bluuebunny/crossref_metadata_2025 huggingface dataset:
Polar Flagellum Biogenesis inAeromonas hydrophila
Authors: Canals Rocío , Ramirez Silvia , Vilches Silvia , Horsburgh Gavin , Shaw Jonathan G. , Tomás Juan M. , Merino Susana
Published: December 2005

MesophilicAeromonasspp. constitutively express a single polar flagellum that helps the bacteria move to more favorable environments and is an important virulence and colonization factor. Certain strains can also produce multiple lateral flagella in semisolid media or over surfaces. We have previously reported 16 genes (flgNtoflgL) that constitute region 1 of theAeromonas hydrophilaAH-3 polar flagellum biogenesis gene clusters. We identified 39 new polar flagellum genes distributed in four noncontiguous chromosome regions (regions 2 to 5). Region 2 contained six genes (flaAtomaf-1), including a modification accessory factor gene (maf-1) that has not been previously reported and is thought to be involved in glycosylation of polar flagellum filament. Region 3 contained 29 genes (fliEtoorf29), most of which are involved in flagellum basal body formation and chemotaxis. Region 4 contained a single gene involved in the motor stator formation (motX), and region 5 contained the three master regulatory genes for theA. hydrophilapolar flagella (flrAtoflrC). Mutations in theflaH,maf-1,fliM,flhA,fliA, andflrCgenes, as well as the double mutantflaA flaB, all caused loss of polar flagella and reduction in adherence and biofilm formation. A defined mutation in thepomBstator gene did not affect polar flagellum motility, in contrast to themotXmutant, which was unable to swim even though it expressed a polar flagellum. Mutations in all of these genes did not affect lateral flagellum synthesis or swarming motility, showing that bothA. hydrophilaflagellum systems are entirely distinct.
Identification ofListeria monocytogenesGenes Contributing to Intracellular Replication by Expression Profiling and Mutant Screening
Authors: Joseph Biju , Przybilla Karin , Stühler Claudia , Schauer Kristina , Slaghuis Jörg , Fuchs Thilo M. , Goebel Werner
Published: December 2005

A successful transition ofListeria monocytogenesfrom the extracellular to the intracellular environment requires a precise adaptation response to conditions encountered in the host milieu. Although many key steps in the intracellular lifestyle of this gram-positive pathogen are well characterized, our knowledge about the factors required for cytosolic proliferation is still rather limited. We used DNA microarray and real-time reverse transcriptase PCR analyses to investigate the transcriptional profile of intracellularL. monocytogenesfollowing epithelial cell infection. Approximately 19% of the genes were differentially expressed by at least 1.6-fold relative to their level of transcription when grown in brain heart infusion medium, including genes encoding transporter proteins essential for the uptake of carbon and nitrogen sources, factors involved in anabolic pathways, stress proteins, transcriptional regulators, and proteins of unknown function. To validate the biological relevance of the intracellular gene expression profile, a random mutant library ofL. monocytogeneswas constructed by insertion-duplication mutagenesis and screened for intracellular-growth-deficient strains. By interfacing the results of both approaches, we provide evidence thatL. monocytogenescan use alternative carbon sources like phosphorylated glucose and glycerol and nitrogen sources like ethanolamine during replication in epithelial cells and that the pentose phosphate cycle, but not glycolysis, is the predominant pathway of sugar metabolism in the host environment. Additionally, we show that the synthesis of arginine, isoleucine, leucine, and valine, as well as a species-specific phosphoenolpyruvate-dependent phosphotransferase system, play a major role in the intracellular growth ofL. monocytogenes.
Novel Mechanism of
Escherichia coli
Porin Regulation
Authors: Castillo-Keller Maria , Vuong Phu , Misra Rajeev
Published: December 2005

A novel mechanism of
Escherichia coli
porin regulation was discovered from multicopy suppressors that permitted growth of cells expressing a mutant OmpC protein in the absence of DegP. Analyses of two suppressors showed that both substantially lowered OmpC expression. Suppression activities were confined to a short DNA sequence, which we designated
ipeX
for
i
nhibition of
p
orin
ex
pression, and to DNA containing a 3′-truncated
ompR
gene. The major effect of
ipeX
on
ompC
expression was exerted posttranscriptionally, whereas the truncated OmpR protein reduced
ompC
transcription.
ipeX
was localized within an untranslated region of 247 base pairs between the stop codon of
nmpC
—a remnant porin gene from the cryptic phage
qsr
′ (DLP12) genome—and its predicted Rho-independent transcriptional terminator. Interestingly, another prophage, PA-2, which encodes a porin similar to NmpC, known as Lc, has sequences downstream from
lc
identical to that of
ipeX
. PA-2 lysogenization leads to Lc expression and OmpC inhibition. Our data show that the synthesis of the
lc
transcript, whose 3′ end contains the corresponding
ipeX
sequence, inhibits OmpC expression. Overexpression of
ipeX
RNA inhibited both OmpC and OmpF expression but not that of OmpA.
ompC
-
phoA
chimeric gene constructs revealed a 248-bp untranslated region of
ompC
required for
ipeX
-mediated inhibition. However, no sequence complementarity was found between
ipeX
and this region of
ompC
, indicating that inhibition may not involve simple base pairing between the two RNA molecules. The effect of
ipeX
on
ompC
, but not on
ompF
, was independent of the RNA chaperone Hfq.
YdgG (TqsA) Controls Biofilm Formation inEscherichia coliK-12 through Autoinducer 2 Transport
Authors: Herzberg Moshe , Kaye Ian K. , Peti Wolfgang , Wood Thomas K.
Published: December 2005

YdgG is an uncharacterized protein that is induced inEscherichia colibiofilms. Here it is shown that deletion ofydgGdecreased extracellular and increased intracellular concentrations of autoinducer 2 (AI-2); hence, YdgG enhances transport of AI-2. Consistent with this hypothesis, deletion ofydgGresulted in a 7,000-fold increase in biofilm thickness and 574-fold increase in biomass in flow cells. Also consistent with the hypothesis, deletion ofydgGincreased cell motility by increasing transcription of flagellar genes (genes induced by AI-2). By expressingydgGintrans, the wild-type phenotypes for extracellular AI-2 activity, motility, and biofilm formation were restored. YdgG is also predicted to be a membrane-spanning protein that is conserved in many bacteria, and it influences resistance to several antimicrobials, including crystal violet and streptomycin (this phenotype could also be complemented). Deletion ofydgGalso caused 31% of the bacterial chromosome to be differentially expressed in biofilms, as expected, since AI-2 controls hundreds of genes. YdgG was found to negatively modulate expression of flagellum- and motility-related genes, as well as other known products essential for biofilm formation, including operons for type 1 fimbriae, autotransporter protein Ag43, curli production, colanic acid production, and production of polysaccharide adhesin. Eighty genes not previously related to biofilm formation were also identified, including those that encode transport proteins (yihNandyihP), polysialic acid production (gutMandgutQ), CP4-57 prophage functions (yfjRandalpA), methionine biosynthesis (metR), biotin and thiamine biosynthesis (bioFandthiDFH), anaerobic metabolism (focB,hyfACDR,ttdA, andfumB), and proteins with unknown function (ybfG,yceO,yjhQ, andyjbE); 10 of these genes were verified through mutation to decrease biofilm formation by 40% or more (yfjR,bioF,yccW,yjbE,yceO,ttdA,fumB,yjiP,gutQ, andyihR). Hence, it appears YdgG controls the transport of the quorum-sensing signal AI-2, and so we suggest the gene nametqsA.
Dissection of the Bacteriophage Mu Strong Gyrase Site (SGS): Significance of the SGS Right Arm in Mu Biology and DNA Gyrase Mechanism
Authors: Oram Mark , Travers Andrew A. , Howells Alison J. , Maxwell Anthony , Pato Martin L.
Published: December 2005

The bacteriophage Mu strong gyrase site (SGS), required for efficient phage DNA replication, differs from other gyrase sites in the efficiency of gyrase binding coupled with a highly processive supercoiling activity. Genetic studies have implicated the right arm of the SGS as a key structural feature for promoting rapid Mu replication. Here, we show that deletion of the distal portion of the right arm abolishes efficient binding, cleavage, and supercoiling by DNA gyrase in vitro. DNase I footprinting analysis of the intact SGS revealed an adenylyl imidodiphosphate-dependent change in protection in the right arm, indicating that this arm likely forms the T segment that is passed through the cleaved G segment during the supercoiling reaction. Furthermore, in an SGS derivative with an altered right-arm sequence, the left arm showed these changes, suggesting that the selection of a T segment by gyrase is determined primarily by the sequences of the arms. Analysis of the sequences of the SGS and other gyrase sites suggests that the choice of T segment correlates with which arm possesses the more extensive set of phased anisotropic bending signals, with the Mu right arm possessing an unusually extended set of such signals. The implications of these observations for the structure of the gyrase-DNA complex and for the biological function of the Mu SGS are discussed.
Roles of Lsr2 in Colony Morphology and Biofilm Formation ofMycobacterium smegmatis
Authors: Chen Jeffrey M. , German Greg J. , Alexander David C. , Ren Huiping , Tan Tracy , Liu Jun
Published: December 2005

The lipid-rich cell wall is a defining feature ofMycobacteriumspecies. Individual cell wall components affect diverse mycobacterial phenotypes including colony morphology, biofilm formation, antibiotic resistance, and virulence. In this study, we describe a transposon insertion mutant ofMycobacterium smegmatismc2155 that exhibits altered colony morphology and defects in biofilm formation. The mutation was localized to thelsr2gene. First identified as an immunodominant T-cell antigen ofMycobacterium leprae,lsr2orthologs have been identified in all sequenced mycobacterial genomes, and homologs are found in many actinomycetes. Although its precise function remains unknown, localization experiments indicate that Lsr2 is a cytosolic protein, and cross-linking experiments demonstrate that it exists as a dimer. Characterization of cell wall lipid components reveals that theM. smegmatis lsr2mutant lacks two previously unidentified apolar lipids. Characterization by mass spectrometry and thin-layer chromatography indicate that these two apolar lipids are novel mycolate-containing compounds, called mycolyl-diacylglycerols (MDAGs), in which a mycolic acid (α- or α′-mycolate) molecule is esterified to a glycerol. Upon complementation with an intactlsr2gene, the mutant reverts to the parental phenotypes and MDAG production is restored. This study demonstrates that due to its impact on the biosynthesis of the hydrophobic MDAGs, Lsr2 plays an important role in the colony morphology and biofilm formation ofM. smegmatis.
Anaerobic Survival ofPseudomonas aeruginosaby Pyruvate Fermentation Requires an Usp-Type Stress Protein
Authors: Schreiber Kerstin , Boes Nelli , Eschbach Martin , Jaensch Lothar , Wehland Juergen , Bjarnsholt Thomas , Givskov Michael , Hentzer Morten , Schobert Max
Published: December 2005

Recently, we identified a pyruvate fermentation pathway inPseudomonas aeruginosasustaining anaerobic survival in the absence of alternative anaerobic respiratory and fermentative energy generation systems (M. Eschbach, K. Schreiber, K. Trunk, J. Buer, D. Jahn, and M. Schobert, J. Bacteriol.186:4596-4604, 2004). Anaerobic long-term survival ofP. aeruginosamight be essential for survival in deeper layers of a biofilm and the persistent infection of anaerobic mucus plaques in the cystic fibrosis lung. Proteome analysis ofP. aeruginosacells during a 7-day period of pyruvate fermentation revealed the induced synthesis of three enzymes involved in arginine fermentation, ArcA, ArcB, and ArcC, and the outer membrane protein OprL. Moreover, formation of two proteins of unknown function, PA3309 and PA4352, increased by factors of 72- and 22-fold, respectively. Both belong to the group of universal stress proteins (Usp). Long-term survival of a PA3309 knockout mutant by pyruvate fermentation was found drastically reduced. The oxygen-sensing regulator Anr controls expression of thePPA3309-lacZreporter gene fusion after a shift to anaerobic conditions and further pyruvate fermentation. PA3309 expression was also found induced during the anaerobic and aerobic stationary phases. This aerobic stationary-phase induction is independent of the regulatory proteins Anr, RpoS, RelA, GacA, RhlR, and LasR, indicating a currently unknown mechanism of stationary-phase-dependent gene activation. PA3309 promoter activity was detected in the deeper layers of aP. aeruginosabiofilm using aPPA3309-gfp(green fluorescent protein gene) fusion and confocal laser-scanning microscopy. This is the first description of an Anr-dependent, anaerobically induced, and functional Usp-like protein in bacteria.
Transcriptional Studies and Regulatory Interactions between thephoR-phoPOperon and thephoU,mtpA, andppkGenes ofStreptomyces lividansTK24
Authors: Ghorbel Sofiane , Kormanec Jan , Artus Alexandra , Virolle Marie-Joelle
Published: December 2005

The PhoR/PhoP two-component system ofStreptomyces lividanswas previously shown to allow the growth of the bacteria at low Piconcentrations and to negatively control antibiotic production. The present study focuses on the transcriptional analysis ofphoRandphoP, along with thephoUandmtpAgenes that are transcribed divergently from thephoRPoperon inS. lividans. The effect ofphoR,phoP,phoU, andppkmutations on transcription of these genes was examined under phosphate-replete and phosphate-limited conditions. We demonstrated thatphoRandphoPwere cotranscribed as a leaderless bicistronic transcript cleaved at discrete sites toward the 3′ end ofphoR. In addition,phoPcould also be transcribed alone from a promoter located at the 3′ end ofphoR. ThephoUandmtpAgenes, predicted to encode metal binding proteins, were shown to be transcribed as monocistronic transcripts. The expression ofphoR-phoP,phoP, andphoUwas found to be induced under conditions of Pilimitation inS. lividansTK24. This induction, requiring both PhoR and PhoP, was significantly weaker in thephoUmutant but much stronger in theppkmutant than in the parental strain. The expression ofmtpAwas also shown to be up-regulated when Piwas limiting but independently of PhoR/PhoP. The induction ofmtpAexpression was much stronger in thephoUmutant strain than in the other strains. This study revealed interesting regulatory interactions between the different genes and allowed us to propose putative roles for PhoU and MtpA in the adaptation to phosphate scarcity.
Phenotype Microarray Profiling of
Staphylococcus aureus menD
and
hemB
Mutants with the Small-Colony-Variant Phenotype
Authors: von Eiff Christof , McNamara Peter , Becker Karsten , Bates Donna , Lei Xiang-He , Ziman Michael , Bochner Barry R. , Peters Georg , Proctor Richard A.
Published: December 2005

Standard biochemical tests have revealed that hemin and menadione auxotrophic
Staphylococcus aureus
small-colony variants (SCVs) exhibit multiple phenotypic changes. To provide a more complete analysis of the SCV phenotype, two genetically defined mutants with a stable SCV phenotype were comprehensively tested. These mutants, generated via mutations in
menD
or
hemB
that yielded menadione and hemin auxotrophs, were subjected to phenotype microarray (PM) analysis of over 1,500 phenotypes (including utilization of different carbon, nitrogen, phosphate, and sulfur sources; growth stimulation or inhibition by amino acids and other nutrients, osmolytes, and metabolic inhibitors; and susceptibility to antibiotics). Compared to parent strain COL, the
hemB
mutant was defective in utilization of a variety of carbon sources, including Krebs cycle intermediates and compounds that ultimately generate ATP via electron transport. The phenotype of the
menD
mutant was similar to that of the
hemB
mutant, but the defects in carbon metabolism were more pronounced than those seen with the
hemB
mutant. In both mutant strains, hexose phosphates and other carbohydrates that provide ATP in the absence of electron transport stimulated growth. Other phenotypes of SCV mutants, such as hypersensitivity to sodium selenite, sodium tellurite, and sodium nitrite, were also uncovered by the PM analysis. Key results of the PM analysis were confirmed in independent growth studies and by using Etest strips for susceptibility testing. PM technology is a new and efficient technology for assessing cellular phenotypes in
S. aureus
.
Identification and Characterization of a Novel Bacterial Sulfite Oxidase with No Heme Binding Domain from
Deinococcus radiodurans
Authors: D'Errico Giovanni , Di Salle Anna , La Cara Francesco , Rossi Mosè , Cannio Raffaele
Published: December 2005

An open reading frame (
draSO
) encoding a putative sulfite oxidase (SO) was identified in the sequence of chromosome II of
Deinococcus radiodurans
; the predicted gene product showed significant amino acid sequence homology to several bacterial and eukaryotic SOs, such as the biochemically and structurally characterized enzyme from
Arabidopsis thaliana
. Cloning of the
Deinococcus
SO gene was performed by PCR amplification from the bacterial genomic DNA, and heterologous gene expression of a histidine-tagged polypeptide was obtained in a molybdopterin-overproducing strain of
Escherichia coli
. The recombinant protein was purified to homogeneity by nickel chelating affinity chromatography, and its main kinetic and chemical physical parameters were determined. Northern blot and enzyme activity analyses indicated that
draSO
gene expression is constitutive in
D. radiodurans
and that there is no increase upon exposure to thiosulfate and/or molybdenum(II).
Electron Transport in the Pathway of Acetate Conversion to Methane in the Marine Archaeon
Methanosarcina acetivorans
Authors: Li Qingbo , Li Lingyun , Rejtar Tomas , Lessner Daniel J. , Karger Barry L. , Ferry James G.
Published: December 2005

A liquid chromatography-hybrid linear ion trap-Fourier transform ion cyclotron resonance mass spectrometry approach was used to determine the differential abundance of proteins in acetate-grown cells compared to that of proteins in methanol-grown cells of the marine isolate
Methanosarcina acetivorans
metabolically labeled with
14
N versus
15
N. The 246 differentially abundant proteins in
M. acetivorans
were compared with the previously reported 240 differentially expressed genes of the freshwater isolate
Methanosarcina mazei
determined by transcriptional profiling of acetate-grown cells compared to methanol-grown cells. Profound differences were revealed for proteins involved in electron transport and energy conservation. Compared to methanol-grown cells, acetate-grown
M. acetivorans
synthesized greater amounts of subunits encoded in an eight-gene transcriptional unit homologous to operons encoding the ion-translocating Rnf electron transport complex previously characterized from the
Bacteria
domain. Combined with sequence and physiological analyses, these results suggest that
M. acetivorans
replaces the H
2
-evolving Ech hydrogenase complex of freshwater
Methanosarcina
species with the Rnf complex, which generates a transmembrane ion gradient for ATP synthesis. Compared to methanol-grown cells, acetate-grown
M. acetivorans
synthesized a greater abundance of proteins encoded in a seven-gene transcriptional unit annotated for the Mrp complex previously reported to function as a sodium/proton antiporter in the
Bacteria
domain. The differences reported here between
M. acetivorans
and
M. mazei
can be attributed to an adaptation of
M. acetivorans
to the marine environment.
Two-Component Systems of
Corynebacterium glutamicum
: Deletion Analysis and Involvement of the PhoS-PhoR System in the Phosphate Starvation Response
Authors: Kočan Martina , Schaffer Steffen , Ishige Takeru , Sorger-Herrmann Ulrike , Wendisch Volker F. , Bott Michael
Published: December 2005

Corynebacterium glutamicum
contains genes for 13 two-component signal transduction systems. In order to test for their essentiality and involvement in the adaptive response to phosphate (P
i
) starvation, a set of 12 deletion mutants was constructed. One of the mutants was specifically impaired in its ability to grow under P
i
limitation, and therefore the genes lacking in this strain were named
phoS
(encoding the sensor kinase) and
phoR
(encoding the response regulator). DNA microarray analyses with the
C. glutamicum
wild type and the Δ
phoRS
mutant supported a role for the PhoRS system in the adaptation to P
i
starvation. In contrast to the wild type, the Δ
phoRS
mutant did not induce the known P
i
starvation-inducible (
psi
) genes within 1 hour after a shift from P
i
excess to P
i
limitation, except for the
pstSCAB
operon, which was still partially induced. This indicates an activator function for PhoR and the existence of at least one additional regulator of the
pst
operon. Primer extension analysis of selected
psi
genes (
pstS
,
ugpA
,
phoR
,
ushA
, and
nucH
) confirmed the microarray data and provided evidence for positive autoregulation of the
phoRS
genes.
The Iron Control Element, Acting in Positive and Negative Control of Iron-RegulatedBradyrhizobium japonicumGenes, Is a Target for the Irr Protein
Authors: Rudolph Gesine , Semini Geo , Hauser Felix , Lindemann Andrea , Friberg Markus , Hennecke Hauke , Fischer Hans-Martin
Published: December 2005

Bradyrhizobium japonicum, the nitrogen-fixing soybean symbiont, possesses a heme uptake system encoded by the gene clusterhmuVUT-hmuR-exbBD-tonB. Transcription of the divergently orientedhmuTandhmuRgenes was previously found to be induced by iron limitation and to depend on a 21-bp promoter-upstream iron control element (ICE). Here, we show by deletion analysis that the full-length ICE is needed for this type of positive control. Additional genes associated with ICE-like motifs were identified in theB. japonicumgenome, of which bll6680 and blr7895 code for bacterioferritin and rubrerythrin homologs, respectively. Transcription start site mapping revealed that their ICEs directly overlap with either the −10 promoter region or the transcription initiation site, suggesting an involvement of the ICE in negative control of both genes. Consistent with this inference was the observed down-regulation of both genes under iron limitation, which in the case of bll6680 was shown to require an intact ICE motif. Using a yeast one-hybrid system, we demonstrated in vivo interaction of the iron response regulator (Irr) with all three ICEs. Moreover, specific in vitro binding of purified Irr protein to the ICE motifs of bll6680 and blr7895 was shown in electrophoretic mobility shift experiments. A genome-wide survey for iron-regulated genes with a custom-made Affymetrix gene chip revealed 17 genes to be induced and 68 to be repressed under iron-replete conditions. Remarkably, ICE-like motifs are associated with a large subset of thoseB. japonicumgenes. We propose the ICE as an importantcis-acting element inB. japonicumwhich represents the DNA-binding site for the Irr protein and, depending on its location within promoter regions, is involved in positive or negative control of the associated iron-regulated genes.
Global Phylogeny ofMycobacterium tuberculosisBased on Single Nucleotide Polymorphism (SNP) Analysis: Insights into Tuberculosis Evolution, Phylogenetic Accuracy of Other DNA Fingerprinting Systems, and Recommendations for a Minimal Standard SNP Set
Authors: Filliol Ingrid , Motiwala Alifiya S. , Cavatore Magali , Qi Weihong , Hazbón Manzour Hernando , Bobadilla del Valle Miriam , Fyfe Janet , García-García Lourdes , Rastogi Nalin , Sola Christophe , Zozio Thierry , Guerrero Marta Inírida , León Clara Inés , Crabtree Jonathan , Angiuoli Sam , Eisenach Kathleen D. , Durmaz Riza , Joloba Moses L. , Rendón Adrian , Sifuentes-Osornio José , Ponce de León Alfredo , Cave M. Donald , Fleischmann Robert , Whittam Thomas S. , Alland David
Published: December 2005

We analyzed a global collection ofMycobacterium tuberculosisstrains using 212 single nucleotide polymorphism (SNP) markers. SNP nucleotide diversity was high (average across all SNPs, 0.19), and 96% of the SNP locus pairs were in complete linkage disequilibrium. Cluster analyses identified six deeply branching, phylogenetically distinct SNP cluster groups (SCGs) and five subgroups. The SCGs were strongly associated with the geographical origin of theM. tuberculosissamples and the birthplace of the human hosts. The most ancestral cluster (SCG-1) predominated in patients from the Indian subcontinent, while SCG-1 and another ancestral cluster (SCG-2) predominated in patients from East Asia, suggesting thatM. tuberculosisfirst arose in the Indian subcontinent and spread worldwide through East Asia. Restricted SCG diversity and the prevalence of less ancestral SCGs in indigenous populations in Uganda and Mexico suggested a more recent introduction ofM. tuberculosisinto these regions. The East African Indian and Beijing spoligotypes were concordant with SCG-1 and SCG-2, respectively; X and Central Asian spoligotypes were also associated with one SCG or subgroup combination. Other clades had less consistent associations with SCGs. Mycobacterial interspersed repetitive unit (MIRU) analysis provided less robust phylogenetic information, and only 6 of the 12 MIRU microsatellite loci were highly differentiated between SCGs as measured byGST. Finally, an algorithm was devised to identify two minimal sets of either 45 or 6 SNPs that could be used in future investigations to enable global collaborations for studies on evolution, strain differentiation, and biological differences ofM. tuberculosis.
Specificity of Acyl-Homoserine Lactone Synthases Examined by Mass Spectrometry
Authors: Gould Ty A. , Herman Jake , Krank Jessica , Murphy Robert C. , Churchill Mair E. A.
Published: December 2005

Many gram-negative bacteria produce a specific set of
N
-acyl-
l
-homoserine-lactone (AHL) signaling molecules for the purpose of quorum sensing, which is a means of regulating coordinated gene expression in a cell-density-dependent manner. AHLs are produced from acylated acyl-carrier protein (acyl-ACP) and
S
-adenosyl-
l
-methionine by the AHL synthase enzyme. The appearance of specific AHLs is due in large part to the intrinsic specificity of the enzyme for subsets of acyl-ACP substrates. Structural studies of the
Pantoea stewartii
enzyme EsaI and AHL-sensitive bioassays revealed that threonine 140 in the acyl chain binding pocket directs the enzyme toward production of 3-oxo-homoserine lactones. Mass spectrometry was used to examine the range of AHL molecular species produced by AHL synthases under a variety of conditions. An AHL selective normal-phase chromatographic purification with addition of a deuterated AHL internal standard was followed by reverse-phase liquid chromatography-tandem mass spectrometry in order to obtain estimates of the relative amounts of different AHLs from biological samples. The AHLs produced by wild-type and engineered EsaI and LasI AHL synthases show that intrinsic specificity and different cellular conditions influence the production of AHLs. The threonine at position 140 in EsaI is important for the preference for 3-oxo-acyl-ACPs, but the role of the equivalent threonine in LasI is less clear. In addition, LasI expressed in
Escherichia coli
produces a high proportion of unusual AHLs with acyl chains consisting of an odd number of carbons. Furthermore, these studies offer additional methods that will be useful for surveying and quantitating AHLs from different sources.
A
Vibrio cholerae
Relaxed (
relA
) Mutant Expresses Major Virulence Factors, Exhibits Biofilm Formation and Motility, and Colonizes the Suckling Mouse Intestine
Authors: Silva Anisia J. , Benitez Jorge A.
Published: December 2005

We have constructed a relaxed mutant of El Tor biotype
Vibrio cholerae
strain C7258 by disruption of the RelA catalytic domain. The ability of the
V. cholerae
relaxed mutant to biosynthesize guanosine tetraphosphate and pentaphosphate was severely affected; the mutant showed a reduced growth rate in minimal medium that could be reversed by the addition of Casamino Acids, and it was thermosensitive. Contrary to published findings, the new
relA
mutant still produced significant cholera toxin and toxin-coregulated pilus. The
V. cholerae relA
mutant was motile, produced normal biofilms, and colonized the suckling mouse intestine. Our data suggest that levels of basal guanosine nucleotides pppGpp and ppGpp, rather than the availability of a stringent response, could influence expression of virulence factors, depending on strain and culture conditions. Production of hemagglutinin (HA)/protease, which requires HapR, RpoS, and the cyclic AMP receptor protein, was not strongly affected. Nevertheless, overexpression of RelA protein from an isopropyl-β-
d
-thiogalactopyranoside-inducible promoter posttranscriptionally diminished production of HA/protease.
Intact Carboxysomes in a Cyanobacterial Cell Visualized by Hilbert Differential Contrast Transmission Electron Microscopy
Authors: Kaneko Yasuko , Danev Radostin , Nagayama Kuniaki , Nakamoto Hitoshi
Published: December 2005

Carboxysomes in rapidly frozen ice-embedded whole cells of the cyanobacterium
Synechococcus
sp. strain PCC 7942 were visualized by the recently developed Hilbert differential contrast transmission electron microscope. Structural details of carboxysomes were especially clearly visualized in the ruptured cells. The novel electron microscopy exhibited the paracrystalline arrays of molecules of the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase in the carboxysomes in much better contrast than conventional transmission electron microscopy with ultrathin sections of cells. The carboxysome was surrounded by a 5- to 6-nm-thick monolayer shell which consisted of orderly arrays of globular particles.
The Quorum-Sensing Negative Regulator RsaL of
Pseudomonas aeruginosa
Binds to the
lasI
Promoter
Authors: Rampioni Giordano , Bertani Iris , Zennaro Elisabetta , Polticelli Fabio , Venturi Vittorio , Leoni Livia
Published: December 2005

A mutation in the
rsaL
gene of
Pseudomonas aeruginosa
produces dramatically higher amounts of
N
-acyl homoserine lactone with respect to the wild type, highlighting the key role of this negative regulator in controlling quorum sensing (QS) in this opportunistic pathogen. The DNA binding site of the RsaL protein on the
rsaL-lasI
bidirectional promoter partially overlaps the binding site of the LasR protein, consistent with the hypothesis that RsaL and LasR could be in binding competition on this promoter. This is the first direct demonstration that RsaL acts as a QS negative regulator by binding to the
lasI
promoter.
AqpZ-Mediated Water Permeability in
Escherichia coli
Measured by Stopped-Flow Spectroscopy
Authors: Mallo Rachael C. , Ashby Michael T.
Published: December 2005

We report that the water permeability of wild-type
Escherichia coli
during exponential growth is comparable to that of an
aqpZ
disruption mutant. In contrast, an increase in permeability is observed for the wild type at the onset of the stationary stage with no significant corresponding change in the permeability of the mutant.
Contribution of the Cyclic Nucleotide Phosphodiesterases PdeA and PdeB to Adaptation of
Myxococcus xanthus
Cells to Osmotic or High-Temperature Stress
Authors: Kimura Yoshio , Nakatuma Hiromi , Sato Naoko , Ohtani Mika
Published: December 2005

A tBLASTn search of the
Myxococcus xanthus
genome database at The Institute for Genomic Research (TIGR) identified three genes (
pdeA
,
pdeB
, and
pdeC
) that encode proteins homologous to 3′,5′-cyclic nucleotide phosphodiesterase.
pdeA
,
pdeB
, and
pdeC
mutants, constructed by replacing a part of the gene with the kanamycin or tetracycline resistance gene, showed normal growth, development, and germination under nonstress conditions. However, the spores of mutants, especially the
pdeA
and
pdeB
mutants, placed under osmotic stress germinated earlier than the wild-type spores. The phenotype was the opposite of that of the receptor-type adenylyl cyclase (
cyaA
or
cyaB
) mutant. Also,
pdeA
and
pdeB
mutants were found to have impaired growth under the condition of high-temperature stress. Intracellular cyclic AMP (cAMP) levels of
pdeA
or
pdeB
mutant cells under these stressful conditions were about 1.3-fold to 2.0-fold higher than those of wild-type cells. These results suggest that PdeA and PdeB may be involved in osmotic adaptation during spore germination and temperature adaptation during vegetative growth through the regulation of cAMP levels.
ON THE CONTINUITY OF LIAO QUALITATIVE FUNCTIONS OF DIFFERENTIAL SYSTEMS AND APPLICATIONS
Authors: DAI XIONGPING
Published: December 2005

Let 𝔛r(M), r ≥ 1, denote the space of all Cr vector fields over a compact, smooth and boundaryless Riemannian manifold M of finite dimension; let [Formula: see text], 1 ≤ ℓ ≤ dim M, be the bundle of orthonormal ℓ-frames of the tangent space TM of M. For any V ∈ 𝔛r(M), Liao defined functions [Formula: see text], k = 1, …, ℓ, on [Formula: see text], which are qualitatively equivalent to the Lyapunov exponents of the differential system V. In this paper, the author shows that every [Formula: see text] depends Cr-1-continuously upon [Formula: see text] and Cr-continuously on [Formula: see text] for any given V. In addition, applying the qualitative functions, the author generalizes Liao's global linearization along a given orbit of V and considers the stochastic stability of Lyapunov spectra of linear skew-product flows based on a given ergodic system.
RATIONAL JORDAN DECOMPOSITION OF BILINEAR FORMS
Authors: ĐOKOVIĆ DRAGOMIR Ž. , ZHAO KAIMING
Published: December 2005

This is a continuation of our previous work on Jordan decomposition of bilinear forms over algebraically closed fields of characteristic 0. In this note, we study Jordan decomposition of bilinear forms over any field K0 of characteristic 0.

Let V0 be an n-dimensional vector space over K0. Denote by [Formula: see text] the space of bilinear forms f : V0 × V0 → K0. We say that f = g + h, where f, g, [Formula: see text], is a rational Jordan decomposition of f if, after extending the field K0 to an algebraic closure K, we obtain a Jordan decomposition over K. By using the Galois group of K/K0, we prove the existence of rational Jordan decompositions and describe a method for constructing all such decompositions. Several illustrative examples of rational Jordan decompositions of bilinear forms are included.

We also show how to classify the unimodular congruence classes of bilinear forms over an algebraically closed field of characteristic different from 2 and over the real field.
ENERGY ESTIMATES FOR WEAKLY HYPERBOLIC SYSTEMS OF THE FIRST ORDER
Authors: DREHER MICHAEL , WITT INGO
Published: December 2005

For a class of first-order weakly hyperbolic pseudo-differential systems with finite time degeneracy, well-posedness of the Cauchy problem is proved in an adapted scale of Sobolev spaces. These Sobolev spaces are constructed in correspondence to the hyperbolic operator under consideration, making use of ideas from the theory of elliptic boundary value problems on manifolds with singularities. In addition, an upper bound for the loss of regularity that occurs when passing from the Cauchy data to the solutions is established. In many examples, this upper bound turns out to be sharp.
INVARIANT SUBSETS OF VOLUME HYPERBOLIC SETS
Authors: AGUILAR I. W. , APAZA E. H. , MORALES C. A.
Published: December 2005

A volume hyperbolic set is a compact invariant set with a dominated splitting whose external bundles uniformly contract and expand the volume respectively [1]. Examples of volume hyperbolic sets for diffeomorphisms or flows are the hyperbolic sets, the geometric Lorenz attractor [3] and the singular horseshoe [6]. We shall prove that no invariant subset of a volume hyperbolic set of a three-dimensional flow is homeomorphic to a closed surface.
NON-RADIALLY SYMMETRIC SOLUTIONS FOR A SUPERLINEAR AMBROSETTI–PRODI TYPE PROBLEM IN A BALL
Authors: DE FIGUEIREDO DJAIRO G. , SRIKANTH P. N. , SANTRA SANJIBAN
Published: December 2005

Using a careful analysis of the Morse indices of the solutions obtained by using the Mountain Pass Theorem applied to the associated Euler–Lagrange functional acting both in the full space [Formula: see text] and in its subspace of radially symmetric functions, we prove the existence of non-radially symmetric solutions of a problem of Ambrosetti–Prodi type in a ball.
FOUNTAIN-LIKE SOLUTIONS FOR NONLINEAR ELLIPTIC EQUATIONS WITH CRITICAL GROWTH AND HARDY POTENTIAL
Authors: FELLI VERONICA , TERRACINI SUSANNA
Published: December 2005

We prove the existence of fountain-like solutions, obtained by superposition of bubbles of different blow-up orders, for a nonlinear elliptic equation with critical growth and Hardy-type potential.
Sterol regulatory element-binding protein 1 is negatively modulated by PKA phosphorylation
Authors: Lu Min , Shyy John Y.-J.
Published: December 2005

Sterol regulatory element-binding protein (SREBP)-1a and -1c are key transcription factors that regulate lipid biosynthesis in cells. We identified that Ser338 located at the NH2 terminus of SREBP-1a is a PKA phosphorylation site in vitro and in HepG2 cells. PKA phosphorylation of this site attenuated DNA occupancy, as revealed by chromatin immunoprecipitation assay, and the ensuing transactivation. In contrast, replacing Ser with Ala [SREBP-1a(N)-S338A] increased transactivation. Although it forms heterodimers with the wild-type SREBP-1a(N) or S338A but not a homodimer with itself, SREBP-1a(N)-S338D (replacing Ser with Asp) decreased DNA binding. Ser314 of SREBP-1c, the counterpart of SREBP-1a Ser338, was also phosphorylated by PKA. Accordingly, the adenovirus-mediated expression of SREBP-1c(N)-S314D in HepG2 cells retarded lipogenesis. Our results indicate that the cAMP-PKA pathway, by phosphorylating SREBP-1, may modulate lipid metabolism in liver cell lines.
Glucose-induced mixed [Ca2+]c oscillations in mouse β-cells are controlled by the membrane potential and the SERCA3 Ca2+-ATPase of the endoplasmic reticulum
Authors: Beauvois Melanie C. , Merezak Charafa , Jonas Jean-Christophe , Ravier Magalie A. , Henquin Jean-Claude , Gilon Patrick
Published: December 2005

Stimulatory concentrations of glucose induce two patterns of cytosolic Ca2+ concentration ([Ca2+]c) oscillations in mouse islets: simple or mixed. In the mixed pattern, rapid oscillations are superimposed on slow ones. In the present study, we examined the role of the membrane potential in the mixed pattern and the impact of this pattern on insulin release. Simultaneous measurement of [Ca2+]c and insulin release from single islets revealed that mixed [Ca2+]c oscillations triggered synchronous oscillations of insulin secretion. Simultaneous recordings of membrane potential in a single β-cell within an islet and of [Ca2+]c in the whole islet demonstrated that the mixed pattern resulted from compound bursting (i.e., clusters of membrane potential oscillations separated by prolonged silent intervals) that was synchronized in most β-cells of the islet. Each slow [Ca2+]c increase during mixed oscillations was due to a progressive summation of rapid oscillations. Digital image analysis confirmed the good synchrony between subregions of an islet. By contrast, islets from sarco(endo)plasmic reticulum Ca2+-ATPase isoform 3 (SERCA3)-knockout mice did not display typical mixed [Ca2+]c oscillations in response to glucose. This results from a lack of progressive summation of rapid oscillations and from altered spontaneous electrical activity, i.e., lack of compound bursting, and membrane potential oscillations characterized by lower-frequency but larger-depolarization phases than observed in SERCA3+/+ β-cells. We conclude that glucose-induced mixed [Ca2+]c oscillations result from compound bursting in all β-cells of the islet. Disruption of SERCA3 abolishes mixed [Ca2+]c oscillations and augments β-cell depolarization. This latter observation indicates that the endoplasmic reticulum participates in the control of the β-cell membrane potential during glucose stimulation.
Spatial and temporal mapping of pacemaker activity in interstitial cells of Cajal in mouse ileum in situ
Authors: Park Kyu Joo , Hennig Grant W. , Lee Hyun-Tai , Spencer Nick J. , Ward Sean M. , Smith Terence K. , Sanders Kenton M.
Published: December 2005

Spontaneous electrical pacemaker activity occurs in tunica muscularis of the gastrointestinal tract and drives phasic contractions. Interstitial cells of Cajal (ICC) are the pacemaker cells that generate and propagate electrical slow waves. We used Ca2+ imaging to visualize spontaneous rhythmicity in ICC in the myenteric region (ICC-MY) of the murine small intestine. ICC-MY, verified by colabeling with Kit antibody, displayed regular Ca2+ transients that occurred after electrical slow waves. ICC-MY formed networks, and Ca2+ transient wave fronts propagated through the ICC-MY networks at ∼2 mm/s and activated attached longitudinal muscle fibers. Nicardipine blocked Ca2+ transients in LM but had no visible effect on the transients in ICC-MY. β-Glycyrrhetinic acid reduced the coherence of propagation, causing single cells to pace independently. Thus, virtually all ICC-MYs are spontaneously active, but normal activity is organized into propagating wave fronts. Inhibitors of dihydropyridine-resistant Ca2+ entry (Ni2+ and mibefradil) and elevated external K+ reduced the coherence and velocity of propagation, eventually blocking all activity. The mitochondrial uncouplers, FCCP, and antimycin and the inositol 1,4,5-trisphosphate receptor-inhibitory drug, 2-aminoethoxydiphenyl borate, abolished rhythmic Ca2+ transients in ICC-MY. These data show that global Ca2+ transients in ICC-MYs are a reporter of electrical slow waves in gastrointestinal muscles. Imaging of ICC networks provides a unique multicellular view of pacemaker activity. The activity of ICC-MY is driven by intracellular Ca2+ handling mechanisms and entrained by voltage-dependent Ca2+ entry and coupling of cells via gap junctions.
Depolarization-induced slow Ca2+ transients stimulate transcription of IL-6 gene in skeletal muscle cells
Authors: Juretić Nevenka , García-Huidobro Paola , Iturrieta Juan Antonio , Jaimovich Enrique , Riveros Nora
Published: December 2005

Contracting skeletal muscle produces and releases interleukin-6 (IL-6) in high amounts. Nevertheless, the mechanisms underlying IL-6 expression are not understood. Because inositol-1,4,5-trisphosphate (IP3)-mediated slow Ca2+ signals evoked by depolarization of skeletal myotubes appears to play a role in the regulation of gene expression, we examined its involvement on IL-6 transcription. With the use of semiquantitative RT-PCR, we have shown that K+ depolarization of myotubes induces a transient increase in IL-6 mRNA level, which peaks at 3–4 h and is independent of extracellular Ca2+. Inhibitors of IP3-dependent Ca2+ signals, like 2-aminoethoxydiphenyl borate (2-APB) and U-73122, decreased activation of IL-6 gene expression as did Ca2+ signals inhibitor BAPTA-AM, whereas ryanodine, a fast Ca2+ transient inhibitor, had no effect on IL-6 induction. Depolarization of myotubes transiently transfected with a reporter gene construct, containing 651 bp of IL-6 promoter, induced a twofold increase in promoter activity, which was abolished by either 2-APB or U-73122 and remained unaffected after ryanodine treatment. Site-directed mutagenesis of parental construct allowed us to identify activator protein-1 and NF-κB sequences as regulatory elements involved in IL-6 upregulation. Our results provide evidence for involvement of IP3-mediated Ca2+ signals on IL-6 transcription in skeletal muscle cells.
Time-restricted feeding entrains daily rhythms of energy metabolism in mice
Authors: Satoh Yoko , Kawai Hiroshi , Kudo Naomi , Kawashima Yoichi , Mitsumoto Atsushi
Published: December 2005

Energy metabolism, oxygen consumption rate (V̇o2), and respiratory quotient (RQ) in mice were monitored continuously throughout 12:12-h light-dark cycles before, during, and after time-restricted feeding (RF). Mice fed ad libitum showed robust daily rhythms in both parameters: high during the dark phase and low during the light phase. The daily profile of energy metabolism in mice under daytime-only feeding was reversed at the beginning of the first fasting night. A few days after daytime-only feeding began, RF also reversed the circadian core body temperature rhythm. Moreover, RF for 6 consecutive days shifted the phases of circadian expression patterns of clock genes in liver significantly by 8–10 h. When mice were fed a high-fat (HF) diet ad libitum, the daily rhythm of RQ dampened day by day and disappeared on the sixth day of RF, whereas V̇o2 showed a robust daily rhythm. Mice fed HF only in the daytime had reversed V̇o2 and RQ rhythms. Similarly, mice fed HF only in the daytime significantly phase shifted the clock gene expression in liver, whereas ad libitum feeding with HF had no significant effect on the expression phases of liver clock genes. These results suggested that V̇o2 is a sensitive indicator of entrainment in the mouse liver. Moreover, physiologically, it can be determined without any surgery or constraint. On the basis of these results, we hypothesize that a change in the daily V̇o2 rhythm, independent of the energy source, might drive phase shifts of circadian oscillators in peripheral tissues, at least in the liver.
Glycine-Gated Chloride Channels Depress Synaptic Transmission in Rat Hippocampus
Authors: Song Weifeng , Chattipakorn Siriporn C. , McMahon Lori L.
Published: December 2005

An inhibitory role for strychnine-sensitive glycine-gated chloride channels (GlyRs) in mature hippocampus is beginning to be appreciated. We have reported previously that CA1 pyramidal cells and GABAergic interneurons recorded in 3- to 4-wk-old rat hippocampal slices express functional GlyRs, dispelling previous misconceptions that GlyR expression ceases in early development. However, the effect of GlyR activation on cell excitability and synaptic circuits in hippocampus has not been fully explored. Using whole cell current-clamp recordings, we show that activation of strychnine-sensitive GlyRs through exogenous glycine application causes a significant decrease in input resistance and prevents somatically generated action potentials in both CA1 pyramidal cells and interneurons. Furthermore, GlyR activation depresses the synaptic network by reducing suprathreshold excitatory postsynaptic potentials (EPSPs) to subthreshold events in both cell types. Blockade of postsynaptic GlyRs with the chloride channel blocker 4, 4′-diisothiocyanatostilbene-2-2′-disulfonic acid (DIDS) or altering the chloride ion driving force in recorded cells attenuates the synaptic depression, strongly indicating that a postsynaptic mechanism is responsible. Increasing the local glycine concentration by blocking reuptake causes a strychnine-sensitive synaptic depression in interneuron recordings, suggesting that alterations in extracellular glycine will impact excitability in hippocampal circuits. Finally, using immunohistochemical methods, we show that glycine and the glycine transporter GlyT2 are co-localized selectively in GABAergic interneurons, indicating that interneurons contain both inhibitory neurotransmitters. Thus we report a novel mechanism whereby activation of postsynaptic GlyRs can function to depress activity in the synaptic network in hippocampus. Moreover, the co-localization of glycine and GABA in hippocampal interneurons, similar to spinal cord, brain stem, and cerebellum, suggests that this property is likely to be a general characteristic of inhibitory interneurons throughout the CNS.