bluuebunny/crossref_metadata_2025_split huggingface dataset:
IcsB, secreted via the type III secretion system, is chaperoned by IpgA and required at the post‐invasion stage of Shigella pathogenicity
Authors: Ogawa Michinaga , Suzuki Toshihiko , Tatsuno Ichiro , Abe Hiroyuki , Sasakawa Chihiro 
Published: May 2003

Summary Shigella deliver a subset of effector proteins such as IpaA, IpaB and IpaC via the type III secretion system (TTSS) into host cells during the infection of colonic epithelial cells. Many bacterial effectors including some from Shigella require specific chaperones for protection from degradation and targeting to the TTSS. In this study, we have investigated the role of the icsB gene located upstream of the ipaBCDA operon in Shigella infection because the role of IcsB as a virulence factor remains unknown. Here, we found that the IcsB protein is secreted via the TTSS of Shigella in vitro and in vivo. We show that IpgA protein encoded by ipgA, the gene immediately downstream of icsB, serves as the chaperone required for the stabilization and secretion of IcsB. We have shown that IcsB binds to IpgA in bacterial cytosol and the binding site is in the middle of the IcsB protein. Intriguingly, although its significance in Shigella pathogenicity is as yet unclear, the icsB gene can be read‐through into the ipgA gene to create a translational fusion protein. Furthermore, the contribution of IcsB to the pathogenicity of Shigella was demonstrated by plaque‐forming assay and the Sereny test. The ability of the icsB mutant to form plaques was greatly reduced compared with that of the wild type in MDCK cell monolayers. Furthermore, when guinea pig eyes were infected with a non‐polar icsB mutant, the bacteria failed to provoke keratoconjunctivitis. These results suggest that IcsB is secreted via the TTSS, chaperoned by IpgA, and required at the post‐invasion stage of Shigella pathogenicity
Development and maturation of Escherichia coli K‐12 biofilms
Authors: Reisner Andreas , Haagensen Janus A. J. , Schembri Mark A. , Zechner Ellen L. , Molin Søren 
Published: May 2003

SummaryThe development and maturation of E. coli biofilms in flow‐chambers was investigated. We found that the presence of transfer constitutive IncF plasmids induced biofilm development forming structures resembling those reported for Pseudomonas aeruginosa. The development occurred in a step‐wise process: (i) attachment of cells to the substratum, (ii) clonal growth and microcolony formation, and (iii) differentiation into expanding structures rising 70–100 µm into the water phase. The first two steps were the same in the plasmid‐carrying and plasmid‐free strains, whereas the third step only occurred in conjugation pilus proficient plasmid‐carrying strains. The final shapes of the expanding structures in the mature biofilm seem to be determined by the pilus configuration, as various mutants affected in the processing and activity of the transfer pili displayed differently structured biofilms. We further provide evidence that flagella, type 1 fimbriae, curli and Ag43 are all dispensable for the observed biofilm maturation. In addition, our results indicate that cell‐to‐cell signalling mediated by autoinducer 2 (AI‐2) is not required for differentiation of E. coli within a biofilm community. We suggest on the basis of these results that E. coli K‐12 biofilm development and maturation is dependent on cell‐cell adhesion factors, which may act as inducers of self‐assembly processes that result in differently structured biofilms depending on the adhesive properties on the cell surface.
Fodrin CaM‐binding domain cleavage by Pet from enteroaggregative Escherichia coli leads to actin cytoskeletal disruption
Authors: Canizalez‐Roman Adrián , Navarro‐García Fernando 
Published: May 2003

SummaryWe have previously shown that the plasmid‐encoded toxin (Pet) of enteroaggregative Escherichia coli produces cytotoxic and enterotoxic effects. Pet‐intoxicated epithelial cells reveal contraction of the cytoskeleton and loss of actin stress fibres. Pet effects require its internalization into epithelial cells. We have also shown that Pet degrades erythroid spectrin. Pet delivery within the intestine suggests that Pet may degrade epithelial fodrin (non‐erythroid spectrin). Here we demonstrate that Pet has affinity for α‐fodrin (formally named αII spectrin) in vitro and in vivo and cleaves epithelial fodrin, causing its redistribution within the cells. When Pet has produced its cytoskeletal effects, fodrin is found in intracellular aggregates as membrane blebs. Pet cleaves recombinant GST‐fodrin, generating two breakdown products of 37 and 72 kDa. Sequencing of the 37 kDa fragment demonstrated that the cleavage site occurred within fodrin's 11th repetitive unit between M1198 and V1199, in the calmodulin binding domain. Site‐directed mutagenesis of these amino acids prevented fodrin degradation by Pet. Pet also cleaves epithelial fodrin from cultured Pet‐treated cells. A mutant in the Pet serine protease motif was unable to cause fodrin redistribution or to cleave GST‐fodrin. This is the first report showing cleavage of α‐fodrin by a bacterial protease. Cleavage occurs in the middle of the calmodulin binding domain, which leads to cytoskeleton disruption.
SarA and not σB is essential for biofilm development by Staphylococcus aureus
Authors: Valle Jaione , Toledo‐Arana Alejandro , Berasain Carmen , Ghigo Jean‐Marc , Amorena Beatriz , Penadés José R. , Lasa Iñigo 
Published: May 2003

Summary Staphylococcus aureus biofilm formation is associated with the production of the polysaccharide intercellular adhesin (PIA/PNAG), the product of the ica operon. The staphylococcal accessory regulator, SarA, is a central regulatory element that controls the production of S. aureus virulence factors. By screening a library of Tn917 insertions in a clinical S. aureus strain, we identified SarA as being essential for biofilm development. Non‐polar mutations of sarA in four genetically unrelated S. aureus strains decreased PIA/PNAG production and completely impaired biofilm development, both in steady state and flow conditions via an agr‐independent mechanism. Accordingly, real‐time PCR showed that the mutation in the sarA gene resulted in downregulation of the ica operon transcription. We also demonstrated that complete deletion of σB did not affect PIA/PNAG production and biofilm formation, although it slightly decreased ica operon transcription. Furthermore, the sarA‐σB double mutant showed a significant decrease of ica expression but an increase of PIA/PNAG production and biofilm formation compared to the sarA single mutant. We propose that SarA activates S. aureus development of biofilm by both enhancing the ica operon transcription and suppressing the transcription of either a protein involved in the turnover of PIA/PNAG or a repressor of its synthesis, whose expression would be σB‐dependent.
Calcineurin A of Candida albicans: involvement in antifungal tolerance, cell morphogenesis and virulence
Authors: Sanglard Dominique , Ischer Françoise , Marchetti Oscar , Entenza José , Bille Jacques 
Published: May 2003

SummaryThe azole antifungal fluconazole possesses only fungistatic activity in Candida albicans and, therefore, this human pathogen is tolerant to this agent. However, tolerance to fluconazole can be inhibited when C. albicans is exposed to fluconazole combined with the immunosuppressive drug cyclosporin A, which is known to inhibit calcineurin activity in yeast. A mutant lacking both alleles of a gene encoding the calcineurin A subunit (CNA) lost viability in the presence of fluconazole, thus making calcineurin essential for fluconazole tolerance. Consistent with this observation, tolerance to fluconazole was modulated by calcium ions or by the expression of a calcineurin A derivative autoactivated by the removal of its C‐terminal inhibitory domain. Interestingly, CNA was also essential for tolerance to other antifungal agents (voriconazole, itraconazole, terbinafine, amorolfine) and to several other metabolic inhibitors (caffeine, brefeldin A, mycophenolic acid, fluphenazine) or cell wall‐perturbing agents (SDS, calcofluor white, Congo red), thus indicating that the calcineurin pathway plays an important role in the survival of C. albicans in the presence of external growth inhibitors. Several genes, including PMC1, a vacuolar calcium P‐type ATPase, were regulated in a calcineurin‐ and fluconazole‐dependent manner. However, PMC1 did not play a direct role in the survival of C. albicans when exposed to fluconazole. In addition to these different properties, calcineurin was found to affect colony morphology in several media known to modulate the C. albicans dimorphic switch. In particular, calcineurin was found to be essential for C. albicans viability in serum‐containing media. Finally, calcineurin was found to be necessary for the virulence of C. albicans in a mice model of infection, thus making calcineurin an important element for adequate adaptation to the conditions of the host environment.
Construction and usage of a onefold‐coverage shotgun DNA microarray to characterize the metabolism of the archaeon Haloferax volcanii
Authors: Zaigler Alexander , Schuster Stephan C. , Soppa Jörg 
Published: May 2003

Summary Haloferax volcanii is a moderately halophilic archaeon that can grow aerobically and anaerobically with a variety of substrates. We undertook a novel approach for the characterization of metabolic adaptations, i.e. transcriptome analysis with a onefold‐coverage shotgun DNA microarray. A genomic library was constructed and converted into a polymerase chain reaction (PCR) product library, which was used to print two DNA microarrays, a 960‐spot test array used for optimization of microarray analysis and a 2880‐spot onefold‐coverage array. H. volcanii cultures were shifted from casamino acid‐based metabolism to glucose‐based metabolism, and the transcriptome changes were analysed with the onefold‐coverage array at five time points covering the transition phase and the onset of exponential growth with the new carbon source. About 10% of all genes were found to be more than 2.5‐fold regulated at at least one time point. The genes fall into five clusters of kinetically co‐regulated genes. For members of all five clusters, the results were verified by Northern blot analyses. The identity of the regulated genes was determined by sequencing. Many co‐regulated genes encode proteins of common functions. Expected as well as a variety of unexpected findings allowed predictions about the central metabolism, the transport capacity and the cellular composition of H. volcanii growing on casamino acids and on glucose. The microarray analyses are in accordance with the growth rates and ribosome contents of H. volcanii growing on the two carbon sources. Analysis of the results revealed that onefold‐coverage shotgun DNA microarrays are well suited to characterize the regulation of metabolic pathways as well as protein complexes in response to changes in environmental conditions.
Mechanisms of homology‐facilitated illegitimate recombination for foreign DNA acquisition in transformable Pseudomonas stutzeri
Authors: Meier Petra , Wackernagel Wilfried 
Published: May 2003

SummaryIntra‐ and interspecific natural transformation has been observed in many prokaryotic species and is considered a fundamental mechanism for the generation of genetic variation. Recently, it has been described in detail how, in transformable Acinetobacter BD413 and Streptococcus pneumoniae, long stretches of nucleotides lacking homology were integrated into recipient genomes when they were linked on one side to a small piece of DNA with homology to resident DNA serving as a recA‐dependent recombination anchor. Now, such homology‐facilitated illegitimate recombination (HFIR) has also been detected in transformable Pseudomonas stutzeri. However, analysis of the recombinants revealed qualitative and quantitative differences in their generation compared with that in Acinetobacter BD413. In P. stutzeri, foreign DNA with an anchor sequence was integrated 105‐ to 106‐fold less frequently than fully homologous DNA, but still at least 200‐fold more frequently than without the anchor. The anchor sequence could be as small as 311 bp. Remarkably, in 98% of the events, the 3′ end was integrated within the homologous anchor, whereas the 5′ end underwent illegitimate fusion. Moreover, about one‐third of the illegitimate fusion sites shared no or only a single identical basepair in foreign and resident DNA. The other fusions occurred within microhomologies of up to 6 bp with a higher GC content on average than the interacting nucleotide sequences. Foreign DNA of 69–1903 bp was integrated, and resident DNA of 22–2345 bp was lost. In a recA mutant, HFIR was not detectable. The findings suggest that genomic acquisition of foreign DNA by HFIR during transformation occurs widely in prokaryotes, but that details of the required recombination and strand fusion mechanisms may differ between organisms from different genera.
TFIIB and subunits of the SAGA complex are involved in transcriptional activation of phospholipid biosynthetic genes by the regulatory protein Ino2 in the yeastSaccharomyces cerevisiae
Authors: Dietz Martin , Heyken Willm‐Thomas , Hoppen Jens , Geburtig Susanne , Schüller Hans‐Joachim 
Published: May 2003

SummaryIn the yeastSaccharomyces cerevisiae, genes involved in phospholipid biosynthesis are activated by ICRE (inositol/choline‐responsive element) up‐stream motifs and the corresponding heterodimeric binding factor, Ino2 + Ino4. Both Ino2 and Ino4 contain basic helix–loop–helix (bHLH) domains required for ICRE binding, whereas transcriptional activation is mediated exclusively by Ino2. In this work, we describe a molecular analysis of functional minimal domains responsible for specific DNA recognition and transcriptional activation (TAD1 and TAD2). We also define the importance of individual amino acids within the more important activation domain TAD1. Random mutagenesis at five amino acid positions showed the importance of acidic as well as hydrophobic residues within this minimal TAD. We also investigated the contribution of known general transcription factors and co‐activators for Ino2‐dependent gene activation. Although anada5single mutant and agal11 paf1double mutant were severely affected, a partial reduction in activation was found forgcn5andsrb2. Ino2 interacts physically with the basal transcription factor Sua7 (TFIIB of yeast). Interestingly, interaction is mediated by the HLH dimerization domain of Ino2 and by two non‐overlapping domains within Sua7. Thus, Sua7 may compete with Ino4 for binding to the Ino2 activator, creating the possibility of positive and negative influence of Sua7 on ICRE‐dependent gene expression.
Dual regulation by phospho‐OmpR of ssrA/B gene expression in Salmonella pathogenicity island 2
Authors: Feng Xiuhong , Oropeza Ricardo , Kenney Linda J. 
Published: May 2003

SummaryExpression of genes located on Salmonella pathogenicity island 2 (SPI‐2) is required for systemic infection in mice. This region encodes a type III secretion system, secreted effectors and the two‐component regulatory system SsrA/B (also referred to as SpiR), as well as additional uncharacterized genes. In the present work, we demonstrate that phospho‐OmpR (OmpR‐P) functions as an activator at the spiC–ssrA/B locus. There are two promoters at spiR; one is upstream of ssrA and the other upstream of ssrB. Our results indicate that, in contrast to many two‐component regulatory systems, regulation of the sensor kinase SsrA appears to be uncoupled and distinct from regulation of the response regulator SsrB. OmpR regulation of ssrA/B is one of only a few examples known in which a two‐component response regulator directly regulates the expression of another two‐component regulatory system.
Cell cycle function of a rice B2‐type cyclin interacting with a B‐type cyclin‐dependent kinase
Authors: Lee Jeongkyung , Das Avijit , Yamaguchi Masatoshi , Hashimoto Junji , Tsutsumi Nobuhiro , Uchimiya Hirofumi , Umeda Masaaki 
Published: May 2003

SummaryCyclin‐dependent kinases (CDKs) are involved in the control of cell cycle progression. Plant A‐type CDKs are functional homologs of yeast Cdc2/Cdc28 and are expressed throughout the cell cycle. In contrast, B‐type CDK (CDKB) is a family of mitotic CDKs expressed during the S/M phase, and its precise function remains unknown. Here, we identified two B2‐type cyclins, CycB2;1 and CycB2;2, as a specific partner of rice CDKB2;1. The CDKB2;1–CycB2 complexes produced in insect cells showed a significant level of kinase activity in vitro, suggesting that CycB2 binds to and activates CDKB2. We then expressed green fluorescent protein (GFP)‐fused CDKB2;1 and CycB2;2 in tobacco BY2 cells to investigate their subcellular localization during mitosis. Surprisingly, the fluorescence signal of CDKB2;1‐GFP was tightly associated with chromosome alignment as well as with spindle structure during the metaphase. During the telophase, the signal was localized to the spindle midzone and the separating sister chromosomes, and then to the phragmoplast. On the other hand, the CycB2;2‐GFP fluorescence signal was detected in nuclei during the interphase and prophase, moved to the metaphase chromosomes, and then disappeared completely after the cells passed through the metaphase. Co‐localization of CDKB2;1‐GFP and CycB2;2‐GFP on chromosomes aligned at the center of the metaphase cells suggests that the CDKB2–CycB2 complex may function in retaining chromosomes at the metaphase plate. Overexpression of CycB2;2 in rice plants resulted in acceleration of root growth without any increase in cell size, indicating that CycB2;2 promoted cell division probably through association with CDKB2 in the root meristem.
The FHY3 and FAR1 genes encode transposase‐related proteins involved in regulation of gene expression by the phytochrome A‐signaling pathway
Authors: Hudson Matthew E. , Lisch Damon R. , Quail Peter H. 
Published: May 2003

SummaryThe Arabidopsis mutants far1 and fhy3 display a phenotype of reduced inhibition of hypocotyl elongation, which is specific to far‐red light and therefore specific to the phytochrome A (phyA)‐signaling pathway. We report that the proteins encoded by the FAR1 and FHY3 genes are both related to the transposases of type II MuDR family transposons. We demonstrate that the FAR1 protein is capable of activating transcription in Arabidopsis, indicating that it may define a type of transcriptional regulator. Using microarray expression analysis, we show that of 293 mRNAs twofold induced in wild‐type Col‐0 plants by continuous far‐red light, 85% show reduced responsiveness in the fhy3 mutant. Notable alterations were observed in the responses of genes encoding certain transcription factors, proteins involved in cell wall extension, and proteins related to redox balance control. We also found genes, including some involved in transcriptional control, which showed altered transcriptional behavior in the dark‐grown mutant plants. Taken together, our data suggest that FAR1 and FHY3 may function ‘permissively’ outside the signal transduction pathway of light‐regulated development, yet be required for the expression of transcriptional regulatory components. An alternative possibility is that their role includes both light‐signal transduction and transcriptional regulation of other genes not responsive to light. We propose that FAR1 and FHY3 control the expression of their target genes by a mechanism that has evolved directly from the way that an ancestral, MuDRA‐like transposase bound to the TIRs of mobile elements.
Neither inverted repeat T‐DNA configurations nor arrangements of tandemly repeated transgenes are sufficient to trigger transgene silencing
Authors: Lechtenberg Berthold , Schubert Daniel , Forsbach Alexandra , Gils Mario , Schmidt Renate 
Published: May 2003

SummaryTransgene expression was analysed in Arabidopsis T‐DNA transformants carrying defined numbers and arrangement of different reporter genes. All transgenes were placed under the control of the strong constitutive CaMV 35S promoter. High, stable transgene expression was observed in plants containing two copies of the β‐glucuronidase (GUS) gene, two or four copies of the green fluorescent protein (GFP) gene and two, four or six copies of the streptomycin phosphotransferase (SPT) gene. Thus, the mere presence of multiple promoter and/or transgene sequences did not result in gene silencing. In none of the cases analysed were tandem repeat arrangements of transgenes and/or inverted repeat (IR) T‐DNA structures sufficient to trigger silencing of the different reporter genes. Instead, post‐transcriptional gene silencing (PTGS) correlated with the copy number of the highly expressed transgenes. Twelve copies of the SPT and four copies of the GUS gene triggered silencing. Silencing is frequently associated with repetitive T‐DNA structures. We favour the idea that in many cases this may be attributed to the high transgene doses rather than the repeat arrangements themselves.
Effect of Modified Eros Centre on Some Reproductive Parameters of the Sow
Authors: Molnar GB , Bilkei G 
Published: May 2003

ContentsThe effect of a modified eros centre on weaning to oestrus interval, follicle size, ovulation and farrowing rate and total born litter size was investigated. In modified eros centre 94.4% and in group housing 79.1% of the sows (p < 0.01) expressed oestrus within 10 days post‐weaning. Weaning to oestrus interval was shorter (p < 0.001) for sows kept in modified eros centre. The interval from onset of oestrus to the time of ovulation was longer for sows in group housing (p=0.05). The time of ovulation was negatively correlated (r=−0.50) with the interval from weaning to oestrus (p=0.005). The time of ovulation after onset of oestrus was significantly (p < 0.05) shorter for sows expressing oestrus within 2–4 days of weaning, compared with the animals that expressed oestrus between days 5 and 6 post‐weaning and was shortest for sows expressing oestrus after day 6 post‐weaning. Farrowing rate was not affected by a modified eros centre. Litter size tended to be smaller in group‐housed weaned sows (p=0.10). The timing of last artificial insemination relative to time of ovulation did not affect litter size (p > 0.10). The implication of these results is that a modified eros centre may improve some of the post‐weaning oestrous parameters of the sow.
Prevalence and clinical significance of community‐acquired penicillin‐resistant pneumococcal pneumonia in Thailand
Authors: SANGTHAWAN Patchanee , CHANTARATCHADA Sudaluck , CHANTHADISAI Nonglak , WATTANATHUM Anan 
Published: May 2003

Objective:  The study aimed to evaluate the prevalence, clinical significance and outcome of community‐acquired penicillin‐resistant Streptococcus pneumoniae (PRSP) pneumonia in Thailand.Methodology:  We conducted a prospective study in culture‐proven pneumococcal pneumonia. Pneumococci were defined as susceptible, intermediate resistance and high resistance according to the definitions of the United States National Committee for Clinical Laboratory Standards (NCCLS).Results:  Forty‐six consecutive patients were enrolled. Of the S. pneumoniae isolates collected, 41.3% were resistant to penicillin (37% intermediate resistance and 4.3% high resistance). Resistance to other antibiotics was 13% to ceftriaxone (8.7% intermediate resistance and 4.3% high resistance), 34.8% to erythromycin, 39.1% to tetracycline, 26.1% to chloramphenicol, and 43.5% to trimethoprim ‐sulfamethoxazole. Prior antibiotic use within 3 months was significantly associated with resistance to penicillin. The overall mortality of pneumococcal pneumonia was 26.1%. Multilobar involvement and requirement for mechanical ventilation proved to be associated with mortality. However, resistance to penicillin or ceftriaxone was not associated with death.Conclusions:  According to the US NCCLS guidelines, we found a high prevalence of drug‐resistant S. pneumoniae in Thai patients with community‐acquired pneumonia. Prior antibiotic use was significantly associated with penicillin resistance. However, the outcome was not related to in vitro penicillin susceptibility of S. pneumoniae isolated from the patients.