bluuebunny/crossref_metadata_2025 huggingface dataset:
Transcription Factors in Light and Circadian Clock Signaling Networks Revealed by Genomewide Mapping of Direct Targets for Neurospora White Collar Complex
Authors: Smith Kristina M. , Sancar Gencer , Dekhang Rigzin , Sullivan Christopher M. , Li Shaojie , Tag Andrew G. , Sancar Cigdem , Bredeweg Erin L. , Priest Henry D. , McCormick Ryan F. , Thomas Terry L. , Carrington James C. , Stajich Jason E. , Bell-Pedersen Deborah , Brunner Michael , Freitag Michael
Published: July 2010

Light signaling pathways and circadian clocks are inextricably linked and have profound effects on behavior in most organisms. Here, we used chromatin immunoprecipitation (ChIP) sequencing to uncover direct targets of the
Neurospora crassa
circadian regulator White Collar Complex (WCC). The WCC is a blue-light receptor and the key transcription factor of the circadian oscillator. It controls a transcriptional network that regulates ∼20% of all genes, generating daily rhythms and responses to light. We found that in response to light, WCC binds to hundreds of genomic regions, including the promoters of previously identified clock- and light-regulated genes. We show that WCC directly controls the expression of 24 transcription factor genes, including the clock-controlled
adv-1
gene, which controls a circadian output pathway required for daily rhythms in development. Our findings provide links between the key circadian activator and effectors in downstream regulatory pathways.
Identification of
GIG1
, a GlcNAc-Induced Gene in Candida albicans Needed for Normal Sensitivity to the Chitin Synthase Inhibitor Nikkomycin Z
Authors: Gunasekera Angelo , Alvarez Francisco J. , Douglas Lois M. , Wang Hong X. , Rosebrock Adam P. , Konopka James B.
Published: July 2010

The amino sugar
N
-acetylglucosamine (GlcNAc) is known to be an important structural component of cells from bacteria to humans, but its roles in cell signaling are less well understood. GlcNAc induces two pathways in the human fungal pathogen
Candida albicans
. One activates cyclic AMP (cAMP) signaling, which stimulates the formation of hyphal cells and the expression of virulence genes, and the other pathway induces genes needed to catabolize GlcNAc. Microarray analysis of gene expression was carried out under four different conditions in order to characterize the transcriptional changes induced by GlcNAc. The most highly induced genes include those that encode a GlcNAc transporter (
NGT1
) and the GlcNAc catabolic enzymes (
HXK1
,
DAC1
, and
NAG1
). GlcNAc also activated most of the genes whose expression is increased when cells are triggered with other stimuli to form hyphae. Surprisingly, GlcNAc also induced a subset of genes that are regulated by galactose (
GAL1
,
GAL7
, and
GAL10
), which may be due to cross talk between signaling pathways. A novel GlcNAc-induced gene,
GIG1
, which is not essential for GlcNAc catabolism or the induction of hyphae, was identified. However, a Gig1-green fluorescent protein (GFP) fusion protein was specifically induced by GlcNAc, and not by other sugars. Gig1-GFP localized to the cytoplasm, where GlcNAc metabolism occurs. Significantly, a
gig1
Δ mutant displayed increased resistance to nikkomycin Z, which inhibits chitin synthase from converting UDP-GlcNAc into cell wall chitin. Gig1 is highly conserved in fungi, especially those that contain GlcNAc catabolic genes. These results implicate Gig1 in GlcNAc metabolism.
The Twin Arginine Transport System Appears To Be Essential for Viability in
Sinorhizobium meliloti
Authors: Pickering Brad S. , Oresnik Ivan J.
Published: July 2010

The twin arginine transport (Tat) system is responsible for transporting prefolded proteins to the periplasmic space. The Tat pathway has been implicated in many bacterial cellular functions, including motility, biofilm formation, and pathogenesis and symbiosis. Since the annotation of
Sinorhizobium meliloti
Rm1021 genome suggests that there may be up to 94 putative Tat substrates, we hypothesized that characterizing the twin arginine transport system in this organism might yield unique data that could help in the understanding of twin arginine transport. To initiate this work we attempted a targeted mutagenesis of the
tat
locus. Despite repeated attempts using a number of different types of media, the attempts at mutation construction were unsuccessful unless the experiment was carried out in a strain that was merodiploid for
tatABC
. In addition, it was shown that a plasmid carrying
tatABC
was stable in the absence of antibiotic selection in a
tat
deletion background. Finally, fluorescence microscopy and live/dead assays of these cultures show a high proportion of dead and irregularly shaped cells, suggesting that the loss of
tatABC
is inversely correlated with viability. Taken together, the results of this work provide evidence that the twin arginine transport system of
S. meliloti
appears to be essential for viability under all the conditions that we had tested.
Characterization of NADH Oxidase/NADPH Polysulfide Oxidoreductase and Its Unexpected Participation in Oxygen Sensitivity in an Anaerobic Hyperthermophilic Archaeon
Authors: Kobori Hiroki , Ogino Masayuki , Orita Izumi , Nakamura Satoshi , Imanaka Tadayuki , Fukui Toshiaki
Published: July 2010

Many genomes of anaerobic hyperthermophiles encode multiple homologs of NAD(P)H oxidase that are thought to function in response to oxidative stress. We investigated one of the seven NAD(P)H oxidase homologs (TK1481) in the sulfur-reducing hyperthermophilic archaeon
Thermococcus kodakarensis
, focusing on the catalytic properties and roles in oxidative-stress defense and sulfur-dependent energy conservation. The recombinant form of TK1481 exhibited both NAD(P)H oxidase and NAD(P)H:polysulfide oxidoreductase activities. The enzyme also possessed low NAD(P)H peroxidase and NAD(P)H:elemental sulfur oxidoreductase activities under anaerobic conditions. A mutant form of the enzyme, in which the putative redox-active residue Cys43 was replaced by Ala, still showed NADH-dependent flavin adenine dinucleotide (FAD) reduction activity. Although it also retained successive oxidase and anaerobic peroxidase activities, the ability to reduce polysulfide and sulfur was completely lost, suggesting the specific reactivity of the Cys43 residue for sulfur. To evaluate the physiological function of TK1481, we constructed a gene deletant, ΔTK1481, and mutant KUTK1481C43A, into which two base mutations altering Cys43 of TK1481 to Ala were introduced. ΔTK1481 exhibited growth properties nearly identical to those of the parent strain, KU216, in sulfur-containing media. Interestingly, in the absence of elemental sulfur, the growth of ΔTK1481 was not affected by dissolved oxygen, whereas the growth of KU216 and KUTK1481C43A was significantly impaired. These results indicate that although TK1481 does not play a critical role in either sulfur reduction or the response to oxidative stress, the NAD(P)H oxidase activity of TK1481 unexpectedly participates in the oxygen sensitivity of the hyperthermophilic archaeon
T. kodakarensis
in the absence of sulfur.
Pyruvate Is Synthesized by Two Pathways in Pea Bacteroids with Different Efficiencies for Nitrogen Fixation
Authors: Mulley Geraldine , Lopez-Gomez Miguel , Zhang Ye , Terpolilli Jason , Prell Jurgen , Finan Turlough , Poole Philip
Published: July 2010

Nitrogen fixation in legume bacteroids is energized by the metabolism of dicarboxylic acids, which requires their oxidation to both oxaloacetate and pyruvate. In alfalfa bacteroids, production of pyruvate requires NAD
+
malic enzyme (Dme) but not NADP
+
malic enzyme (Tme). However, we show that
Rhizobium leguminosarum
has two pathways for pyruvate formation from dicarboxylates catalyzed by Dme and by the combined activities of phosphoenolpyruvate (PEP) carboxykinase (PckA) and pyruvate kinase (PykA). Both pathways enable N
2
fixation, but the PckA/PykA pathway supports N
2
fixation at only 60% of that for Dme. Double mutants of
dme
and
pckA/pykA
did not fix N
2
. Furthermore,
dme pykA
double mutants did not grow on dicarboxylates, showing that they are the only pathways for the production of pyruvate from dicarboxylates normally expressed. PckA is not expressed in alfalfa bacteroids, resulting in an obligate requirement for Dme for pyruvate formation and N
2
fixation. When PckA was expressed from a constitutive
nptII
promoter in alfalfa
dme
bacteroids, acetylene was reduced at 30% of the wild-type rate, although this level was insufficient to prevent nitrogen starvation. Dme has N-terminal, malic enzyme (Me), and C-terminal phosphotransacetylase (Pta) domains. Deleting the Pta domain increased the peak acetylene reduction rate in 4-week-old pea plants to 140 to 150% of the wild-type rate, and this was accompanied by increased nodule mass. Plants infected with Pta deletion mutants did not have increased dry weight, demonstrating that there is not a sustained change in nitrogen fixation throughout growth. This indicates a complex relationship between pyruvate synthesis in bacteroids, nitrogen fixation, and plant growth.
Unexpected Abundance of Coenzyme F
420
-Dependent Enzymes in
Mycobacterium tuberculosis
and Other Actinobacteria
Authors: Selengut Jeremy D. , Haft Daniel H.
Published: July 2010

Regimens targeting
Mycobacterium tuberculosis
, the causative agent of tuberculosis (TB), require long courses of treatment and a combination of three or more drugs. An increase in drug-resistant strains of
M. tuberculosis
demonstrates the need for additional TB-specific drugs. A notable feature of
M. tuberculosis
is coenzyme F
420
, which is distributed sporadically and sparsely among prokaryotes. This distribution allows for comparative genomics-based investigations. Phylogenetic profiling (comparison of differential gene content) based on F
420
biosynthesis nominated many actinobacterial proteins as candidate F
420
-dependent enzymes. Three such families dominated the results: the luciferase-like monooxygenase (LLM), pyridoxamine 5′-phosphate oxidase (PPOX), and deazaflavin-dependent nitroreductase (DDN) families. The DDN family was determined to be limited to F
420
-producing species. The LLM and PPOX families were observed in F
420
-producing species as well as species lacking F
420
but were particularly numerous in many actinobacterial species, including
M. tuberculosis
. Partitioning the LLM and PPOX families based on an organism's ability to make F
420
allowed the application of the SIMBAL (sites inferred by metabolic background assertion labeling) profiling method to identify F
420
-correlated subsequences. These regions were found to correspond to flavonoid cofactor binding sites. Significantly, these results showed that
M. tuberculosis
carries at least 28 separate F
420
-dependent enzymes, most of unknown function, and a paucity of flavin mononucleotide (FMN)-dependent proteins in these families. While prevalent in mycobacteria, markers of F
420
biosynthesis appeared to be absent from the normal human gut flora. These findings suggest that
M. tuberculosis
relies heavily on coenzyme F
420
for its redox reactions. This dependence and the cofactor's rarity may make F
420
-related proteins promising drug targets.
Transcriptional Profiling of XdrA, a New Regulator ofspaTranscription inStaphylococcus aureus
Authors: McCallum N. , Hinds J. , Ender M. , Berger-Bächi B. , Stutzmann Meier P.
Published: July 2010

Transcription ofspa, encoding the virulence factor protein A inStaphylococcus aureus, is tightly controlled by a complex regulatory network, ensuring its temporal expression over growth and at appropriate stages of the infection process. Transcriptomic profiling of XdrA, a DNA-binding protein that is conserved in allS. aureusgenomes and shares similarity with the XRE family of helix-turn-helix, antitoxin-like proteins, revealed it to be a previously unidentified activator ofspatranscription. To assess how XdrA fits into the complex web ofsparegulation, a series of regulatory mutants were constructed; consisting of single, double, triple, and quadruple mutants lacking XdrA and/or the three key regulators previously shown to influencespatranscription directly (SarS, SarA, and RNAIII). A series oflacZreporter gene fusions containing nested deletions of thespapromoter identified regions influenced by XdrA and the other three regulators. XdrA had almost as strong an activating effect onspaas SarS and acted on the samespaoperator regions as SarS, or closely overlapping regions. All data from microarrays, Northern and Western blot analyses, and reporter gene fusion experiments indicated that XdrA is a major activator ofspaexpression that appears to act directly on thespapromoter and not through previously characterized regulators.
Staphylococcus aureusNrdH Redoxin Is a Reductant of the Class Ib Ribonucleotide Reductase
Authors: Rabinovitch Inbal , Yanku Michaela , Yeheskel Adva , Cohen Gerald , Borovok Ilya , Aharonowitz Yair
Published: July 2010

Staphylococci contain a class Ib NrdEF ribonucleotide reductase (RNR) that is responsible, under aerobic conditions, for the synthesis of deoxyribonucleotide precursors for DNA synthesis and repair. The genes encoding that RNR are contained in an operon consisting of three genes,nrdIEF, whereas many other class Ib RNR operons contain a fourth gene,nrdH, that determines a thiol redoxin protein, NrdH. We identified a 77-amino-acid open reading frame inStaphylococcus aureusthat resembles NrdH proteins. However,S. aureusNrdH differs significantly from the canonical NrdH both in its redox-active site, C-P-P-C instead of C-M/V-Q-C, and in the absence of the C-terminal [WF]SGFRP[DE] structural motif. We show thatS. aureusNrdH is a thiol redox protein. It is not essential for aerobic or anaerobic growth and appears to have a marginal role in protection against oxidative stress.In vitro,S. aureusNrdH was found to be an efficient reductant of disulfide bonds in low-molecular-weight substrates and proteins using dithiothreitol as the source of reducing power and an effective reductant for the homologous class Ib RNR employing thioredoxin reductase and NADPH as the source of the reducing power. Its ability to reduce NrdEF is comparable to that of thioredoxin-thioredoxin reductase. Hence,S. aureuscontains two alternative thiol redox proteins, NrdH and thioredoxin, with both proteins being able to functionin vitrowith thioredoxin reductase as the immediate hydrogen donors for the class Ib RNR. It remains to be clarified under whichin vivophysiological conditions the two systems are used.
The E2 Domain of OdhA of
Corynebacterium glutamicum
Has Succinyltransferase Activity Dependent on Lipoyl Residues of the Acetyltransferase AceF
Authors: Hoffelder Melanie , Raasch Katharina , van Ooyen Jan , Eggeling Lothar
Published: July 2010

Oxoglutarate dehydrogenase (ODH) and pyruvate dehydrogenase (PDH) complexes catalyze key reactions in central metabolism, and in
Corynebacterium glutamicum
there is indication of an unusual supercomplex consisting of AceE (E1), AceF (E2), and Lpd (E3) together with OdhA. OdhA is a fusion protein of additional E1 and E2 domains, and
odhA
orthologs are present in all
Corynebacterineae
, including, for instance,
Mycobacterium tuberculosis
. Here we show that deletion of any of the individual domains of OdhA in
C. glutamicum
resulted in loss of ODH activity, whereas PDH was still functional. On the other hand, deletion of AceF disabled both PDH activity and ODH activity as well, although isolated AceF protein had solely transacetylase activity and no transsuccinylase activity. Surprisingly, the isolated OdhA protein was inactive with 2-oxoglutarate as the substrate, but it gained transsuccinylase activity upon addition of dihydrolipoamide. Further enzymatic analysis of mutant proteins and mutant cells revealed that OdhA specifically catalyzes the E1 and E2 reaction to convert 2-oxoglutarate to succinyl-coenzyme A (CoA) but fully relies on the lipoyl residues provided by AceF involved in the reactions to convert pyruvate to acetyl-CoA. It therefore appears that in the putative supercomplex in
C. glutamicum
, in addition to dihydrolipoyl dehydrogenase E3, lipoyl domains are also shared, thus confirming the unique evolutionary position of bacteria such as
C. glutamicum
and
M. tuberculosis
.
Catabolic Function of Compartmentalized Alanine Dehydrogenase in the Heterocyst-Forming Cyanobacterium
Anabaena
sp. Strain PCC 7120
Authors: Pernil Rafael , Herrero Antonia , Flores Enrique
Published: July 2010

In the diazotrophic filaments of heterocyst-forming cyanobacteria, an exchange of metabolites takes place between vegetative cells and heterocysts that results in a net transfer of reduced carbon to the heterocysts and of fixed nitrogen to the vegetative cells. Open reading frame
alr2355
of the genome of
Anabaena
sp. strain PCC 7120 is the
ald
gene encoding alanine dehydrogenase. A strain carrying a green fluorescent protein (GFP) fusion to the N terminus of Ald (Ald-N-GFP) showed that the
ald
gene is expressed in differentiating and mature heterocysts. Inactivation of
ald
resulted in a lack of alanine dehydrogenase activity, a substantially decreased nitrogenase activity, and a 50% reduction in the rate of diazotrophic growth. Whereas production of alanine was not affected in the
ald
mutant,
in vivo
labeling with [
14
C]alanine (in whole filaments and isolated heterocysts) or [
14
C]pyruvate (in whole filaments) showed that alanine catabolism was hampered. Thus, alanine catabolism in the heterocysts is needed for normal diazotrophic growth. Our results extend the significance of a previous work that suggested that alanine is transported from vegetative cells into heterocysts in the diazotrophic
Anabaena
filament.
Inclusion Membrane Proteins ofProtochlamydia amoebophilaUWE25 Reveal a Conserved Mechanism for Host Cell Interaction among theChlamydiae
Authors: Heinz Eva , Rockey Daniel D. , Montanaro Jacqueline , Aistleitner Karin , Wagner Michael , Horn Matthias
Published: July 2010

Chlamydiaeare a group of obligate intracellular bacteria comprising several important human pathogens. Inside the eukaryotic cell, chlamydiae remain within a host-derived vesicular compartment, termed the inclusion. They modify the inclusion membrane through insertion of unique proteins, which are involved in interaction with and manipulation of the host cell. Among chlamydiae, inclusion membrane proteins have been exclusively found in members of the familyChlamydiaceae, which predominantly infect mammalian and avian hosts. Here, the presence of inclusion membrane proteins inProtochlamydia amoebophilaUWE25, a chlamydial endosymbiont of free-living amoebae, is reported. A genome-wide screening for secondary structure motifs resulted in the identification of 23 putative inclusion membrane proteins for this organism. Immunofluorescence analysis demonstrated that five of these proteins were expressed, and four of them could be localized to a halo surrounding the intracellular bacteria. Colocalization studies showed an almost complete overlap of the signals obtained for the four putative inclusion membrane proteins, and immuno-transmission electron microscopy unambiguously demonstrated their location in the inclusion membrane. The presence of inclusion membrane proteins (designated IncA, IncQ, IncR, and IncS) inP. amoebophilashows that this strategy for host cell interaction is conserved among the chlamydiae and is used by chlamydial symbionts and pathogens alike.
ICEEc2, a New Integrative and Conjugative Element Belonging to the pKLC102/PAGI-2 Family, Identified inEscherichia coliStrain BEN374
Authors: Roche David , Fléchard Maud , Lallier Nathalie , Répérant Maryline , Brée Annie , Pascal Géraldine , Schouler Catherine , Germon Pierre
Published: July 2010

The diversity of theEscherichia colispecies is in part due to the large number of mobile genetic elements that are exchanged between strains. We report here the identification of a new integrative and conjugative element (ICE) of the pKLC102/PAGI-2 family located downstream of the tRNA genepheUin theE. colistrain BEN374. Indeed, this new region, which we called ICEEc2, can be transferred by conjugation from strain BEN374 to theE. colistrain C600. We were also able to transfer this region into aSalmonella entericaserovar Typhimurium strain and into aYersinia pseudotuberculosisstrain. This transfer was then followed by the integration of ICEEc2into the host chromosome downstream of aphetRNA gene. Our data indicated that this transfer involved a set of three genes encoding DNA mobility enzymes and a type IV pilus encoded by genes present on ICEEc2. Given the wide distribution of members of this family, these mobile genetic elements are likely to play an important role in the diversification of bacteria.
Inhibiting the Initiation ofClostridium difficileSpore Germination using Analogs of Chenodeoxycholic Acid, a Bile Acid
Authors: Sorg Joseph A. , Sonenshein Abraham L.
Published: July 2010

To cause disease,Clostridium difficilespores must germinate in the host gastrointestinal tract. Germination is initiated upon exposure to glycine and certain bile acids, e.g., taurocholate. Chenodeoxycholate, another bile acid, inhibits taurocholate-mediated germination. By applying Michaelis-Menten kinetic analysis toC. difficilespore germination, we found that chenodeoxycholate is a competitive inhibitor of taurocholate-mediated germination and appears to interact with the spores with greater apparent affinity than does taurocholate. We also report that several analogs of chenodeoxycholate are even more effective inhibitors. Some of these compounds resist 7α-dehydroxylation byClostridium scindens, a core member of the normal human colonic microbiota, suggesting that they are more stable than chenodeoxycholate in the colonic environment.
A Group IIC-Type Intron Interrupts the rRNA Methylase Gene of
Geobacillus stearothermophilus
Strain 10
Authors: Moretz Samuel E. , Lampson Bert C.
Published: July 2010

Group IIC introns insert next to the stem-loop structure of rho-independent transcription terminators, thus avoiding intact genes. The insertion sites of 17 copies of the G.st.I1 intron from
Geobacillus stearothermophilus
were compared. One copy of the intron was found to interrupt an open reading frame (ORF) encoding an rRNA methylase.
Mycobacterial Biofilms Facilitate Horizontal DNA Transfer between Strains ofMycobacterium smegmatis
Authors: Nguyen Kiet T. , Piastro Kristina , Gray Todd A. , Derbyshire Keith M.
Published: July 2010

Conjugal transfer of chromosomal DNA between strains ofMycobacterium smegmatisoccurs by a novel mechanism. In a transposon mutagenesis screen, three transfer-defective insertions were mapped to thelsr2gene of the donor strain mc2155. Becauselsr2encodes a nonspecific DNA-binding protein, mutations oflsr2give rise to a variety of phenotypes, including an inability to form biofilms. In this study, we show that efficient DNA transfer between strains ofM. smegmatisoccurs in a mixed biofilm and that the process requires expression oflsr2in the donor but not in the recipient strain. Testing cells from different strata of standing cultures showed that transfer occurred predominantly at the biofilm air-liquid interface, as other strata containing higher cell densities produced very few transconjugants. These data suggest that the biofilm plays a role beyond mere facilitation of cell-cell contact. Surprisingly, we found that under standard assay conditions the recipient strain does not form a biofilm. Taking these results together, we conclude that for transfer to occur, the recipient strain is actively recruited into the biofilm. In support of this idea, we show that donor and recipient cells are present in almost equal numbers in biofilms that produce transconjugants. Our demonstration of genetic exchange between mycobacteria in a mixed biofilm suggests that conjugation occurs in the environment. Since biofilms are considered to be the predominant natural microhabitat for bacteria, our finding emphasizes the importance of studying biological and physical processes that occur between cells in mixed biofilms.
The Delta Subunit of RNA Polymerase, RpoE, Is a Global Modulator ofStreptococcus mutansEnvironmental Adaptation
Authors: Xue Xiaoli , Tomasch Jürgen , Sztajer Helena , Wagner-Döbler Irene
Published: July 2010

The delta subunit of RNA polymerase, RpoE, is widespread in low-G+C Gram-positive bacteria and is thought to play a role in enhancing transcriptional specificity by blocking RNA polymerase binding at weak promoter sites and stimulating RNA synthesis by accelerating core enzyme recycling. Despite the well-studied biochemical properties of RpoE, a role for this proteinin vivohas not been defined in depth. In this study, we show that inactivation ofrpoEin the human dental caries pathogenStreptococcus mutanscauses impaired growth and loss of important virulence traits, including biofilm formation, resistance to antibiotics, and tolerance to environmental stresses. Complementation of the mutant withrpoEexpressed intransrestored its phenotype to wild type. The luciferase fusion reporter showed thatrpoEwas highly transcribed throughout growth and that acid and hydrogen peroxide stresses repressedrpoEexpression. Transcriptome profiling of wild-type and ΔrpoEcells in the exponential and early stationary phase of growth, under acid and hydrogen peroxide stress and under both stresses combined, revealed that genes involved in histidine synthesis, malolactic fermentation, biofilm formation, and antibiotic resistance were downregulated in the ΔrpoEmutant under all conditions. Moreover, the loss of RpoE resulted in dramatic changes in transport and metabolism of carbohydrates and amino acids. Interestingly, differential expression, mostly upregulation, of 330 noncoding regions was found. In conclusion, this study demonstrates that RpoE is an important global modulator of gene expression inS. mutanswhich is required for optimal growth and environmental adaptation.
Repair of DNA Double-Strand Breaks following UV Damage in ThreeSulfolobussolfataricusStrains
Authors: Rolfsmeier Michael L. , Laughery Marian F. , Haseltine Cynthia A.
Published: July 2010

DNA damage repair mechanisms have been most thoroughly explored in the eubacterial and eukaryotic branches of life. The methods by which members of the archaeal branch repair DNA are significantly less well understood but have been gaining increasing attention. In particular, the approaches employed by hyperthermophilic archaea have been a general source of interest, since these organisms thrive under conditions that likely lead to constant chromosomal damage. In this work we have characterized the responses of threeSulfolobus solfataricusstrains to UV-C irradiation, which often results in double-strand break formation. We examinedS. solfataricusstrain P2 obtained from two different sources andS. solfataricusstrain 98/2, a popular strain for site-directed mutation by homologous recombination. Cellular recovery, as determined by survival curves and the ability to return to growth after irradiation, was found to be strain specific and differed depending on the dose applied. Chromosomal damage was directly visualized using pulsed-field gel electrophoresis and demonstrated repair rate variations among the strains following UV-C irradiation-induced double-strand breaks. Several genes involved in double-strand break repair were found to be significantly upregulated after UV-C irradiation. Transcript abundance levels and temporal expression patterns for double-strand break repair genes were also distinct for each strain, indicating that theseSulfolobus solfataricusstrains have differential responses to UV-C-induced DNA double-strand break damage.
Regulation of a Novel Gene Cluster Involved in Secondary Metabolite Production in
Streptomyces coelicolor
Authors: Hindra  , Pak Patricia , Elliot Marie A.
Published: July 2010

Antibiotic biosynthesis in the streptomycetes is a complex and highly regulated process. Here, we provide evidence for the contribution of a novel genetic locus to antibiotic production in
Streptomyces coelicolor
. The overexpression of a gene cluster comprising four protein-encoding genes (
abeABCD
) and an antisense RNA-encoding gene (α-
abeA
) stimulated the production of the blue-pigmented metabolite actinorhodin on solid medium. Actinorhodin production also was enhanced by the overexpression of an adjacent gene (
abeR
) encoding a predicted
Streptomyces
antibiotic regulatory protein (SARP), while the deletion of this gene impaired actinorhodin production. We found the
abe
genes to be differentially regulated and controlled at multiple levels. Upstream of
abeA
was a promoter that directed the transcription of
abeABCD
at a low but constitutive level. The expression of
abeBCD
was, however, significantly upregulated at a time that coincided with the initiation of aerial development and the onset of secondary metabolism; this expression was activated by the binding of AbeR to four heptameric repeats upstream of a promoter within
abeA
. Expressed divergently to the
abeBCD
promoter was α-
abeA
, whose expression mirrored that of
abeBCD
but did not require activation by AbeR. Instead, α-
abeA
transcript levels were subject to negative control by the double-strand-specific RNase, RNase III.
Complete Genome Sequence of
Lactobacillus salivarius
CECT 5713, a Probiotic Strain Isolated from Human Milk and Infant Feces
Authors: Jiménez Esther , Martín Rocío , Maldonado Antonio , Martín Virginia , Gómez de Segura Aranzazu , Fernández Leonides , Rodríguez Juan M.
Published: July 2010

Lactobacillus salivarius
is a homofermentative lactic acid bacterium and is frequently isolated from mucosal surfaces of healthy humans.
L. salivarius
CECT 5713, a strain isolated simultaneously from breast milk and infant feces of a healthy mother-infant pair, has immunomodulatory, anti-inflammatory, and anti-infectious properties, as revealed by several
in vitro
and
in vivo
assays. Here, we report its complete and annotated genome sequence.
StyA1 and StyA2B from
Rhodococcus opacus
1CP: a Multifunctional Styrene Monooxygenase System
Authors: Tischler Dirk , Kermer René , Gröning Janosch A. D. , Kaschabek Stefan R. , van Berkel Willem J. H. , Schlömann Michael
Published: July 2010

Two-component flavoprotein monooxygenases are emerging biocatalysts that generally consist of a monooxygenase and a reductase component. Here we show that
Rhodococcus opacus
1CP encodes a multifunctional enantioselective flavoprotein monooxygenase system composed of a single styrene monooxygenase (SMO) (StyA1) and another styrene monooxygenase fused to an NADH-flavin oxidoreductase (StyA2B). StyA1 and StyA2B convert styrene and chemical analogues to the corresponding epoxides at the expense of FADH
2
provided from StyA2B. The StyA1/StyA2B system presents the highest monooxygenase activity in an equimolar ratio of StyA1 and StyA2B, indicating (transient) protein complex formation. StyA1 is also active when FADH
2
is supplied by StyB from
Pseudomonas
sp. VLB120 or PheA2 from
Rhodococcus opacus
1CP. However, in both cases the reductase produces an excess of FADH
2
, resulting in a high waste of NADH. The epoxidation rate of StyA1 heavily depends on the type of reductase. This supports that the FADH
2
-induced activation of StyA1 requires interprotein communication. We conclude that the StyA1/StyA2B system represents a novel type of multifunctional flavoprotein monooxygenase. Its unique mechanism of cofactor utilization provides new opportunities for biotechnological applications and is highly relevant from a structural and evolutionary point of view.
The PpaA/AerR Regulators of Photosynthesis Gene Expression from Anoxygenic Phototrophic Proteobacteria Contain Heme-Binding SCHIC Domains
Authors: Moskvin Oleg V. , Gilles-Gonzalez Marie-Alda , Gomelsky Mark
Published: July 2010

The SCHIC domain of the B
12
-binding domain family present in the
Rhodobacter sphaeroides
AppA protein binds heme and senses oxygen. Here we show that the predicted SCHIC domain PpaA/AerR regulators also bind heme and respond to oxygen
in vitro
, despite their low sequence identity with AppA.
Draft Genome Sequences of Two
Neisseria meningitidis
Serogroup C Clinical Isolates
Authors: Lavezzo Enrico , Toppo Stefano , Barzon Luisa , Cobelli Claudio , Di Camillo Barbara , Finotello Francesca , Franchin Elisa , Peruzzo Denis , Toffolo Gianna Maria , Trevisan Marta , Palù Giorgio
Published: July 2010

Neisseria meningitidis
is a human-specific pathogen known for its capability to cause sepsis and meningitis. Here we report the availability of 2 draft genome sequences obtained from patients infected during the same epidemic outbreak. Both bacterial isolates belong to serogroup C, but their genome sequences show local and remarkable differences compared with each other or with the reference genome of strain FAM18.
Complete Genome Sequence of
Lactobacillus casei
Zhang, a New Probiotic Strain Isolated from Traditional Homemade Koumiss in Inner Mongolia, China
Authors: Zhang Wenyi , Yu Dongliang , Sun Zhihong , Wu Rina , Chen Xia , Chen Wei , Meng He , Hu Songnian , Zhang Heping
Published: July 2010

Lactobacillus casei
Zhang is a new probiotic bacterium isolated from koumiss collected in Inner Mongolia, China. Here, we report the main genome features of
L. casei
Zhang and the identification of several predicted proteins implicated in interactions with the host.
20-HETE increases NADPH oxidase-derived ROS production and stimulates the L-type Ca2+ channel via a PKC-dependent mechanism in cardiomyocytes
Authors: Zeng Qinghua , Han Yong , Bao Yuyan , Li Wei , Li Xingting , Shen Xin , Wang Xu , Yao Fanrong , O'Rourke Stephen T. , Sun Chengwen
Published: July 2010

The production of 20-hydroxyeicosatetraenoic acid (20-HETE) is increased during ischemia-reperfusion, and inhibition of 20-HETE production has been shown to reduce infarct size caused by ischemia. This study was aimed to discover the molecular mechanism underlying the action of 20-HETE in cardiac myocytes. The effect of 20-HETE on L-type Ca2+ currents ( ICa,L) was examined in rat isolated cardiomyocytes by patch-clamp recording in the whole cell mode. Superfusion of cardiomyocytes with 20-HETE (10–100 nM) resulted in a concentration-dependent increase in ICa,L, and this action of 20-HETE was attenuated by a specific NADPH oxidase inhibitor, gp91ds-tat (5 μM), or a superoxide scavenger, polyethylene glycol-superoxide dismutase (25 U/ml), suggesting that NADPH-oxidase-derived superoxide is involved in the stimulatory action of 20-HETE on ICa,L. Treatment of cardiomyocytes with 20-HETE (100 nM) increased both NADPH oxidase activity and superoxide production by approximately twofold. To study the molecular mechanism mediating the 20-HETE-induced increase in NADPH oxidase activity, PKC activity was measured in cardiomyocytes. Incubation of the cells with 20-HETE (100 nM) significantly increased PKC activity, and pretreatment of cardiomyocytes with a selective PKC inhibitor, GF-109203 (1 μM), attenuated the 20-HETE-induced increases in ICa,L and in NADPH oxidase activity. In summary, 20-HETE stimulates NADPH oxidase-derived superoxide production, which activates L-type Ca2+ channels via a PKC-dependent mechanism in cardiomyocytes. 20-HETE and 20-HETE-producing enzymes could be novel targets for the treatment of cardiac ischemic diseases.
Early activation of matrix metalloproteinases underlies the exacerbated systolic and diastolic dysfunction in mice lacking TIMP3 following myocardial infarction
Authors: Kandalam Vijay , Basu Ratnadeep , Abraham Thomas , Wang Xiuhua , Awad Ahmed , Wang Wei , Lopaschuk Gary D. , Maeda Nobuyo , Oudit Gavin Y. , Kassiri Zamaneh
Published: July 2010

Extracellular matrix (ECM) remodeling is a critical aspect of cardiac remodeling following myocardial infarction. Tissue inhibitors of metalloproteinases (TIMPs) are physiological inhibitors of matrix metalloproteinases (MMPs) that degrade the ECM proteins. TIMP3 is highly expressed in the heart, and is markedly downregulated in patients with ischemic cardiomyopathy. We therefore examined the time- and region-dependent role of TIMP3 in the cardiac response to myocardial infarction (MI). TIMP3−/− and wild-type (WT) mice were subjected to MI by ligation of the left anterior descending artery. TIMP3−/−-MI mice exhibited a significantly compromised rate of survival compared with WT-MI mice, primarily due to increased left ventricular (LV) rupture, greater infarct expansion, exacerbated LV dilation, and greater systolic and diastolic dysfunction. Second harmonic generation imaging of unfixed and unstained hearts revealed greater collagen disarray and reduced density in the TIMP3−/− infarct myocardium compared with the WT group. Gelatinolytic and collagenolytic activities increased in TIMP3−/− compared with WT hearts at 1 day post-MI but not at 3 days or 1 wk post-MI. Neutrophil infiltration and inflammatory MMPs were significantly increased in the infarct and peri-infarct regions of TIMP3−/−-MI hearts. Treatment of TIMP3−/− mice with a broad-spectrum MMP inhibitor (PD-166793) for 2 days before and 2 days after MI markedly improved post-MI infarct expansion, LV rupture incident, LV dilation, and systolic dysfunction in these mice up to 1 wk post-MI. Our data demonstrate that the initial rise in proteolytic activities early post-MI is a triggering factor for subsequent LV adverse remodeling, LV rupture, and dilated cardiomyopathy. Hence, timing of treatments to improve cardiac response to MI may be critical in producing favorable outcome.
Differential mechanisms of inhibition of glyceraldehyde-3-phosphate dehydrogenase byS-nitrosothiols and NO in cellular and cell-free conditions
Authors: Broniowska Katarzyna A. , Hogg Neil
Published: July 2010

S-nitrosothiols are nitric oxide (NO)-derived molecules found in biological systems. They have been variously discussed as both NO reservoirs and as major actors in NO-dependent, but cGMP-independent, signal transduction. Although S-nitrosation of specific cysteine residues has been suggested to represent a novel redox-based signaling mechanism, the exact mechanisms of S-nitrosothiol formation under (patho)physiological conditions and the determinants of signaling specificity have not yet been established. Here we examined the sensitivity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to inhibition by S-nitrosocysteine (CysNO) and NO both intracellularly and in isolation. Bovine aortic endothelial cells (BAECs) and purified GAPDH preparations were treated with CysNO or NO, and enzymatic activity was monitored. Intracellular GAPDH was irreversibly inhibited upon CysNO administration, whereas treatment with NO resulted in a DTT-reversible inhibition of the enzyme. Purified GAPDH was inhibited by both CysNO and NO, but the inhibition pattern was diametrically opposite to that observed in the cells; CysNO-dependent inhibition was reversed with DTT, whereas NO-dependent inhibition was not. In the presence of GSH, NO inhibited purified GAPDH in a DTT-reversible way. Our data suggest that in response to CysNO treatment, cellular GAPDH undergoes S-nitrosation, which results in an irreversible inhibition of the enzyme under turnover conditions. In contrast, NO inhibits the enzyme via oxidative mechanisms that do not involve S-nitrosation and are reversible. In summary, our data show that GAPDH is a target for CysNO- and NO-dependent inhibition; however, these two agents inhibit the enzyme via different mechanisms both inside the cell and in isolation. Additionally, the differences observed between the cellular system and purified protein strongly imply that the intracellular environment dictates the mechanism of inhibition.
Resveratrol improves left ventricular diastolic relaxation in type 2 diabetes by inhibiting oxidative/nitrative stress: in vivo demonstration with magnetic resonance imaging
Authors: Zhang Hanrui , Morgan Brandon , Potter Barry J. , Ma Lixin , Dellsperger Kevin C. , Ungvari Zoltan , Zhang Cuihua
Published: July 2010

Resveratrol is a natural phytophenol that exhibits cardioprotective effects. This study was designed to elucidate the mechanisms by which resveratrol protects against diabetes-induced cardiac dysfunction. Normal control ( m-Leprdb) mice and type 2 diabetic ( Leprdb) mice were treated with resveratrol orally for 4 wk. In vivo MRI showed that resveratrol improved cardiac function by increasing the left ventricular diastolic peak filling rate in Leprdbmice. This protective role is partially explained by resveratrol's effects in improving nitric oxide (NO) production and inhibiting oxidative/nitrative stress in cardiac tissue. Resveratrol increased NO production by enhancing endothelial NO synthase (eNOS) expression and reduced O2·−production by inhibiting NAD(P)H oxidase activity and gp91phoxmRNA and protein expression. The increased nitrotyrosine (N-Tyr) protein expression in Leprdbmice was prevented by the inducible NO synthase (iNOS) inhibitor 1400W. Resveratrol reduced both N-Tyr and iNOS expression in Leprdbmice. Furthermore, TNF-α mRNA and protein expression, as well as NF-κB activation, were reduced in resveratrol-treated Leprdbmice. Both Leprdbmice null for TNF-α ( dbTNF−/ dbTNF−mice) and Leprdbmice treated with the NF-κB inhibitor MG-132 showed decreased NAD(P)H oxidase activity and iNOS expression as well as elevated eNOS expression, whereas m-Leprdbmice treated with TNF-α showed the opposite effects. Thus, resveratrol protects against cardiac dysfunction by inhibiting oxidative/nitrative stress and improving NO availability. This improvement is due to the role of resveratrol in inhibiting TNF-α-induced NF-κB activation, therefore subsequently inhibiting the expression and activation of NAD(P)H oxidase and iNOS as well as increasing eNOS expression in type 2 diabetes.
Differential effects on nitric oxide-mediated vasodilation in mesenteric and uterine arteries from cytomegalovirus-infected mice
Authors: Gombos Randi B. , Hemmings Denise G.
Published: July 2010

Chronic cytomegalovirus (CMV) infections are implicated in vascular diseases. Recently, we showed that an active mouse CMV (mCMV) infection in nonpregnant mice increased endothelial-dependent vasodilation in isolated mesenteric and uterine arteries. In late pregnancy, while increased vasodilation was found in mesenteric arteries from infected mice, there was a dramatic decrease in uterine arteries. Understanding the mechanisms for these vascular changes during CMV infections is important for pregnancy outcomes and long-term consequences of this chronic infection. Increased nitric oxide (NO) is implicated in CMV-associated atherosclerosis, and CMV replication is dependent on prostaglandin H synthase (PGHS) activity. Alternatively, CMV infections decrease NO under inflammatory conditions. We therefore hypothesized that changes in the contribution by NO or PGHS-induced vasodilators would explain the increased or decreased endothelial-dependent vasodilation in arteries from nonpregnant and late pregnant mice, respectively. We found that the contribution by NO to methacholine-induced vasodilation was significantly increased in mesenteric, but not uterine, arteries isolated from nonpregnant and pregnant mCMV-infected mice. Prostaglandin inhibition did not affect endothelial-dependent vasodilation in any group. Vasodilation responses to sodium nitroprusside, an NO donor, were increased in mesenteric and uterine arteries isolated only from mCMV-infected nonpregnant mice. These results explain the increased vasodilation responses observed in mesenteric arteries from mCMV-infected mice; however, the decreased vasodilation in uterine arteries from pregnant mice could not be explained by these mechanisms. Thus CMV infection affects the contribution of NO differently in endothelial-dependent vasodilation in pregnant compared with nonpregnant mice and also in the mesenteric compared with the uterine vascular bed.
Critical role for lactate dehydrogenase A in aerobic glycolysis that sustains pulmonary microvascular endothelial cell proliferation
Authors: Parra-Bonilla Glenda , Alvarez Diego F. , Al-Mehdi Abu-Bakr , Alexeyev Mikhail , Stevens Troy
Published: July 2010

Pulmonary microvascular endothelial cells possess both highly proliferative and angiogenic capacities, yet it is unclear how these cells sustain the metabolic requirements essential for such growth. Rapidly proliferating cells rely on aerobic glycolysis to sustain growth, which is characterized by glucose consumption, glucose fermentation to lactate, and lactic acidosis, all in the presence of sufficient oxygen concentrations. Lactate dehydrogenase A converts pyruvate to lactate necessary to sustain rapid flux through glycolysis. We therefore tested the hypothesis that pulmonary microvascular endothelial cells express lactate dehydrogenase A necessary to utilize aerobic glycolysis and support their growth. Pulmonary microvascular endothelial cell (PMVEC) growth curves were conducted over a 7-day period. PMVECs consumed glucose, converted glucose into lactate, and acidified the media. Restricting extracellular glucose abolished the lactic acidosis and reduced PMVEC growth, as did replacing glucose with galactose. In contrast, slow-growing pulmonary artery endothelial cells (PAECs) minimally consumed glucose and did not develop a lactic acidosis throughout the growth curve. Oxygen consumption was twofold higher in PAECs than in PMVECs, yet total cellular ATP concentrations were twofold higher in PMVECs. Glucose transporter 1, hexokinase-2, and lactate dehydrogenase A were all upregulated in PMVECs compared with their macrovascular counterparts. Inhibiting lactate dehydrogenase A activity and expression prevented lactic acidosis and reduced PMVEC growth. Thus PMVECs utilize aerobic glycolysis to sustain their rapid growth rates, which is dependent on lactate dehydrogenase A.
The Protection of Minorities and Indigenous Peoples in International Law: A Comparative Temporal Analysis
Authors: Castellino Joshua
Published: July 2010

It is easy to detect a sense of achievement with the extent to which the human rights regime has progressed 60 years after the Universal Declaration of Human Rights. The relative international successes suggest a bright outlook for the future of the human rights regime. However, an important lacuna remains in the attention that ought to be paid to minorities, indigenous peoples and others in vulnerable situations, including in some instances, women. This paper argues that despite the creation of sophisticated systems of international human rights law, the regimes for the protection of minority rights were stronger before the United Nations (UN) era. In support of this argument it seeks to assess regimes that existed at three different times, attempting to extrapolate and analyse the snapshots presented by these through the lens of evolving human rights law.
Minorities and Hatred: Protections and Implications
Authors: Ghanea Nazila
Published: July 2010

The international concern with minorities has benefitted from a range of rationales and gone through a number of permutations over recent decades. Within these are included a wide spectrum of objectives from concern with their very obliteration covered under genocide instruments to soft law instruments concerned with their positive flourishing. This article will address just one aspect of those concerns – those protecting minorities from hate speech.