bluuebunny/crossref_metadata_2025 huggingface dataset:
Two Genetic Loci Produce Distinct Carbohydrate-Rich Structural Components of thePseudomonas aeruginosaBiofilm Matrix
Authors: Friedman Lisa , Kolter Roberto
Published: July 2004

Pseudomonas aeruginosaforms biofilms, which are cellular aggregates encased in an extracellular matrix. Molecular genetics studies of three common autoaggregative phenotypes, namely wrinkled colonies, pellicles, and solid-surface-associated biofilms, led to the identification of two loci,pelandpsl, that are involved in the production of carbohydrate-rich components of the biofilm matrix. Thepelgene cluster is involved in the production of a glucose-rich matrix material inP. aeruginosastrain PA14 (L. Friedman and R. Kolter, Mol. Microbiol.51:675-690, 2004). Here we investigate the role of thepelgene cluster inP. aeruginosastrain ZK2870 and identify a second genetic locus, termedpsl, involved in the production of a mannose-rich matrix material. The 11 predicted protein products of thepslgenes are homologous to proteins involved in carbohydrate processing.P. aeruginosais thus able to produce two distinct carbohydrate-rich matrix materials. Either carbohydrate-rich matrix component appears to be sufficient for mature biofilm formation, and at least one of them is required for mature biofilm formation inP. aeruginosastrains PA14 and ZK2870.
Identification ofpsl, a Locus Encoding a Potential Exopolysaccharide That Is Essential forPseudomonas aeruginosaPAO1 Biofilm Formation
Authors: Jackson Kara D. , Starkey Melissa , Kremer Stefanie , Parsek Matthew R. , Wozniak Daniel J.
Published: July 2004

Bacteria inhabiting biofilms usually produce one or more polysaccharides that provide a hydrated scaffolding to stabilize and reinforce the structure of the biofilm, mediate cell-cell and cell-surface interactions, and provide protection from biocides and antimicrobial agents. Historically, alginate has been considered the major exopolysaccharide of thePseudomonas aeruginosabiofilm matrix, with minimal regard to the different functions polysaccharides execute. Recent chemical and genetic studies have demonstrated that alginate is not involved in the initiation of biofilm formation inP. aeruginosastrains PAO1 and PA14. We hypothesized that there is at least one other polysaccharide gene cluster involved in biofilm development. Two separate clusters of genes with homology to exopolysaccharide biosynthetic functions were identified from the annotated PAO1 genome. Reverse genetics was employed to generate mutations in genes from these clusters. We discovered that one group of genes, designatedpsl, are important for biofilm initiation. A PAO1 strain with a disruption of the first two genes of thepslcluster (PA2231 and PA2232) was severely compromised in biofilm initiation, as confirmed by static microtiter and continuous culture flow cell and tubing biofilm assays. This impaired biofilm phenotype could be complemented with the wild-typepslsequences and was not due to defects in motility or lipopolysaccharide biosynthesis. These results implicate an as yet unknown exopolysaccharide as being required for the formation of the biofilm matrix. Understandingpsl-encoded exopolysaccharide expression and protection in biofilms will provide insight into the pathogenesis ofP. aeruginosain cystic fibrosis and other infections involving biofilms.
SadB Is Required for the Transition from Reversible to Irreversible Attachment during Biofilm Formation by
Pseudomonas aeruginosa
PA14
Authors: Caiazza Nicky C. , O'Toole George A.
Published: July 2004

Current models of biofilm formation by
Pseudomonas aeruginosa
propose that (i) planktonic cells become surface associated in a monolayer, (ii) surface-associated cells form microcolonies by clonal growth and/or aggregation, (iii) microcolonies transition to a mature biofilm comprised of exopolysaccharide-encased macrocolonies, and (iv) cells exit the mature biofilm and reenter the planktonic state. Here we report a new class of
P. aeruginosa
biofilm mutant that defines the transition from reversible to irreversible attachment and is thus required for monolayer formation. The transposon insertion carried by the
sadB199
mutant was mapped to open reading frame PA5346 of
P. aeruginosa
PA14 and encodes a protein of unknown function. Complementation analysis and phage-mediated transduction demonstrated that the transposon insertion in PA5346 was the cause of the biofilm-defective phenotype. Examination of flow cell-grown biofilms showed that the
sadB199
mutant could initiate surface attachment but failed to form microcolonies despite being proficient in both twitching and swimming motility. Closer examination of early attachment revealed an increased number of the
sadB199
mutant cells arrested at reversible attachment, functionally defined as adherence via the cell pole. A positive correlation among biofilm formation, irreversible attachment, and SadB level was demonstrated, and furthermore, RpoN and FleR appear to negatively affect SadB levels. Fractionation studies showed that the SadB protein is localized to the cytoplasm, and with the use of GPS-linker scanning mutagenesis, the C-terminal portion of SadB was shown to be dispensable for function, whereas the two putative domains of unknown function and the linker region spanning these domains were required for function. We discuss the results presented here in the context of microbial development as it applies to biofilm formation.
Genes Regulated by TorR, the Trimethylamine Oxide Response Regulator of
Shewanella oneidensis
Authors: Bordi Christophe , Ansaldi Mireille , Gon Stéphanie , Jourlin-Castelli Cécile , Iobbi-Nivol Chantal , Méjean Vincent
Published: July 2004

The
torECAD
operon encoding the trimethylamine oxide (TMAO) respiratory system of
Shewanella oneidensis
is positively controlled by the TorS/TorR two-component system when TMAO is available. Activation of the
tor
operon occurs upon binding of the phosphorylated response regulator TorR to a single operator site containing the direct repeat nucleotide sequence TTCATAN
4
TTCATA. Here we show that the replacement of any nucleotide of one TTCATA hexamer prevented TorR binding in vitro, meaning that TorR specifically interacts with this DNA target. Identical direct repeat sequences were found in the promoter regions of
torR
and of the new gene
torF
(
SO4694
), and they allowed TorR binding to both promoters. Real-time PCR experiments revealed that
torR
is negatively autoregulated, whereas
torF
is strongly induced by TorR in response to TMAO. Transcription start site location and footprinting analysis indicate that the operator site at
torR
overlaps the promoter −10 box, whereas the operator site at
torF
is centered at −74 bp from the start site, in agreement with the opposite role of TorR in the regulation of the two genes. Since
torF
and
torECAD
are positively coregulated by TorR, we propose that the TorF protein plays a role related to TMAO respiration.
Flexibility in the Receptor-Binding Domain of the Enzymatic Colicin E9 Is Required for Toxicity against
Escherichia coli
Cells
Authors: Penfold Christopher N. , Healy Bryan , Housden Nicholas G. , Boetzel Ruth , Vankemmelbeke Mireille , Moore Geoffrey R. , Kleanthous Colin , James Richard
Published: July 2004

The events that occur after the binding of the enzymatic E colicins to
Escherichia coli
BtuB receptors that lead to translocation of the cytotoxic domain into the periplasmic space and, ultimately, cell killing are poorly understood. It has been suggested that unfolding of the coiled-coil BtuB receptor binding domain of the E colicins may be an essential step that leads to the loss of immunity protein from the colicin and immunity protein complex and then triggers the events of translocation. We introduced pairs of cysteine mutations into the receptor binding domain of colicin E9 (ColE9) that resulted in the formation of a disulfide bond located near the middle or the top of the R domain. After dithiothreitol reduction, the ColE9 protein with the mutations L359C and F412C (ColE9 L359C-F412C) and the ColE9 protein with the mutations Y324C and L447C (ColE9 Y324C-L447C) were slightly less active than equivalent concentrations of ColE9. On oxidation with diamide, no significant biological activity was seen with the ColE9 L359C-F412C and the ColE9 Y324C-L447C mutant proteins; however diamide had no effect on the activity of ColE9. The presence of a disulfide bond was confirmed in both of the oxidized, mutant proteins by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The loss of biological activity of the disulfide-containing mutant proteins was not due to an indirect effect on the properties of the translocation or DNase domains of the mutant colicins. The data are consistent with a requirement for the flexibility of the coiled-coil R domain after binding to BtuB.
The
Bacillus subtilis yqjI
Gene Encodes the NADP
+
-Dependent 6-P-Gluconate Dehydrogenase in the Pentose Phosphate Pathway
Authors: Zamboni Nicola , Fischer Eliane , Laudert Dietmar , Aymerich Stéphane , Hohmann Hans-Peter , Sauer Uwe
Published: July 2004

Despite the importance of the oxidative pentose phosphate (PP) pathway as a major source of reducing power and metabolic intermediates for biosynthetic processes, almost no direct genetic or biochemical evidence is available for
Bacillus subtilis
. Using a combination of knockout mutations in known and putative genes of the oxidative PP pathway and
13
C-labeling experiments, we demonstrated that
yqjI
encodes the NADP
+
-dependent 6-P-gluconate dehydrogenase, as was hypothesized previously from sequence similarities. Moreover, YqjI was the predominant isoenzyme during glucose and gluconate catabolism, and its role in the oxidative PP pathway could not be played by either of two homologues, GntZ and YqeC. This conclusion is in contrast to the generally held view that GntZ is the relevant isoform; hence, we propose a new designation for
yqjI
,
gndA
, the monocistronic gene encoding the principal 6-P-gluconate dehydrogenase. Although we demonstrated the NAD
+
-dependent 6-P-gluconate dehydrogenase activity of GntZ,
gntZ
mutants exhibited no detectable phenotype on glucose, and GntZ did not contribute to PP pathway fluxes during growth on glucose. Since
gntZ
mutants grew normally on gluconate, the functional role of GntZ remains obscure, as does the role of the third homologue, YqeC. Knockout of the glucose-6-P dehydrogenase-encoding
zwf
gene was primarily compensated for by increased glycolytic fluxes, but about 5% of the catabolic flux was rerouted through the gluconate bypass with glucose dehydrogenase as the key enzyme.
Long-Term Anaerobic Survival of the Opportunistic Pathogen
Pseudomonas aeruginosa
via Pyruvate Fermentation
Authors: Eschbach Martin , Schreiber Kerstin , Trunk Katharina , Buer Jan , Jahn Dieter , Schobert Max
Published: July 2004

Denitrification and arginine fermentation are central metabolic processes performed by the opportunistic pathogen
Pseudomonas aeruginosa
during biofilm formation and infection of lungs of patients with cystic fibrosis. Genome-wide searches for additional components of the anaerobic metabolism identified potential genes for pyruvate-metabolizing NADH-dependent lactate dehydrogenase (
ldhA
), phosphotransacetylase (
pta
), and acetate kinase (
ackA
). While pyruvate fermentation alone does not sustain significant anaerobic growth of
P. aeruginosa
, it provides the bacterium with the metabolic capacity for long-term survival of up to 18 days. Detected conversion of pyruvate to lactate and acetate is dependent on the presence of intact
ldhA
and
ackA-pta
loci, respectively. DNA microarray studies in combination with reporter gene fusion analysis and enzyme activity measurements demonstrated the
anr
- and
ihfA-
dependent anaerobic induction of the
ackA-pta
promoter. Potential Anr and integration host factor binding sites were localized. Pyruvate-dependent anaerobic long-term survival was found to be significantly reduced in
anr
and
ihfA
mutants. No obvious
ldhA
regulation by oxygen tension was observed. Pyruvate fermentation is pH dependent. Nitrate respiration abolished pyruvate fermentation, while arginine fermentation occurs independently of pyruvate utilization.
The CorA Mg
2+
Transporter Is a Homotetramer
Authors: Warren Mary Ann , Kucharski Lisa M. , Veenstra Alexander , Shi Liang , Grulich Paul F. , Maguire Michael E.
Published: July 2004

CorA is a primary Mg
2+
transporter for
Bacteria
and
Archaea
. The C-terminal domain of ∼80 amino acids forms three transmembrane (TM) segments, which suggests that CorA is a homo-oligomer. A Cys residue was added to the cytoplasmic C terminus (C317) of
Salmonella enterica
serovar Typhimurium CorA with or without mutation of the single periplasmic Cys191 to Ser; each mutant retained function. Oxidation of the Cys191Ser Cys317 CorA gave a dimer. Oxidation of Cys317 CorA showed a dimer plus an additional band, apparently cross-linked via both Cys317 and C191. To determine oligomer order, intact cells or purified membranes were treated with formaldehyde or carbon disulfide. Higher-molecular-mass bands formed, consistent with the presence of a tetramer. Cross-linking of the
Bacillus subtilis
CorA expressed in
Salmonella
serovar Typhimurium similarly indicated a tetramer. CorA periplasmic soluble domains from both
Salmonella
serovar Typhimurium and the archaeon
Methanococcus jannaschii
were purified and shown to retain structure. Formaldehyde treatment showed formation of a tetramer. Finally, previous mutagenesis of the CorA membrane domain identified six intramembrane residues forming an apparent pore that interacts with Mg
2+
during transport. Each was mutated to Cys. In mutants carrying a single intramembrane Cys residue, spontaneous disulfide bond formation that was enhanced by oxidation with Cu(II)-1,10-phenanthroline was observed between monomers, indicating that these Mg
2+
-interacting residues within the membrane are very close to their cognate residue on another monomer. Thus, CorA appears to be a homotetramer with a TM segment of one monomer physically close to the same TM segment of another monomer.
The
Vibrio cholerae
FlgM Homologue Is an Anti-σ
28
Factor That Is Secreted through the Sheathed Polar Flagellum
Authors: Correa Nidia E. , Barker Jeffrey R. , Klose Karl E.
Published: July 2004

Vibrio cholerae
has a single polar sheathed flagellum that propels the cells of this bacterium. Flagellar synthesis, motility, and chemotaxis have all been linked to virulence in this human pathogen.
V. cholerae
expresses flagellar genes in a hierarchy consisting of σ
54
- and σ
28
-dependent transcription. In other bacteria, σ
28
transcriptional activity is controlled by an anti-σ
28
factor, FlgM. We demonstrate that the
V. cholerae
FlgM homologue (i) physically interacts with σ
28
, (ii) has a repressive effect on some
V. cholerae
σ
28
-dependent flagellar promoters, and (iii) is secreted through the polar sheathed flagellum, consistent with anti-σ
28
activity. Interestingly, FlgM does not have a uniform repressive effect on all σ
28
-dependent promoters, as determined by measurement of σ
28
-dependent transcription in cells either lacking FlgM (Δ
flgM
) or incapable of secretion (Δ
fliF
). Further analysis of a Δ
fliF
strain revealed that this flagellar assembly block causes a decrease in class III (FlrC- and σ
54
-dependent) and class IV (σ
28
-dependent), but not class II (FlrA- and σ
54
-dependent), flagellar transcription.
V. cholerae flgM
and
fliA
(encodes σ
28
) mutants were only modestly affected in their ability to colonize the infant mouse intestine, a measure of virulence. Our results demonstrate that
V. cholerae
FlgM functions as an anti-σ
28
factor and that the sheathed flagellum is competent for secretion of nonstructural proteins.
Structure and Electrophysiological Properties of the YscC Secretin from the Type III Secretion System of
Yersinia enterocolitica
Authors: Burghout Peter , van Boxtel Ria , Van Gelder Patrick , Ringler Philippe , Müller Shirley A. , Tommassen Jan , Koster Margot
Published: July 2004

YscC is the integral outer membrane component of the type III protein secretion machinery of
Yersinia enterocolitica
and belongs to the family of secretins. This group of proteins forms stable ring-like oligomers in the outer membrane, which are thought to function as transport channels for macromolecules. The YscC oligomer was purified after solubilization from the membrane with a nonionic detergent. Sodium dodecyl sulfate did not dissociate the oligomer, but it caused a change in electrophoretic mobility and an increase in protease susceptibility, indicating partial denaturation of the subunits within the oligomer. The mass of the homo-oligomer, as determined by scanning transmission electron microscopy, was approximately 1 MDa. Analysis of the angular power spectrum from averaged top views of negatively stained YscC oligomers revealed a 13-fold angular order, suggesting that the oligomer consists of 13 subunits. Reconstituted in planar lipid bilayers, the YscC oligomer displayed a constant voltage-independent conductance of approximately 3 nS, thus forming a stable pore. However, in vivo, the expression of YscC did not lead to an increased permeability of the outer membrane. Electron microscopy revealed that the YscC oligomer is composed of three domains, two stacked rings attached to a conical domain. This structure is consistent with the notion that the secretin forms the upper part of the basal body of the needle structure of the type III secreton.
Response ofBacillus subtilisto Nitric Oxide and the Nitrosating Agent Sodium Nitroprusside
Authors: Moore Charles M. , Nakano Michiko M. , Wang Tao , Ye Rick W. , Helmann John D.
Published: July 2004

We examined the effects of nitric oxide (NO) and sodium nitroprusside (SNP) onBacillus subtilisphysiology and gene expression. In aerobically growing cultures, cell death was most pronounced when NO gas was added incrementally rather than as a single bolus, suggesting that the length of exposure was important in determining cell survival. DNA microarrays, Northern hybridizations, and RNA slot blot analyses were employed to characterize the global transcriptional response ofB. subtilisto NO and SNP. Under both aerobic and anaerobic conditions the gene most highly induced by NO washmp, a flavohemoglobin known to protect bacteria from NO stress. Anaerobically, NO also induced genes repressed by the Fe(II)-containing metalloregulators, Fur and PerR, consistent with the known ability of NO to nitrosylate the Fe(II) center in Fur. In support of this model, we demonstrate that NO fails to induce PerR-regulated genes under growth conditions that favor the formation of PerR:Mn(II) rather than PerR:Fe(II). Aerobically, NO gas inducedhmp, the σBgeneral stress regulon, and, to a lesser extent, the Fur and PerR regulons. Surprisingly, NO gas induced the σBregulon via the energy branch of the σBregulatory cascade while induction by SNP was mediated by the environmental stress branch. This emphasizes that NO and SNP elicit genetically distinct stress responses.
Global Gene Expression in
Staphylococcus aureus
Biofilms
Authors: Beenken Karen E. , Dunman Paul M. , McAleese Fionnuala , Macapagal Daphne , Murphy Ellen , Projan Steven J. , Blevins Jon S. , Smeltzer Mark S.
Published: July 2004

We previously demonstrated that mutation of the staphylococcal accessory regulator (
sarA
) in a clinical isolate of
Staphylococcus aureus
(UAMS-1) results in an impaired capacity to form a biofilm in vitro (K. E. Beenken, J. S. Blevins, and M. S. Smeltzer, Infect. Immun. 71:4206-4211, 2003). In this report, we used a murine model of catheter-based biofilm formation to demonstrate that a UAMS-1
sarA
mutant also has a reduced capacity to form a biofilm in vivo. Surprisingly, mutation of the UAMS-1
ica
locus had little impact on biofilm formation in vitro or in vivo. In an effort to identify additional loci that might be relevant to biofilm formation and/or the adaptive response required for persistence of
S. aureus
within a biofilm, we isolated total cellular RNA from UAMS-1 harvested from a biofilm grown in a flow cell and compared the transcriptional profile of this RNA to RNA isolated from both exponential- and stationary-phase planktonic cultures. Comparisons were done using a custom-made Affymetrix GeneChip representing the genomic complement of six strains of
S. aureus
(COL, N315, Mu50, NCTC 8325, EMRSA-16 [strain 252], and MSSA-476). The results confirm that the sessile lifestyle associated with persistence within a biofilm is distinct by comparison to the lifestyles of both the exponential and postexponential phases of planktonic culture. Indeed, we identified 48 genes in which expression was induced at least twofold in biofilms over expression under both planktonic conditions. Similarly, we identified 84 genes in which expression was repressed by a factor of at least 2 compared to expression under both planktonic conditions. A primary theme that emerged from the analysis of these genes is that persistence within a biofilm requires an adaptive response that limits the deleterious effects of the reduced pH associated with anaerobic growth conditions.
Inactivation ofsll1556inSynechocystisStrain PCC 6803 Impairs Isoprenoid Biosynthesis from Pentose Phosphate Cycle Substrates In Vitro
Authors: Poliquin Kelly , Ershov Yuri V. , Cunningham Francis X. , Woreta Tinsay T. , Gantt R. Raymond , Gantt Elisabeth
Published: July 2004

In cyanobacteria many compounds, including chlorophylls, carotenoids, and hopanoids, are synthesized from the isoprenoid precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate. Isoprenoid biosynthesis in extracts of the cyanobacteriumSynechocystisstrain PCC 6803 grown under photosynthetic conditions, stimulated by pentose phosphate cycle substrates, does not appear to require methylerythritol phosphate pathway intermediates. Thesll1556gene, distantly related to type 2 IPP isomerase genes, was disrupted by insertion of a Kanrcassette. The mutant was fully viable under photosynthetic conditions although impaired in the utilization of pentose phosphate cycle substrates. Compared to the parental strain the Δsll1556mutant (i) is deficient in isoprenoid biosynthesis in vitro with substrates including glyceraldehyde-3-phosphate, fructose-6-phosphate, and glucose-6-phosphate; (ii) has smaller cells (diameter ca. 13% less); (iii) has fewer thylakoids (ca. 30% less); and (iv) has a more extensive fibrous outer wall layer. Isoprenoid biosynthesis is restored with pentose phosphate cycle substrates plus the recombinant Sll1556 protein in the Δsll1556supernatant fraction. IPP isomerase activity could not be demonstrated for the purified Sll1556 protein under our in vitro conditions. The reduction of thylakoid area and the effect on outer wall layer components are consistent with an impairment of isoprenoid biosynthesis in the mutant, possibly via hopanoid biosynthesis. Our findings are consistent with an alternate metabolic shunt for biosynthesis of isoprenoids.
Altering Toluene 4-Monooxygenase by Active-Site Engineering for the Synthesis of 3-Methoxycatechol, Methoxyhydroquinone, and Methylhydroquinone
Authors: Tao Ying , Fishman Ayelet , Bentley William E. , Wood Thomas K.
Published: July 2004

Wild-type toluene 4-monooxygenase (T4MO) of
Pseudomonas mendocina
KR1 oxidizes toluene to
p
-cresol (96%) and oxidizes benzene sequentially to phenol, to catechol, and to 1,2,3-trihydroxybenzene. In this study T4MO was found to oxidize
o
-cresol to 3-methylcatechol (91%) and methylhydroquinone (9%), to oxidize
m
-cresol and
p
-cresol to 4-methylcatechol (100%), and to oxidize
o
-methoxyphenol to 4-methoxyresorcinol (87%), 3-methoxycatechol (11%), and methoxyhydroquinone (2%). Apparent
V
max
values of 6.6 ± 0.9 to 10.7 ± 0.1 nmol/min/ mg of protein were obtained for
o
-,
m
-, and
p
-cresol oxidation by wild-type T4MO, which are comparable to the toluene oxidation rate (15.1 ± 0.8 nmol/min/mg of protein). After these new reactions were discovered, saturation mutagenesis was performed near the diiron catalytic center at positions I100, G103, and A107 of the alpha subunit of the hydroxylase (TmoA) based on directed evolution of the related toluene
o-
monooxygenase of
Burkholderia cepacia
G4 (K. A. Canada, S. Iwashita, H. Shim, and T. K. Wood, J. Bacteriol.
184
:
344-349, 2002) and a previously reported T4MO G103L regiospecific mutant (K. H. Mitchell, J. M. Studts, and B. G. Fox, Biochemistry
41
:
3176-3188, 2002). By using
o
-cresol and
o
-methoxyphenol as model substrates, regiospecific mutants of T4MO were created; for example, TmoA variant G103A/A107S produced 3-methylcatechol (98%) from
o
-cresol twofold faster and produced 3-methoxycatechol (82%) from 1 mM
o
-methoxyphenol seven times faster than the wild-type T4MO (1.5 ± 0.2 versus 0.21 ± 0.01 nmol/min/mg of protein). Variant I100L produced 3-methoxycatechol from
o
-methoxyphenol four times faster than wild-type T4MO, and G103S/A107T produced methylhydroquinone (92%) from
o
-cresol fourfold faster than wild-type T4MO and there was 10 times more in terms of the percentage of the product. Variant G103S produced 40-fold more methoxyhydroquinone from
o
-methoxyphenol than the wild-type enzyme produced (80 versus 2%) and produced methylhydroquinone (80%) from
o
-cresol. Hence, the regiospecific oxidation of
o
-methoxyphenol and
o
-cresol was changed for significant synthesis of 3-methoxycatechol, methoxyhydroquinone, 3-methylcatechol, and methylhydroquinone. The enzyme variants also demonstrated altered monohydroxylation regiospecificity for toluene; for example, G103S/A107G formed 82%
o
-cresol, so saturation mutagenesis converted T4MO into an
ortho
-hydroxylating enzyme. Furthermore, G103S/A107T formed 100%
p
-cresol from toluene; hence, a better
para-
hydroxylating enzyme than wild-type T4MO was formed. Structure homology modeling suggested that hydrogen bonding interactions of the hydroxyl groups of altered residues S103, S107, and T107 influence the regiospecificity of the oxygenase reaction.
Identification ofCampylobacter jejuniATCC 43431-Specific Genes by Whole Microbial Genome Comparisons
Authors: Poly Frédéric , Threadgill Deborah , Stintzi Alain
Published: July 2004

This study describes a novel approach to identify unique genomic DNA sequences from the unsequenced strainC. jejuniATCC 43431 by comparison with the sequenced strainC. jejuniNCTC 11168. A shotgun DNA microarray was constructed by arraying 9,600 individual DNA fragments from aC. jejuniATCC 43431 genomic library onto a glass slide. DNA fragments unique toC. jejuniATCC 43431 were identified by competitive hybridization to the array with genomic DNA ofC. jejuniNCTC 11168. The plasmids containing unique DNA fragments were sequenced, allowing the identification of up to 130 complete and incomplete genes. Potential biological roles were assigned to 66% of the unique open reading frames. The mean G+C content of these unique genes (26%) differs significantly from the G+C content of the entireC. jejunigenome (30.6%). This suggests that they may have been acquired through horizontal gene transfer from an organism with a G+C content lower than that ofC. jejuni. Because the twoC. jejunistrains differ by Penner serotype, a large proportion of the unique ATCC 43431 genes encode proteins involved in lipooligosaccharide and capsular biosynthesis, as expected. Several unique open reading frames encode enzymes which may contribute to genetic variability, i.e., restriction-modification systems and integrases. Interestingly, many of the uniqueC. jejuniATCC 43431 genes show identity with a possible pathogenicity island fromHelicobacter hepaticusand components of a potential type IV secretion system. In conclusion, this study provides a valuable resource to further investigateCampylobacterdiversity and pathogenesis.
Transcriptional Analysis of the
groES
-
groEL1
,
groEL2
, and
dnaK
genes in
Corynebacterium glutamicum
: Characterization of Heat Shock-Induced Promoters
Authors: Barreiro Carlos , González-Lavado Eva , Pátek Miroslav , Martín Juan-Francisco
Published: July 2004

The appropriate conditions to switch on the heat shock promoters in
Corynebacterium glutamicum
were defined by Northern blot analysis. Transcriptional patterns were characterized for the
groEL2
gene and the
groES-groEL1
and
dnaK
operons. Transcriptional start points of these genes were determined by primer extension analysis, allowing the identification of CIRCE and HAIR boxes close to the −10 and −35 regions of the promoters. The presence of both CIRCE and HAIR sequences within a single promoter (P-
groEL2
) in bacteria is described for the first time. In addition, the
dnaK
promoter showed −10 and −35 sequences similar to those recognized by SigH of
Mycobacterium
and SigR of
Streptomyces
close to a second transcription start region with −10 and −35 boxes typical of promoters for housekeeping genes.
Reverse Gyrase Is Not a Prerequisite for Hyperthermophilic Life
Authors: Atomi Haruyuki , Matsumi Rie , Imanaka Tadayuki
Published: July 2004

We disrupted the reverse gyrase gene from a hyperthermophilic archaeon,
Thermococcus kodakaraensis
KOD1. An apparent positive supercoiling activity that was observed in the host strain was not found in the disruptant strain. We found that a lack of reverse gyrase led to a retardation in growth that was more striking at higher temperatures. However, the disruption of the reverse gyrase gene did not lead to a lethal phenotype at 90°C. This study provides experimental evidence that reverse gyrase is not a prerequisite for hyperthermophilic life.
STRINGS IN BARYONS AND MATRIX COORDINATES
Authors: FATOLLAHI AMIR H.
Published: July 2004

It is argued that the internal dynamics of a baryon, as a bound state of QCD-strings and quarks, may be captured by a theory of matrix coordinates.
ANALYSIS OF D→ππ BEYOND NAIVE FACTORIZATION
Authors: WU XIANG-YAO , YIN XIN-GUO , CHEN DE-BAO , GUO YI-QING , ZENG YU
Published: July 2004

We analyze the D→ππ decay channels at the leading order, αs corrections with the QCD factorization and the soft-gluon corrections with the light cone QCD sum rule. Comparing with the experimental result, the calculations of the D0→π+π- and D+→π+π0 decay are much better under the consideration of the contributions of factorization parts, αs corrections and soft-gluon effects. However, the calculation result is one order less than the experimental result in D0→π0π0 decay. It may have large effects of final state interaction in the decay channel.
WEB BASED LECTURES PRODUCED BY AI SUPPORTED CLASSROOM TEACHING
Authors: FRIEDLAND GERALD , KNIPPING LARS , TAPIA ERNESTO
Published: July 2004

This article presents a system that maps classroom lectures directly into web based education lessons. In the classroom, the lecturer writes on a wide, pen sensitive display. The system tracks all actions and makes it possible for the lecture to be replayed from the web any time. The remote viewer can follow the progress of the lecture: Audio, the creation of the board content, and an optional video image of the instructor is transmitted. In addition to usual drawing functionality the board can handle a range of multimedia elements from the Internet. The board can integrate different kinds of modules, invoked by board drawings. One of these modules is described here: A computer algebra system that evaluates mathematical expressions or plots functions is placed at the lecturers disposal by a handwriting recognition.
USING LINEAR REGRESSION TECHNIQUES WITH CENSORED DATA
Authors: SMITH PETER J.
Published: July 2004

In survival analysis, proportional hazards models are commonly used in a distribution-free way for regression where the response variable has been right-censored. However, often a simple linear model may be more appropriate for the data. We present an overview of the linear regression method of Buckley and James (1979) as an interesting distribution-free alternative. It is a method providing consistent parameter estimates, and, through simulation, has been successfully appraised in the literature in comparison to other regression methods. The model is fitted iteratively, and censored points in the scatterplot are moved to estimated positions as if they had been observed without censoring. In this paper we will concentrate on scatterplot effects, model fitting and diagnostics.
REDUCTION OF DISCRETE DYNAMICAL SYSTEMS OVER GRAPHS
Authors: MORTVEIT H. S. , REIDYS C. M.
Published: July 2004

In this paper we study phase space relations in a certain class of discrete dynamical systems over graphs. The systems we investigate are called Sequential Dynamical Systems (SDSs), which are a class of dynamical systems that provide a framework for analyzing computer simulations. Specifically, an SDS consists of (i) a finite undirected graph Y with vertex set {1,2,…,n} where each vertex has associated a binary state, (ii) a collection of Y-local functions (Fi,Y)i∈ v [Y] with [Formula: see text] and (iii) a permutation π of the vertices of Y. The SDS induced by (i)–(iii) is the map [Formula: see text] The paper is motivated by a general reduction theorem for SDSs which guarantees the existence of a phase space embedding induced by a covering map between the base graphs of two SDSs. We use this theorem to obtain information about phase spaces of certain SDSs over binary hypercubes from the dynamics of SDSs over complete graphs. We also investigate covering maps over binary hypercubes, [Formula: see text], and circular graphs, Circ n. In particular we show that there exists a covering map [Formula: see text] if and only if 2n≡0 mod n+1. Furthermore, we provide an interpretation of a class of invertible SDSs over circle graphs as right-shifts of length n-2 over {0,1}2n-2. The paper concludes with a brief discussion of how we can extend a given covering map to a covering map over certain extended graphs.
AUDITING THE MANAGEMENT OF TECHNOLOGICAL INNOVATION IN CHINESE COMPANIES
Authors: CHEN JIN , GENG XUESONG
Published: July 2004

Technological innovation audit can help companies to improve innovation performance by identifying key factors in innovation management and provide the benchmarking guidance. Combining extant audit theories, the paper develops an audit framework that is more comprehensive and more suitable to Chinese firms. Using beta testing method in a medium-size sample of cross-sectional Chinese companies, the authors test the applicability of the operational audit scoreboard in the sample firms. Moreover, the authors identify qualitatively the key advantages and disadvantages in technological innovation in Chinese companies that can differentiate successful and less successful innovation management practice. Three types of innovation management are found in our sample and possible implication for technological innovation development strategy in Chinese firms is proposed.
EMBODYING CORPORATE ENTREPRENEURSHIP IN STRATEGIC ORIENTATION FOR TECHNOLOGY FIRMS
Authors: YIM-TEO TIEN HUA , CHAO CHRISTIAN JANG TAO
Published: July 2004

The study of entrepreneurial activities and behavior in corporations is considered important as it is viewed as a means for sustained corporate competitiveness. This challenge is particularly crucial for technology firms which face dynamic competition due to rapid technological changes. This highlights the need to adopt a suitable strategic orientation which embraces entrepreneurship. However, there appears to be a fragmented understanding as to what constitutes corporate entrepreneurship. This paper therefore attempts to provide clarity to the definition of corporate entrepreneurship by first determining what essentially 'entrepreneurship' is and subsequently contextualizing entrepreneurship in the corporate setting. Based on a clear understanding of corporate entrepreneurship, the paper then conceptually develops a strategic orientation that embodies corporate entrepreneurship applicable to technology firms.
FROM SUBCONTRACTED R&D TO JOINT COLLABORATION: THE ROLE OF TRUST IN FACILITATING THIS PROCESS
Authors: RYAN PAUL , GIBLIN MAJELLA , WALSHE EDEL
Published: July 2004

High-technology companies operate in a dynamic and unstable environment due to rapidly changing technologies and consumer tastes. An increasing number of companies are engaging in joint R&D projects to subvent the constraints to competitiveness in this turbulent environment. Collaborative R&D activity has been studied from the perspectives of strategic management [Dodgson (1992)], organizational behavior [Powell et al. (1996)], operations management [Nooteboom et al. (2000)] and business-to-business marketing [Turnbull et al. (1996)]. Within the literature, trust has been identified as highly significant to alliance effectiveness, governance and the development of a long-term mutually beneficial relationship. [Perry et al. (2002); Ramaseshan and Loo (1998); Morgan and Hunt (1994); Ganesan (1994); Nooteboom et al. (2000)].

This paper describes a collaborative R&D project between two high-tech companies to trace the development of trust between the partners. It specifically considers the role of trust in facilitating the progression of their relationship from subcontracting to joint collaboration. Reaffirming existing theories, the findings of the paper identify partner compatibility and an effective selection process as well as openness and honesty as the foundations for trust. Moreover, sound management practices were found to foster high levels of co-operation and commitment to the relationship. The main contribution of the paper is the identification of opportunistic behavior as a possible positive phenomenon. This provides new insight to existing literature which traditionally assumes a negative relationship between trust and exploitative behavior.
Indinavir impairs protein synthesis and phosphorylations of MAPKs in mouse C2C12 myocytes
Authors: Hong-Brown Ly Q. , Brown C. Randell , Lang Charles H.
Published: July 2004

Anti-retroviral therapy promotes clinical, immunologic, and virologic improvement in human immunodeficiency virus-infected patients. Whereas this therapy adversely affects carbohydrate and lipid metabolism, the effects of anti-retroviral drugs on muscle protein synthesis and degradation have not been reported. To examine these processes, we treated C2C12 myocytes with increasing concentrations of the protease inhibitor indinavir for 1 or 2 days. Treatment of myocytes with a therapeutic concentration of indinavir (20 μM) for 24 h decreased basal protein synthesis by 18%, whereas a 42% decline was observed after 48 h. A similar decrement, albeit quantitatively smaller, was detected with other protease inhibitors. Indinavir did not alter the rate of proteolysis. Likewise, indinavir did not impair the anabolic effect of insulin-like growth factor-I on protein synthesis. Mechanistically, indinavir decreased the phosphorylation of the S6 ribosomal protein (rpS6), and this reduction was associated with a decreased phosphorylation of p70S6 kinase and p90rsk as well as the upstream regulators ERK1/2 and MEK1/2. Indinavir also decreased the phosphorylation of Mnk1 and its upstream effectors, p38 MAPK and ERK1/2. Indinavir did not affect the phosphorylation of mTOR or 4E-BP1, but it did decrease the amount of the active eukaryotic initiation factor eIF4G-eIF4E complex. In conclusion, indinavir decreased protein synthesis in myocytes. This decrease was associated with the disruption of the ERK1/2 and p38 MAPK pathways and a reduction in both the level of functional eIF4F complex and rpS6 phosphorylation.
ATP-induced mitogenesis is mediated by cyclic AMP response element-binding protein-enhanced TRPC4 expression and activity in human pulmonary artery smooth muscle cells
Authors: Zhang Shen , Remillard Carmelle V. , Fantozzi Ivana , Yuan Jason X.-J.
Published: July 2004

Extracellular ATP and intracellular cyclic AMP response element-binding protein (CREB, a transcription factor) promote cell proliferation in many cell types. The canonical transient receptor potential (TRPC) channels, which putatively participate in forming store- and receptor-operated Ca2+ channels, have been implicated in the pulmonary vascular remodeling processes. A link between extracellular ATP, CREB activation, and TRPC4 channel expression and activity has not been shown in human pulmonary artery smooth muscle cells (PASMC). Long-term (24–48 h) treatment of human PASMC with a low dose (100 μM) of ATP, which did not trigger a transient rise in free cytosolic Ca2+ concentration ([Ca2+]i) when applied acutely to the cells, caused marked increases in CREB phosphorylation and TRPC4 protein expression. The time course indicated that the ATP-mediated CREB phosphorylation preceded TRPC4 upregulation, whereas transfection of a nonphosphorylatable CREB mutant abolished ATP-mediated TRPC4 expression. Furthermore, treatment of human PASMC with ATP also enhanced the amplitude of capacitative Ca2+ entry (CCE) induced by passive store depletion, whereas the small interfering RNA specifically targeting TRPC4 attenuated ATP-mediated increases in TRPC4 expression and CCE amplitude and inhibited ATP-induced PASMC proliferation. These data suggest that low-dose ATP exerts part of its mitogenic effect in human PASMC via CREB-mediated upregulation of TRPC4 channel expression and activity and the subsequent increase in CCE and [Ca2+]i.
Mechanism of activation of Xenopus CFTR by stimulation of PKC
Authors: Chen Yongyue , Altenberg Guillermo A. , Reuss Luis
Published: July 2004

PKA-mediated phosphorylation of the regulatory (R) domain plays a major role in the activation of the human cystic fibrosis transmembrane conductance regulator (hCFTR). In contrast, the effect of PKC-mediated phosphorylation is controversial, smaller than that of PKA, and dependent on the cell type. In the present study, we expressed Xenopus CFTR ( XCFTR) and hCFTR in Xenopus oocytes and examined their responses (i.e., macroscopic membrane conductance) to maximal stimulation by PKC and PKA agonists. With XCFTR, the average response to PKC was approximately sixfold that of PKA stimulation. In contrast, with hCFTR, the response to PKC was ∼90% of the response to PKA stimulation. The reason for these differences was the small response of XCFTR to PKA stimulation. Using the substituted cysteine accessibility method, we found no evidence for insertion of functional CFTR channels in the plasma membrane in response to PKC stimulation. The increase in macroscopic conductance in response to PKC stimulation of XCFTR was due to an approximately fivefold increase in single-channel open probability, with a minor (∼30%) increase in single-channel conductance. The responses of XCFTR to PKC stimulation and of hCFTR to PKA stimulation were mediated by similar increases in Po. In both instances, there were no changes in the number of channels in the membrane. We speculate that in animals other than humans, PKC stimulation may be the dominant mechanism for activation of CFTR.
Skeletal muscle adaptation in response to voluntary running in Ca2+/calmodulin-dependent protein kinase IV-deficient mice
Authors: Akimoto Takayuki , Ribar Thomas J. , Williams R. Sanders , Yan Zhen
Published: July 2004

Mammalian skeletal muscles undergo adaptation in response to alteration in functional demands by means of a variety of cellular signaling events. Previous experiments in transgenic mice showed that an active form of Ca2+/calmodulin-dependent protein kinase IV (CaMKIV) is capable of stimulating peroxisome proliferator-activated receptor γ-coactivator 1α (PGC-1α) gene expression, promoting fast-to-slow fiber type switching and augmenting mitochondrial biogenesis in skeletal muscle. However, a role for endogenous CaMKIV in skeletal muscle has not been investigated rigorously. We report that genetically modified mice devoid of CaMKIV have normal fiber type composition and mitochondrial enzyme expression in fast-twitch skeletal muscles and responded to long-term (4 wk) voluntary running with increased expression of myosin heavy chain type IIa, myoglobin, PGC-1α, and cytochrome c oxidase IV proteins in plantaris muscle in a manner similar to that of wild-type mice. Short-term motor nerve stimulation (2 h at 10 Hz) likewise increased PGC-1α mRNA expression in tibialis anterior muscles in both Camk4−/− and wild-type mice. In addition, we have confirmed that no detectable CaMKIV protein is expressed in murine skeletal muscle. Thus CaMKIV is not required for the maintenance of slow-twitch muscle phenotype and endurance training-induced mitochondrial biogenesis and IIb-to-IIa fiber type switching in murine skeletal muscle. Other protein kinases sharing substrates with constitutively active CaMKIV may function as endogenous mediators of activity-dependent changes in myofiber phenotype.
Human breast milk suppresses the transcriptional regulation of IL-1β-induced NF-κB signaling in human intestinal cells
Authors: Minekawa Ryoko , Takeda Takashi , Sakata Masahiro , Hayashi Masami , Isobe Aki , Yamamoto Toshiya , Tasaka Keiichi , Murata Yuji
Published: July 2004

Neonatal necrotizing enterocolitis (NEC), which is a disease with a poor prognosis, is considered to be caused by the coincidence of intestinal ischemia-reperfusion injury and systemic inflammation due to the colonization of pathogenic bacteria. Interleukin (IL)-8, a proinflammatory cytokine, plays an important role in the pathophysiology of NEC. It was recently reported that IL-1β activates the IL-8 gene by regulating the transcriptional nuclear factor κB (NF-κB) signaling pathways in intestinal cells. The protective role of maternal milk in NEC pathogenesis has been reported in both human and animal studies. In this study, we show that human breast milk dramatically suppressed the IL-1β-induced activation of the IL-8 gene promoter by inhibiting the activation pathway of NF-κB. Moreover, we also show that human breast milk induced the production of IκBα. These results suggest that human breast milk could be protective and therapeutic in neonates with NEC by inhibiting the activation pathway of NF-κB.
Molecular analysis of PIP2 regulation of HERG and IKr
Authors: Bian Jin-Song , Kagan Anna , McDonald Thomas V.
Published: July 2004

We previously reported that cloned human ether-à-go-go-related gene (HERG) K+ channels are regulated by changes in phosphatidylinositol 4,5-bisphosphate (PIP2) concentration. Here we investigated the molecular determinants of PIP2 interactions with HERG channel protein. To establish the molecular nature of the PIP2-HERG interaction, we examined a segment of the HERG COOH terminus with a high concentration of positively charged amino acids (nos. 883–894) as a possible site of interaction with negatively charged PIP2. When we excised deletion-HERG (D-HERG) or mutated methionine-substituted-HERG (M-HERG) this segment of HERG to neutralize the amino acid charge, the mutant channels produced current that was indistinguishable from wild-type HERG. Elevating internal PIP2, however, no longer accelerated the activation kinetics of the mutant HERG. Moreover, PIP2-dependent hyperpolarizing shifts in the voltage dependence of activation were abolished with both mutants. PIP2 effects on channel-inactivation kinetics remained intact, which suggests an uncoupling of inactivation and activation regulation by PIP2. The specific binding of radiolabeled PIP2 to both mutant channel proteins was nearly abolished. Stimulation of α1A-adrenergic receptors produced a reduction in current amplitude of the rapidly activating delayed rectifier K+ current (the current carried by ERG protein) from rabbit ventricular myocytes. The α-adrenergic-induced current reduction was accentuated by PKC blockers and also unmasked a depolarizing shift in the voltage dependence of activation, which supports the conclusion that receptor activation of PLC results in PIP2 consumption that alters channel activity. These results support a physiological role for PIP2 regulation of the rapidly activating delayed rectifier K+ current during autonomic stimulation and localize a site of interaction to the COOH-terminal tail of the HERG K+ channel.