboratories on a rotating basis and analyzed by most probable number (MPN) for total and pathogenic
Vibrio parahaemolyticus
and
V. vulnificus
numbers and for the presence of toxigenic
V. cholerae
,
Salmonella
spp., norovirus (NoV), and hepatitis A virus (HAV). Levels of indicator organisms, including fecal coliforms (MPN),
Escherichia coli
(MPN), male-specific bacteriophage, and aerobic plate counts, were also determined.
V. parahaemolyticus
and
V. vulnificus
levels were distributed seasonally and geographically by harvest region and were similar to levels observed in a previous study conducted in 1998-1999. Levels of pathogenic
V. parahaemolyticus
were typically several logs lower than total
V. parahaemolyticus
levels regardless of season or region. Pathogenic
V. parahaemolyticus
levels in the Gulf and Mid-Atlantic regions were about two logs greater than the levels observed in the Pacific and North Atlantic regions. Pathogens generally associated with fecal pollution were detected sporadically or not at all (toxigenic
V. cholerae
, 0%;
Salmonella
, 1.5%; NoV, 3.9%; HAV, 4.4%). While seasonal prevalences of NoV and HAV were generally greater in oysters harvested from December to March, the low detection frequency obscured any apparent seasonal effects. Overall, there was no relationship between the levels of indicator microorganisms and the presence of enteric viruses. These data provide a baseline that can be used to further validate risk assessment predictions, determine the effectiveness of new control measures, and compare the level of protection provided by the U.S. shellfish sanitation system to those in other countries.
Differential Effects of Rare Specific Flavonoids on Compatible and Incompatible Strains in the
Myrica gale
-
Frankia
Actinorhizal Symbiosis
Authors: Popovici Jean , Comte Gilles , Bagnarol Émilie , Alloisio Nicole , Fournier Pascale , Bellvert Floriant , Bertrand Cédric , Fernandez Maria P.
Published: February 2010

Plant secondary metabolites, and specifically phenolics, play important roles when plants interact with their environment and can act as weapons or positive signals during biotic interactions. One such interaction, the establishment of mutualistic nitrogen-fixing symbioses, typically involves phenolic-based recognition mechanisms between host plants and bacterial symbionts during the early stages of interaction. While these mechanisms are well studied in the rhizobia-legume symbiosis, little is known about the role of plant phenolics in the symbiosis between actinorhizal plants and
Frankia
genus strains. In this study, the responsiveness of
Frankia
strains to plant phenolics was correlated with their symbiotic compatibility. We used
Myrica gale
, a host species with narrow symbiont specificity, and a set of compatible and noncompatible
Frankia
strains.
M. gale
fruit exudate phenolics were extracted, and 8 dominant molecules were purified and identified as flavonoids by high-resolution spectroscopic techniques. Total fruit exudates, along with two purified dihydrochalcone molecules, induced modifications of bacterial growth and nitrogen fixation according to the symbiotic specificity of strains, enhancing compatible strains and inhibiting incompatible ones. Candidate genes involved in these effects were identified by a global transcriptomic approach using ACN14a strain whole-genome microarrays. Fruit exudates induced differential expression of 22 genes involved mostly in oxidative stress response and drug resistance, along with the overexpression of a
whiB
transcriptional regulator. This work provides evidence for the involvement of plant secondary metabolites in determining symbiotic specificity and expands our understanding of the mechanisms, leading to the establishment of actinorhizal symbioses.
Intervention of Bro1 in pH-Responsive Rim20 Localization in Saccharomyces cerevisiae
Authors: Boysen Jacob H. , Subramanian Shoba , Mitchell Aaron P.
Published: February 2010

Yeast cells contain two Bro1 domain proteins: Bro1, which is required for endosomal trafficking, and Rim20, which is required for the response to the external pH via the Rim101 pathway. Rim20 associates with endosomal structures under alkaline growth conditions, when it promotes activation of Rim101 through proteolytic cleavage. We report here that the pH-dependent localization of Rim20 is contingent on the amount of Bro1 in the cell. Cells that lack Bro1 have increased endosomal Rim20-green fluorescent protein (GFP) under acidic conditions; cells that overexpress Bro1 have reduced endosomal Rim20-GFP under acidic or alkaline conditions. The novel endosomal association of Rim20-GFP in the absence of Bro1 requires ESCRT components including Vps27 but not specific Rim101 pathway components such as Dfg16. Vps27 influences the localization of Bro1 but is not required for RIM101 pathway activation in wild-type cells, thus suggesting that Rim20 enters the Bro1 localization pathway when a vacancy exists. Despite altered localization of Rim20, the lack of Bro1 does not bypass the need for signaling protein Dfg16 to activate Rim101, as evidenced by the expression levels of the Rim101 target genes
RIM8
and
SMP1
. Therefore, endosomal association of Rim20 is not sufficient to promote Rim101 activation.
Major Role for Cysteine Proteases during the Early Phase of Acanthamoeba castellanii Encystment
Authors: Leitsch David , Köhsler Martina , Marchetti-Deschmann Martina , Deutsch Andrea , Allmaier Günter , Duchêne Michael , Walochnik Julia
Published: February 2010

Acanthamoeba castellanii
is a facultative pathogen that has a two-stage life cycle comprising the vegetatively growing trophozoite stage and the dormant cyst stage. Cysts are formed when the cell encounters unfavorable conditions, such as environmental stress or food deprivation. Due to their rigid double-layered wall,
Acanthamoeba
cysts are highly resistant to antiamoebic drugs. This is problematic as cysts can survive initially successful chemotherapeutic treatment and cause relapse of the disease. We studied the
Acanthamoeba
encystment process by using two-dimensional gel electrophoresis (2DE) and found that most changes in the protein content occur early in the process. Truncated actin isoforms were found to abound in the encysting cell, and the levels of translation elongation factor 2 (EF2) were sharply decreased, indicating that the rate of protein synthesis must be low at this stage. In the advanced stage of encystment, however, EF2 levels and the trophozoite proteome were partly restored. The protease inhibitors PMSF (phenylmethylsulfonyl fluoride) and E64d [(2S,3S)-
trans
-epoxysuccinyl-
l
-leucylamido-3-methylbutane ethyl ester] inhibited the onset of encystment, whereas the protein synthesis inhibitor cycloheximide was ineffective. Changes in the protein profile, similar to those of encysting cells, could be observed with trophozoite homogenates incubated at room temperature for several hours. Interestingly, these changes could be inhibited significantly by cysteine protease inhibitors but not by inhibitors against other proteases. Taken together, we conclude that the encystment process in
A. castellanii
is of a bipartite nature consisting of an initial phase of autolysis and protein degradation and an advanced stage of restoration accompanied by the expression of encystment-specific genes.
A Trk/HKT-Type K
+
Transporter from Trypanosoma brucei
Authors: Mosimann Marc , Goshima Shinobu , Wenzler Tanja , Lüscher Alexandra , Uozumi Nobuyuki , Mäser Pascal
Published: February 2010

The molecular mechanisms of K
+
homeostasis are only poorly understood for protozoan parasites.
Trypanosoma brucei
subsp. parasites, the causative agents of human sleeping sickness and nagana, are strictly extracellular and need to actively concentrate K
+
from their hosts’ body fluids. The
T. brucei
genome contains two putative K
+
channel genes, yet the trypanosomes are insensitive to K
+
antagonists and K
+
channel-blocking agents, and they do not spontaneously depolarize in response to high extracellular K
+
concentrations. However, the trypanosomes are extremely sensitive to K
+
ionophores such as valinomycin. Surprisingly,
T. brucei
possesses a member of the Trk/HKT superfamily of monovalent cation permeases which so far had only been known from bacteria, archaea, fungi, and plants. The protein was named TbHKT1 and functions as a Na
+
-independent K
+
transporter when expressed in
Escherichia coli
,
Saccharomyces cerevisiae
, or
Xenopus laevis
oocytes. In trypanosomes,
TbHKT1
is expressed in both the mammalian bloodstream stage and the Tsetse fly midgut stage; however, RNA interference (RNAi)-mediated silencing of
TbHKT1
expression did not produce a growth phenotype in either stage. The presence of
HKT
genes in trypanosomatids adds a further piece to the enigmatic phylogeny of the Trk/HKT superfamily of K
+
transporters. Parsimonial analysis suggests that the transporters were present in the first eukaryotes but subsequently lost in several of the major eukaryotic lineages, in at least four independent events.
Use of the Plant Defense Protein Osmotin To Identify Fusarium oxysporum Genes That Control Cell Wall Properties
Authors: Lee Hyeseung , Damsz Barbara , Woloshuk Charles P. , Bressan Ray A. , Narasimhan Meena L.
Published: February 2010

Fusarium oxysporum
is the causative agent of fungal wilt disease in a variety of crops. The capacity of a fungal pathogen such as
F. oxysporum
f. sp.
nicotianae
to establish infection on its tobacco (
Nicotiana tabacum
) host depends in part on its capacity to evade the toxicity of tobacco defense proteins, such as osmotin.
Fusarium
genes that control resistance to osmotin would therefore reflect coevolutionary pressures and include genes that control mutual recognition, avoidance, and detoxification. We identified
FOR
(

F
usarium

O

smotin

R

esistance) genes on the basis of their ability to confer osmotin resistance to an osmotin-sensitive strain of
Saccharomyces cerevisiae
.
FOR1
encodes a putative cell wall glycoprotein.
FOR2
encodes the structural gene for glutamine:fructose-6-phosphate amidotransferase, the first and rate-limiting step in the biosynthesis of hexosamine and cell wall chitin.
FOR3
encodes
a
homolog of
SSD1
, which controls cell wall composition, longevity, and virulence in
S. cerevisiae
. A
for3
null mutation increased osmotin sensitivity of conidia and hyphae of
F. oxysporum
f. sp.
nicotianae
and also reduced cell wall β-1,3-glucan content. Together our findings show that conserved fungal genes that determine cell wall properties play a crucial role in regulating fungal susceptibility to the plant defense protein osmotin.
Basidiomycete Mating Type Genes and Pheromone Signaling
Authors: Raudaskoski Marjatta , Kothe Erika
Published: February 2010

The genome sequences of the basidiomyceteAgaricomycetesspeciesCoprinopsis cinerea,Laccaria bicolor,Schizophyllum commune,Phanerochaete chrysosporium, andPostia placenta, as well as ofCryptococcus neoformansandUstilago maydis, are now publicly available. Out of these fungi,C. cinerea,S. commune, andU. maydis, together with the budding yeastSaccharomyces cerevisiae, have been investigated for years genetically and molecularly for signaling in sexual reproduction. The comparison of the structure and organization of mating type genes in fungal genomes reveals an amazing conservation of genes regulating the sexual reproduction throughout the fungal kingdom. In agaricomycetes, two mating type loci,A, coding for homeodomain type transcription factors, andB, encoding a pheromone/receptor system, regulate the four typical mating interactions of tetrapolar species. Evidence for bothAandBmating type genes can also be identified in basidiomycetes with bipolar systems, where only two mating interactions are seen. In some of these fungi, theBlocus has lost its self/nonself discrimination ability and thus its specificity while retaining the other regulatory functions in development.In silicoanalyses now also permit the identification of putative components of the pheromone-dependent signaling pathways. Induction of these signaling cascades leads to development of dikaryotic mycelia, fruiting body formation, and meiotic spore production. In pheromone-dependent signaling, the role of heterotrimeric G proteins, components of a mitogen-activated protein kinase (MAPK) cascade, and cyclic AMP-dependent pathways can now be defined. Additionally, the pheromone-dependent signaling through monomeric, small GTPases potentially involved in creating the polarized cytoskeleton for reciprocal nuclear exchange and migration during mating is predicted.
SprB Is the Molecular Link between
Salmonella
Pathogenicity Island 1 (SPI1) and SPI4
Authors: Saini Supreet , Rao Christopher V.
Published: February 2010

Salmonella
pathogenicity island 1 (SPI1) and SPI4 have previously been shown to be jointly regulated. We report that SPI1 and SPI4 gene expression is linked through a transcriptional activator, SprB, encoded within SPI1 and regulated by HilA. SprB directly activates SPI4 gene expression and weakly represses SPI1 gene expression through HilD.
A Novel Sialic Acid Utilization and Uptake System in the Periodontal Pathogen
Tannerella forsythia
Authors: Roy Sumita , Douglas C. W. Ian , Stafford Graham P.
Published: February 2010

Tannerella forsythia
is a key contributor to periodontitis, but little is known of its virulence mechanisms. In this study we have investigated the role of sialic acid in biofilm growth of this periodontal pathogen. Our data show that biofilm growth of
T. forsythia
is stimulated by sialic acid, glycolyl sialic acid, and sialyllactose, all three of which are common sugar moieties on a range of important host glycoproteins. We have also established that growth on sialyllactose is dependent on the sialidase of
T. forsythia
since the sialidase inhibitor oseltamivir suppresses growth on sialyllactose. The genome of
T. forsythia
contains a sialic acid utilization locus, which also encodes a putative inner membrane sialic acid permease (NanT), and we have shown this is functional when it is expressed in
Escherichia coli
. This genomic locus also contains a putatively novel TonB-dependent outer membrane sialic acid transport system (TF0033-TF0034). In complementation studies using an
Escherichia coli
strain devoid of its outer membrane sialic acid transporters, the cloning and expression of the
TF0033-TF0034
genes enabled an
E. coli nanR nanC ompR
strain to utilize sialic acid as the sole carbon and energy source. We have thus identified a novel sialic acid uptake system that couples an inner membrane permease with a TonB-dependent outer membrane transporter, and we propose to rename these novel sialic acid uptake genes
nanO
and
nanU
, respectively. Taken together, these data indicate that sialic acid is a key growth factor for this little-characterized oral pathogen and may be key to its physiology
in vivo
.
Complete Genome Sequence of
Bifidobacterium animalis
subsp.
lactis
BB-12, a Widely Consumed Probiotic Strain
Authors: Garrigues Christel , Johansen Eric , Pedersen Martin B.
Published: February 2010

Bifidobacterium animalis
subsp.
lactis
BB-12 is a commercially available probiotic strain used throughout the world in a variety of functional foods and dietary supplements. The benefits of BB-12 have been documented in a number of independent clinical trials. Determination of the complete genome sequence reveals a single circular chromosome of 1,942,198 bp with 1,642 predicted protein-encoding genes, 4 rRNA operons, and 52 tRNA genes. Knowledge of this sequence will lead to insight into the specific features which give this strain its probiotic properties.
Purification and Characterization of the Folate Catabolic Enzyme
p
-Aminobenzoyl-Glutamate Hydrolase from
Escherichia coli
Authors: Green Jacalyn M. , Hollandsworth Ryan , Pitstick Lenore , Carter Eric L.
Published: February 2010

The
abg
locus of the
Escherichia coli
chromosome includes three genes encoding proteins (AbgA, AbgB, and AbgT) that enable uptake and utilization of the folate breakdown product,
p
-aminobenzoyl-glutamate (PABA-GLU). We report on the purification and characterization of the
p
-aminobenzoyl-glutamate hydrolase (PGH) holoenzyme encoded by
abgA
and
abgB
. One-step purification was accomplished using a plasmid carrying
abgAB
with a hexahistidine tag on the carboxyl terminus of AbgB and subsequent metal affinity chromatography (MAC). Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed two subunits (∼53-kDa and ∼47-kDa proteins) of the expected masses of AbgB and AbgA; N-terminal sequencing confirmed the subunit identification, and amino acid analysis yielded a 1:1 ratio of the subunits. Size exclusion chromatography coupled with light-scattering analysis of purified PGH revealed a predominant molecular mass of 206 kDa and a minor component of 400 to 500 kDa. Both peaks contained PGH activity, and SDS-PAGE revealed that fractions containing activity were composed of both AbgA and AbgB. MAC-purified PGH was highly stimulated by manganese chloride. Kinetic analysis of MAC-purified PGH revealed a

K
m

value for PABA-GLU of 60 ± 0.08 μM and a specific activity of 63,300 ± 600 nmol min
−1
mg
−1
. Folic acid and a variety of dipeptides served as poor substrates of PGH. This locus of the
E. coli
chromosome may encode a portion of a folate catabolism pathway.
Transcriptome Analysis of
Pseudomonas syringae
Identifies New Genes, Noncoding RNAs, and Antisense Activity
Authors: Filiatrault Melanie J. , Stodghill Paul V. , Bronstein Philip A. , Moll Simon , Lindeberg Magdalen , Grills George , Schweitzer Peter , Wang Wei , Schroth Gary P. , Luo Shujun , Khrebtukova Irina , Yang Yong , Thannhauser Theodore , Butcher Bronwyn G. , Cartinhour Samuel , Schneider David J.
Published: February 2010

To fully understand how bacteria respond to their environment, it is essential to assess genome-wide transcriptional activity. New high-throughput sequencing technologies make it possible to query the transcriptome of an organism in an efficient unbiased manner. We applied a strand-specific method to sequence bacterial transcripts using Illumina's high-throughput sequencing technology. The resulting sequences were used to construct genome-wide transcriptional profiles. Novel bioinformatics analyses were developed and used in combination with proteomics data for the qualitative classification of transcriptional activity in defined regions. As expected, most transcriptional activity was consistent with predictions from the genome annotation. Importantly, we identified and confirmed transcriptional activity in areas of the genome inconsistent with the annotation and in unannotated regions. Further analyses revealed potential RpoN-dependent promoter sequences upstream of several noncoding RNAs (ncRNAs), suggesting a role for these ncRNAs in RpoN-dependent phenotypes. We were also able to validate a number of transcriptional start sites, many of which were consistent with predicted promoter motifs. Overall, our approach provides an efficient way to survey global transcriptional activity in bacteria and enables rapid discovery of specific areas in the genome that merit further investigation.
Characterization of Acp, a Peptidoglycan Hydrolase of
Clostridium perfringens
with
N
-Acetylglucosaminidase Activity That Is Implicated in Cell Separation and Stress-Induced Autolysis
Authors: Camiade Emilie , Peltier Johann , Bourgeois Ingrid , Couture-Tosi Evelyne , Courtin Pascal , Antunes Ana , Chapot-Chartier Marie-Pierre , Dupuy Bruno , Pons Jean-Louis
Published: February 2010

This work reports the characterization of the first known peptidoglycan hydrolase (Acp) produced mainly during vegetative growth of
Clostridium perfringens
. Acp has a modular structure with three domains: a signal peptide domain, an N-terminal domain with repeated sequences, and a C-terminal catalytic domain. The purified recombinant catalytic domain of Acp displayed lytic activity on the cell walls of several Gram-positive bacterial species. Its hydrolytic specificity was established by analyzing the
Bacillus subtilis
peptidoglycan digestion products by coupling reverse phase-high-pressure liquid chromatography (RP-HPLC) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis, which displayed an
N
-acetylglucosaminidase activity. The study of
acp
expression showed a constant expression during growth, which suggested an important role of Acp in growth of
C. perfringens
. Furthermore, cell fractionation and indirect immunofluorescence staining using anti-Acp antibodies revealed that Acp is located at the septal peptidoglycan of vegetative cells during exponential growth phase, indicating a role in cell separation or division of
C. perfringens
. A knockout
acp
mutant strain was obtained by using the insertion of mobile group II intron strategy (ClosTron). The microscopic examination indicated a lack of vegetative cell separation in the
acp
mutant strain, as well as the wild-type strain incubated with anti-Acp antibodies, demonstrating the critical role of Acp in cell separation. The comparative responses of wild-type and
acp
mutant strains to stresses induced by Triton X-100, bile salts, and vancomycin revealed an implication of Acp in autolysis induced by these stresses. Overall, Acp appears as a major cell wall
N
-acetylglucosaminidase implicated in both vegetative growth and stress-induced autolysis.
Utilization of Lactose and Galactose by
Streptococcus mutans
: Transport, Toxicity, and Carbon Catabolite Repression
Authors: Zeng Lin , Das Satarupa , Burne Robert A.
Published: February 2010

Abundant in milk and other dairy products, lactose is considered to have an important role in oral microbial ecology and can contribute to caries development in both adults and young children. To better understand the metabolism of lactose and galactose by
Streptococcus mutans
, the major etiological agent of human tooth decay, a genetic analysis of the tagatose-6-phosphate (
lac
) and Leloir (
gal
) pathways was performed in strain UA159. Deletion of each gene in the
lac
operon caused various alterations in expression of a

P
lacA

-cat
promoter fusion and defects in growth on either lactose (
lacA
,
lacB
,
lacF
,
lacE
, and
lacG
), galactose (
lacA
,
lacB
,
lacD
, and
lacG
) or both sugars (
lacA
,
lacB
, and
lacG
). Failure to grow in the presence of galactose or lactose by certain
lac
mutants appeared to arise from the accumulation of intermediates of galactose metabolism, particularly galatose-6-phosphate. The glucose- and lactose-PTS permeases, EII
Man
and EII
Lac
, respectively, were shown to be the only effective transporters of galactose in
S. mutans
. Furthermore, disruption of
manL
, encoding EIIAB
Man
, led to increased resistance to glucose-mediated CCR when lactose was used to induce the
lac
operon, but resulted in reduced
lac
gene expression in cells growing on galactose. Collectively, the results reveal a remarkably high degree of complexity in the regulation of lactose/galactose catabolism.
Functional Diversity of Four Glycoside Hydrolase Family 3 Enzymes from the Rumen Bacterium
Prevotella bryantii
B
1
4
Authors: Dodd Dylan , Kiyonari Shinichi , Mackie Roderick I. , Cann Isaac K. O.
Published: February 2010

Prevotella bryantii
B
1
4 is a member of the phylum
Bacteroidetes
and contributes to the degradation of hemicellulose in the rumen. The genome of
P. bryantii
harbors four genes predicted to encode glycoside hydrolase (GH) family 3 (GH3) enzymes. To evaluate whether these genes encode enzymes with redundant biological functions, each gene was cloned and expressed in
Escherichia coli
. Biochemical analysis of the recombinant proteins revealed that the enzymes exhibit different substrate specificities. One gene encoded a cellodextrinase (CdxA), and three genes encoded β-xylosidase enzymes (Xyl3A, Xyl3B, and Xyl3C) with different specificities for either
para
-nitrophenyl (
p
NP)-linked substrates or substituted xylooligosaccharides. To identify the amino acid residues that contribute to catalysis and substrate specificity within this family of enzymes, the roles of conserved residues (R177, K214, H215, M251, and D286) in Xyl3B were probed by site-directed mutagenesis. Each mutation led to a severely decreased catalytic efficiency without a change in the overall structure of the mutant enzymes. Through amino acid sequence alignments, an amino acid residue (E115) that, when mutated to aspartic acid, resulted in a 14-fold decrease in the
k
cat
/

K
m

for
p
NP-β-
d
-xylopyranoside (
p
NPX) with a concurrent 1.1-fold increase in the
k
cat
/

K
m

for
p
NP-β-
d
-glucopyranoside (
p
NPG) was identified. Amino acid residue E115 may therefore contribute to the discrimination between β-xylosides and β-glucosides. Our results demonstrate that each of the four GH3 enzymes has evolved to perform a specific role in lignopolysaccharide hydrolysis and provide insight into the role of active-site residues in catalysis and substrate specificity for GH3 enzymes.
Back to the Future: Studying Cholera Pathogenesis Using Infant Rabbits
Authors: Ritchie Jennifer M. , Rui Haopeng , Bronson Roderick T. , Waldor Matthew K.
Published: March 2010

Cholera is a severe diarrheal disease, caused by
Vibrio cholerae
, for which there has been no reproducible, nonsurgical animal model. Here, we report that orogastric inoculation of
V. cholerae
into 3-day-old rabbits pretreated with cimetidine led to lethal, watery diarrhea in virtually all rabbits. The appearance and chemical composition of the rabbit diarrheal fluid were comparable to those of the “rice-water stool” produced by cholera patients. As in humans,
V. cholerae
mutants that do not produce cholera toxin (CT) and toxin-coregulated pilus (TCP) did not induce cholera-like disease in rabbits. CT induced extensive exocytosis of mucin from intestinal goblet cells, and wild-type
V. cholerae
was predominantly found in close association with mucin. Large aggregates of mucin-embedded
V. cholerae
were observed, both attached to the epithelium and floating within the diarrheal fluid. These findings suggest that CT-dependent mucin secretion significantly influences
V. cholerae
’s association with the host intestine and its exit from the intestinal tract. Our model should facilitate identification and analyses of factors that may govern
V. cholerae
infection, survival, and transmission, such as mucin. In addition, our results using nontoxigenic
V. cholerae
suggest that infant rabbits will be useful for study of the reactogenicity of live attenuated-
V. cholerae
vaccines.

IMPORTANCE
Cholera remains a significant threat to populations in developing nations. Currently, there is no reproducible, nonsurgical animal model of cholera, the secretory diarrheal disease caused by
Vibrio cholerae
. We found that oral infection of infant rabbits with
V. cholerae
led to lethal, watery diarrhea in most rabbits. Using this disease model, we discovered a new role for cholera toxin (CT) during infection. This toxin not only caused secretory diarrhea but also profoundly influenced how
V. cholerae
associates with the intestine and how the pathogen exits from the host. Rabbits inoculated with
V. cholerae
that does not produce CT developed mild diarrhea, suggesting that this model may prove useful for generating improved live attenuated-
V. cholerae
vaccine candidates. Overall, our findings suggest that the infant rabbit model will enable pursuit of several new avenues for research on cholera pathogenesis, as well as serve as a vehicle for testing new therapeutics.
FRACTIONAL DIRAC OPERATORS AND LEFT-RIGHT FRACTIONAL CHAMSEDDINE–CONNES SPECTRAL BOSONIC ACTION PRINCIPLE IN NONCOMMUTATIVE GEOMETRY
Authors: EL-NABULSI RAMI AHMAD
Published: March 2010

The generalization of the Chamseddine–Connes spectral triples action to its (left and right) fractional counterpart is constructed within the context of the Riemann–Liouville and Erdelyi–Kober (left and right) fractional operators. In the fractional approach, the Dirac operators [Formula: see text] is approximated by [Formula: see text] and the spectral triple [Formula: see text] is replaced by its fractional equivalent [Formula: see text], [Formula: see text], [Formula: see text], 0 < α < 1. When the (left) fractional action is applied to the noncommutative space defined by the spectrum of the Standard Model, one obtains many attractive characteristics including time-dependent gauge couplings constants ([Formula: see text]), a time-dependent cosmological constant (Λ cos ), a time-dependent scalar Ricci curvature (R), a time-dependent Newton's coupling constant, and a time-dependent Higgs square mass [Formula: see text]. Furthermore, [Formula: see text], Λ cos , R, and [Formula: see text] were found to be nonsingulars at the Planck's time. When the (left and right) fractional bosonic action is taken into account, all the previous functions are found to be complexified, including gravity. Many additional interesting features are discussed and explored in some details.
SOLUTION TO $\overline{\partial}$ PROBLEM WITH EXACT SUPPORT AND REGULARITY FOR THE $\overline{\partial}$-NEUMANN OPERATOR ON WEAKLY q-CONVEX DOMAINS
Authors: SABER SAYED
Published: March 2010

Let Ω be a weakly q-convex domain in ℂn. We establish the L2 existence theorem for the [Formula: see text]-Neumann operator N when the boundary of Ω is C1. Using this result, we study the [Formula: see text] problem with exact support on such domains. Furthermore, there exists a number ℓ0 > 0 such that the operators N, [Formula: see text] and the Bergman projection are regular in the Sobolev space Wℓ(Ω) for ℓ < ℓ0 when the boundary of Ω is C∞.
Re–Os molybdenite dates from the Ballachulish and Kilmelford Igneous Complexes (Scottish Highlands): age constraints for late Caledonian magmatism
Authors: Conliffe J. , Selby D. , Porter S.J. , Feely M.
Published: March 2010

New Re–Os molybdenite geochronology is presented from the Ballachulish Igneous Complex (433.5 ± 1.8 Ma) and the Kilmelford Igneous Complex (425.8 ± 1.7 Ma) of the SW Scottish Highlands. The sulphide mineralization in the Ballachulish Igneous Complex is related to the latest phase of granite magmatism, and therefore the new Re–Os age provides a minimum crystallization age,
c
. 3.7 Ma earlier than previous crystallization age estimates. This Re–Os age overlaps U–Pb ages obtained from subduction-related granitic and appinitic magmatism north of the Great Glen Fault, and an origin related to active subduction rather than slab breakoff is proposed for the Ballachulish Igneous Complex. Molybdenite mineralization in the Kilmelford Igneous Complex is spatially and genetically associated with porphyry Cu mineralization, which is consistent with the rapid ascent of volatile-rich magma during early rebound following the breakoff of subducted oceanic lithosphere. The Kilmelford Igneous Complex and the coeval Lorn Lava Pile may represent the earliest of the igneous bodies predicted by slab breakoff, indicating that slab breakoff occurred at
c
. 426 Ma.
The middle Darriwilian (Ordovician) δ
13
C excursion (MDICE) discovered in the Yangtze Platform succession in China: implications of its first recorded occurrences outside Baltoscandia
Authors: Schmitz Birger , Bergström Stig M. , Xiaofeng Wang
Published: March 2010

The middle Darriwilian δ
13
C excursion (MDICE), one of the least known of the Ordovician δ
13
C excursions, has previously been recorded only from the Middle Ordovician of Baltoscandia. Analysis of many recently collected limestone samples from the Guniutan Formation at two Yangtze Platform localities show elevated δ
13
C values in the same biostratigraphic interval (
Microzarkodina ozarkodella
Conodont Subzone) as the MDICE in Baltoscandia, which justifies identification of the Chinese δ
13
C excursion as the MDICE. These occurrences, which are in strata that show striking lithological and conodont faunal similarity to the Swedish Holen Limestone and some coeval units in Estonia, indicate that the MDICE, the stratigraphically oldest of the named Ordovician δ
13
C excursions, is likely to have a world-wide distribution and to have great potential for local and long-range chemostratigraphic correlations.
Differential AMPK phosphorylation sites associated with phenylephrine vs. antihypertrophic effects of adenosine agonists in neonatal rat ventricular myocytes
Authors: Pang Theresa , Rajapurohitam Venkatesh , Cook Michael A. , Karmazyn Morris
Published: February 2010

Stimulation of cardiac AMP-activated protein kinase (AMPK) has been demonstrated in both prohypertrophic and antihypertrophic settings, although the reasons for such discrepant results are not well understood. We determined how AMPK is regulated in response to phenylephrine-induced cardiomyocyte hypertrophy and assessed whether AMPK activity may be a factor underlying the antihypertrophic effect of adenosine receptor agonists. The role of AMPK in hypertrophic responses was determined by assessing the effect of the AMPK activator 5-aminoimidazole-4-carboxyamide ribonucleoside on three hypertrophic indexes, including protein synthesis, cell surface area, and fetal gene expression. The changes in phosphorylation of the catalytic α-subunit of AMPK at two different sites, Thr172 and Ser485/491, in response to phenylephrine and adenosine receptor agonists were also examined. 5-Aminoimidazole-4-carboxyamide ribonucleoside completely abolished phenylephrine-induced increases in protein synthesis, cell surface area, and fetal gene expression. AMPK phosphorylation time course studies revealed that phenylephrine induced a time-dependent activation at site Ser485/491, in contrast to adenosine receptor agonists, which demonstrated rapid AMPK phosphorylation at Thr172. Furthermore, the phosphorylation at Ser485/491 by phenylephrine was not affected by the addition of adenosine receptor agonists, although, conversely, phosphorylation of AMPK at Thr172 by adenosine receptor agonists was abrogated by the addition of phenylephrine. We propose from these results that cardiomyocyte hypertrophic and antihypertrophic responses, at least with respect to inhibition of phenylephrine-induced hypertrophy by adenosine receptor agonists, are mediated by multisite AMPK regulation. The latter are reflected by increased phosphorylation at Ser485/491 and at Thr172, associated with prohypertrophic and antihypertrophic responses, respectively.
Heterogeneous ventricular sympathetic innervation, altered β-adrenergic receptor expression, and rhythm instability in mice lacking the p75 neurotrophin receptor
Authors: Lorentz Christina U. , Alston Eric N. , Belcik Todd , Lindner Jonathan R. , Giraud George D. , Habecker Beth A.
Published: February 2010

Sympathetic nerves stimulate cardiac function through the release of norepinephrine and the activation of cardiac β1-adrenergic receptors. The sympathetic innervation of the heart is sculpted during development by chemoattractive factors including nerve growth factor (NGF) and the chemorepulsive factor semaphorin 3a. NGF acts through the TrkA receptor and the p75 neurotrophin receptor (p75NTR) in sympathetic neurons. NGF stimulates sympathetic axon extension into the heart through TrkA, but p75NTRmodulates multiple coreceptors that can either stimulate or inhibit axon outgrowth. In mice lacking p75NTR, the sympathetic innervation density in target tissues ranges from denervation to hyperinnervation. Recent studies have revealed significant changes in the sympathetic innervation density of p75NTR-deficient (p75NTR−/−) atria between early postnatal development and adulthood. We examined the innervation of adult p75NTR−/−ventricles and discovered that the subendocardium of the p75NTR−/−left ventricle was essentially devoid of sympathetic nerve fibers, whereas the innervation density of the subepicardium was normal. This phenotype is similar to that seen in mice overexpressing semaphorin 3a, and we found that sympathetic axons lacking p75NTRare more sensitive to semaphorin 3a in vitro than control neurons. The lack of subendocardial innervation was associated with decreased dP/d t, altered cardiac β1-adrenergic receptor expression and sensitivity, and a significant increase in spontaneous ventricular arrhythmias. The lack of p75NTRalso resulted in increased tyrosine hydroxylase content in cardiac sympathetic neurons and elevated norepinephrine in the right ventricle, where innervation density was normal.
Increased late sodium current in left atrial myocytes of rabbits with left ventricular hypertrophy: its role in the genesis of atrial arrhythmias
Authors: Guo Donglin , Young Lindon , Wu Ying , Belardinelli Luiz , Kowey Peter R. , Yan Gan-Xin
Published: February 2010

Left ventricular hypertrophy (LVH) is frequently associated with clinical atrial arrhythmias, but little is known about how it causes those arrhythmias. Our previous studies have shown that LVH increases the late sodium current ( INa-L) that plays an important role in the genesis of ventricular arrhythmias. We hypothesize that LVH may also induce an upregulation of the INa-L in atrial myocytes, leading to atrial electrical abnormalities. The renovascular hypertension model was used to induce LVH in rabbits. Action potential and membrane current recordings were performed in single myocytes. At a pacing cycle length of 2,000 ms, spontaneous phase-2 early afterdepolarizations (EADs) could be recorded from the left atrial myocytes in 10 of 12 LVH rabbits, whereas no EADs could be elicited in right atrial myocytes of LVH rabbits or atrial myocytes from any of the 12 control rabbits. Spontaneous automaticity (SA) from left atrial myocytes was observed in 9 out of 12 LVH rabbits, but none in right atrial myocytes of LVH rabbits or control rabbits, at a pacing rate of 8,000 ms. The left atrial myocytes of LVH rabbits had a significantly higher density of the INa-L compared with those of control rabbits (0.90 ± 0.12 in LVH vs. 0.50 ± 0.08 pA/pF in control, n = 8, P < 0.01). Tetrodotoxin, an INa-L blocker, abolished all atrial EADs and SA at 10 μM. Our results demonstrate that LVH induction results in a significant increase of INa-L in the left atrial myocytes that may render these cells susceptible to the genesis of EADs and SA. The INa-L may serve as a potentially useful ionic target for antiarrhythmic drugs for the treatment of atrial arrhythmias in the setting of LVH.
Cystic fibrosis remodels the regulation of purinergic signaling by NTPDase1 (CD39) and NTPDase3
Authors: Fausther Michel , Pelletier Julie , Ribeiro Carla M. , Sévigny Jean , Picher Maryse
Published: February 2010

Airway defenses are regulated by a complex purinergic signaling network located on the epithelial surfaces, where ATP stimulates the clearance of mucin and pathogens. The present study shows that the obstructive disease cystic fibrosis (CF) affects the activity, expression, and tissue distribution of two ectonucleotidases found critical for the regulation of ATP on airway surfaces: NTPDase1 and NTPDase3. Functional polarities and mRNA expression levels were determined on primary cultures of human bronchial epithelial (HBE) cells from healthy donors and CF patients. The in vitro model of the disease was completed by exposing CF HBE cultures for 4 days to supernatant of the mucopurulent material (SMM) collected from the airways of CF patients. We report that NTPDase1 and NTPDase3 are coexpressed on HBE cultures, where they regulate physiological and excess nucleotide concentrations, respectively. In aseptic conditions, CF epithelia exhibit >50% lower NTPDase1 activity, protein, and mRNA levels than normal epithelia, whereas these parameters are threefold higher for NTPDase3. Exposure to SMM induced opposite polarity shifts of the two NTPDases on both normal and CF epithelia, apical NTPDase1 being mobilized to basolateral surfaces and bilateral NTPDase3 to the apical surface. Their immunolocalization in human tissue revealed that NTPDase1 is expressed in epithelial, inflammatory, and endothelial cells, whereas NTPDase3 is restricted to epithelial cells. Furthermore, the SMM-exposed CF HBE cultures reproduced the impact of the disease on their in vivo distribution. This study provides evidence that an extensive remodeling of the enzymatic network regulating clearance occurs in the airways of CF patients.
Pulmonary artery smooth muscle hypertrophy: roles of glycogen synthase kinase-3β and p70 ribosomal S6 kinase
Authors: Deng Huan , Hershenson Marc B. , Lei Jing , Anyanwu Anuli C. , Pinsky David J. , Bentley J. Kelley
Published: February 2010

Increased medial arterial thickness is a structural change in pulmonary arterial hypertension (PAH). The role of smooth muscle hypertrophy in this process has not been well studied. Bone morphogenetic proteins (BMPs), transforming growth factor (TGF)-β1, serotonin (or 5-hydroxytryptamine; 5-HT), and endothelin (ET)-1 have been implicated in PAH pathogenesis. We examined the effect of these mediators on human pulmonary artery smooth muscle cell size, contractile protein expression, and contractile function, as well on the roles of glycogen synthase kinase (GSK)-3β and p70 ribosomal S6 kinase (p70S6K), two proteins involved in translational control, in this process. Unlike epidermal growth factor, BMP-4, TGF-β1, 5-HT, and ET-1 each increased smooth muscle cell size, contractile protein expression, fractional cell shortening, and GSK-3β phosphorylation. GSK-3β inhibition by lithium or SB-216763 increased cell size, protein synthesis, and contractile protein expression. Expression of a non-phosphorylatable GSK-3β mutant blocked BMP-4-, TGF-β1-, 5-HT-, and ET-1-induced cell size enlargement, suggesting that GSK-3β phosphorylation is required and sufficient for cellular hypertrophy. However, BMP-4, TGF-β1, 5-HT, and ET-1 stimulation was accompanied by an increase in serum response factor transcriptional activation but not eIF2 phosphorylation, suggesting that GSK-3β-mediated hypertrophy occurs via transcriptional, not translational, control. Finally, BMP-4, TGF-β1, 5-HT, and ET-1 treatment induced phosphorylation of p70S6K and ribosomal protein S6, and siRNAs against p70S6K and S6 blocked the hypertrophic response. We conclude that mediators implicated in the pathogenesis of PAH induce pulmonary arterial smooth muscle hypertrophy. Identification of the signaling pathways regulating vascular smooth muscle hypertrophy may define new therapeutic targets for PAH.