bluuebunny/crossref_metadata_2025 huggingface dataset:
Poor host plant quality causes omnivore to consume predator eggs
Authors: Janssen Arne , Willemse Ellen , Van Der Hammen Tessa
Published: May 2003

Omnivorous arthropods are known to change their diet when host plant quality is low. Consequently, it has been suggested that decreased plant quality has a twofold negative effect on herbivore populations: (1) a decrease in growth rate of herbivores; (2) omnivores include more herbivores in their diet. We hypothesized that decreased host plant quality may also cause omnivores to feed on predators, including their own enemies.

We tested this hypothesis, using the omnivorous western flower thrips. This species is known to feed on many plant species, but also on the eggs of another herbivore, the two‐spotted spider mite. Previous research has shown that a decrease in plant quality leads to increased feeding on spider mite eggs by western flower thrips. Western flower thrips also kill the eggs of various predatory mites, including those of the specialist predator of spider mites and those of a predatory mite that attacks western flower thrips itself.

In this paper we investigate whether thrips larvae kill predator eggs to feed on them and whether this predation depends on host plant quality.

Larval survival as well as developmental rate increased when plant tissue of low quality (sweet pepper) was supplemented with eggs of two predatory mite species or when it was supplemented with pollen, a high‐quality food type.

Supplementing high quality leaf tissue (cucumber) with predator eggs did not lead to increased survival and developmental rate. Thrips larvae fed significantly less on predatory mite eggs when pollen was available.

Thus, thrips larvae indeed feed on predator eggs, including those of their predator, and they feed more on predator eggs when host plants are of low quality.
Geometry of the species–area relationship in central European birds: testing the mechanism
Authors: Storch David , Izling Arnošt L. , Gaston Kevin J.
Published: May 2003

The species–area relationship (SAR) is one of the major patterns in community ecology, but the mechanisms that contribute to its exact shape have remained obscure. In continuous mainland areas, the SAR has been attributed to sampling effects (large areas contain species that are too rare to be present in small areas), habitat heterogeneity (large areas contain more types of habitat allowing more species to coexist), and population and metapopulation processes causing spatial aggregation. We tested the contribution of these effects to SARs using data on breeding bird distributions in the Czech Republic, their total population sizes and spatial distributions of their preferred habitats.

2. The relationship between number of species and sampled area is more or less linear on a log–log scale within the Czech Republic, although it reveals saturation when the area is expanded to the whole of central Europe.

Neither sampling effect nor habitat heterogeneity alone explain the observed SAR shape: both models predict much higher species richness within any area and a SAR of much lower slope than observed.

A combined model based on random sampling constrained by the amount of suitable habitat within an area gives quite realistic predictions of species numbers within different sample areas. Nevertheless, the observed pattern reveals much higher variance of species richness amongst areas, species often being significantly more spatially aggregated than predicted by habitat distribution.

Moreover, the relationship between the amount of suitable habitat and the probability of quadrat occupancy is actually nonsignificant for about two‐thirds of species, indicating that assumptions of the combined model are unrealistic. Therefore, the shape and slope of SARs are actually affected both by habitat heterogeneity that represents the major driver of distribution of some species, and by spatial aggregation that is not attributable to habitat heterogeneity in other species.
Small non‐coding RNAs, co‐ordinators of adaptation processes in Escherichia coli: the RpoS paradigm
Authors: Repoila F. , Majdalani N. , Gottesman S.
Published: May 2003

Adaptation to the changing environment requires both the integration of external signals and the co‐ordination of internal responses. Around 50 non‐coding small RNAs (sRNAs) have been described in Escherichia coli; the levels of many of these vary with changing environmental conditions. This suggests that they play a role in cell adaptation. In this review, we use the regulation of RpoS (σ38) translation as a paradigm of sRNA‐mediated response to environmental conditions; rpoS is currently the only known gene regulated post‐transcriptionally by at least three sRNAs. DsrA and RprA stimulate RpoS translation in response to low temperature and cell surface stress, respectively, whereas OxyS represses RpoS translation in response to oxidative shock. However, in addition to regulating RpoS translation, DsrA represses the translation of HNS (a global regulator of gene expression), whereas OxyS represses the translation of FhlA (a transcriptional activator), allowing the cell to co‐ordinate different pathways involved in cell adaptation. Environmental cues affect the synthesis and stability of specific sRNAs, resulting in specific sRNA‐dependent translational control.
Evidence that acetyl phosphate functions as a global signal during biofilm development
Authors: Wolfe Alan J. , Chang Dong‐Eun , Walker Jason D. , Seitz‐Partridge Jeanine E. , Vidaurri Michael D. , Lange Charles F. , Prüß Birgit M. , Henk Margaret C. , Larkin John C. , Conway Tyrrell
Published: May 2003

We used DNA macroarray analysis to identify genes that respond to the status of the intracellular acetyl phosphate (acP) pool. Genes whose expression correlated negatively with the ability to synthesize acP (i.e. negatively regulated genes) function primarily in flagella biosynthesis, a result consistent with observations that we published previously (Prüß and Wolfe, 1994, Mol Microbiol 12: 973–984). In contrast, genes whose expression correlated positively with the ability to synthesize acP (i.e. positively regulated genes) include those for type 1 pilus assembly, colanic acid (capsule) biosynthesis and certain stress effectors. To our knowledge, this constitutes the first report that these genes may respond to the status of the intracellular acP pool. Previously, other researchers have implicated flagella, type 1 pili, capsule and diverse stress effectors in the formation of biofilms. We therefore tested whether cells altered in their ability to metabolize acP could construct normal biofilms, and found that they could not. Cells defective for the production of acP and cells defective for the degradation of acP could both form biofilms, but these biofilms exhibited characteristics substantially different from each other and from biofilms formed by their wild‐type parent. We confirmed the role of individual cell surface structures, the expression of which appears to correlate with acP levels, in fim or fli mutants that cannot assemble type 1 pili or flagella respectively. Thus, the information gained by expression profiling of cells with altered acP metabolism indicates that acP may help to co‐ordinate the expression of surface structures and cellular processes involved in the initial stages of wild‐type biofilm development.
PY54, a linear plasmid prophage of Yersinia enterocolitica with covalently closed ends
Authors: Hertwig Stefan , Klein Iris , Lurz Rudi , Lanka Erich , Appel Bernd
Published: May 2003

PY54 is a temperate phage isolated from Yersinia enterocolitica. Lysogenic Yersinia strains harbour the PY54 prophage as a plasmid (pY54). The plasmid has the same size (46 kb) as the PY54 genome isolated from phage particles. By electron microscopy, restriction analysis and DNA sequencing, it was demonstrated that the phage and the plasmid DNAs are linear, circularly permuted molecules. Unusually for phages of Gram‐negative bacteria, the phage genome has 3′‐protruding ends. The linear plasmid pY54 has covalently closed ends forming telomere‐like hairpins. The equivalent DNA sequence of the phage genome is a 42 bp perfect palindrome. Downstream from the palindrome, an open reading frame (ORF) was identified that revealed strong DNA homology to the telN gene of Escherichia coli phage N15 encoding a protelomerase. Similar to PY54, the N15 prophage is a linear plasmid with telomeres. The N15 protelomerase has cleaving/joining activity generating the telomeres by processing a 56 bp palindrome (telomere resolution site tel RL). To study the activity of the PY54 protein, the telN‐like gene was cloned and expressed in E. coli. A 77 kDa protein was obtained and partially purified. The protein was found to process recombinant plasmids containing the 42 bp palindrome. Telomere resolution of plasmids under in vivo conditions was also investigated in Yersinia infected with PY54. Processing required a plasmid containing the palindrome as well as adjacent DNA sequences from the phage including an additional inverted repeat. Regions on the phage genome important for plasmid maintenance were defined by the construction of linear and circular miniplasmid derivatives of pY54, of which the smallest miniplasmid comprises a 4.5 kb DNA fragment of the plasmid prophage.
Region 4 of σ as a target for transcription regulation
Authors: Dove Simon L. , Darst Seth A. , Hochschild Ann
Published: May 2003

Bacterial σ factors play a key role in promoter recognition, making direct contact with conserved promoter elements. Most σ factors belong to the σ70 family, named for the primary σ factor in Escherichia coli. Members of the σ70 family typically share four conserved regions and, here, we focus on region 4, which is directly involved in promoter recognition and serves as a target for a variety of regulators of transcription initiation. We review recent advances in the understanding of the mechanism of action of regulators that target region 4 of σ.
Identification and macrophage‐activating activity of glycolipids released from intracellular Mycobacterium bovis BCG
Authors: Rhoades E. , Hsu F‐. F. , Torrelles J. B. , Turk J. , Chatterjee D. , Russell D. G.
Published: May 2003

Intracellular mycobacteria release cell wall glycolipids into the endosomal network of infected macrophages. Here, we characterize the glycolipids of Mycobacterium bovis BCG (BCG) that are released into murine bone marrow‐derived macrophages (BMMØ). Intracellularly released mycobacterial lipids were harvested from BMMØ that had been infected with 14C‐labelled BCG. Released BCG lipids were resolved by thin‐layer chromatography, and they migrated similarly to phosphatidylinositol dimannosides (PIM2), mono‐ and diphosphatidylglycerol, phosphatidylethanolamine, trehalose mono‐ and dimycolates and the phenolic glycolipid, mycoside B. Culture‐derived BCG lipids that co‐migrated with the intracellularly released lipids were purified and identified by electrospray ionization mass spectrometry. When delivered on polystyrene microspheres, fluorescently tagged BCG lipids were also released into the BMMØ, in a manner similar to release from viable or heat‐killed BCG bacilli. To determine whether the released lipids elicited macrophage responses, BCG lipid‐coated microspheres were delivered to interferon gamma‐primed macrophages (BMMØ or thioglycollate‐elicited peritoneal macrophages), and reactive nitrogen intermediates as well as tumour necrosis factor‐alpha and monocyte chemoattractant protein‐1 production were induced. When fractionated BCG lipids were delivered on the microspheres, PIM2 species reproduced the macrophage‐activating activity of total BCG lipids. These results demonstrate that intracellular mycobacteria release a heterogeneous mix of lipids, some of which elicit the production of proinflammatory cytokines from macrophages that could potentially contribute to the granulomatous response in tuberculous diseases.
Suppression of defective ribosome assembly in a rbfA deletion mutant by overexpression of Era, an essential GTPase in Escherichia coli
Authors: Inoue Koichi , Alsina Janivette , Chen Jingqiu , Inouye Masayori
Published: May 2003

Era is a small GTP‐binding protein and essential for cell growth in Escherichia coli. It consists of two domains: N‐terminal GTP‐binding and C‐terminal RNA‐binding KH domains. It has been shown to bind to 16S rRNAs and 30S ribosomal subunits in vitro. Here, we report that a precursor of 16S rRNA accumulates in Era‐depleted cells. The accumulation of the precursors is also seen in a cold‐sensitive mutant, E200K, in which the mutation site is located in the C‐terminal domain. The major precursor molecule accumulated seems to be 17S rRNA, containing extra sequences at both 5′ and 3′ ends of 16S rRNA. Moreover, the amounts of both 30S and 50S ribosomal subunits relative to the amount of 70S monosomes increase in Era‐depleted and E200K mutant cells. The C‐terminal KH domain has a high structural similarity to the RbfA protein, a cold shock protein that also specifically associates with 30S ribosomal subunits. RbfA is essential for cell growth at low temperature, and a precursor of 16S rRNA accumulates in an rbfA deletion strain. The 16S rRNA precursor seems to be identical in size to that accumulated in Era mutant cells. Surprisingly, the cold‐sensitive cell growth of the rbfA deletion cells was partially suppressed by overproduction of the wild‐type Era. The C‐terminal domain alone was not able to suppress the cold‐sensitive phenotype, whereas Era‐dE, which has a 10‐residue deletion in a putative effector region of the N‐terminal domain, functioned as a more efficient suppressor than the wild‐type Era. It was found that Era‐dE suppressed defective 16S rRNA maturation, resuming a normal polysome profile to reduce highly accumulated free 30S and 50S subunits in the rbfA deletion cells. These results indicate that Era is involved in 16S rRNA maturation and ribosome assembly.
Antagonism of PII signalling by the AmtB protein of Escherichia coli
Authors: Blauwkamp Timothy A. , Ninfa Alexander J.
Published: May 2003

Escherichia coli AmtB is a member of the MEP/Amt family of ammonia transporters found in archaea, eubacteria, fungi, plants and animals. In prokaryotes, AmtB homologues are co‐transcribed with a PII paralogue, GlnK, in response to nitrogen limitation. Here, we show that AmtB antagonizes PII signalling through NRII and that co‐expression of GlnK with AmtB overcomes this antagonism. In cells lacking GlnK, expression of AmtB during nitrogen starvation prevented deinduction of Ntr gene expression when a nitrogen source became available. The absence of AmtB in cells lacking GlnK allowed rapid reduction of Ntr gene expression during this transition, indicating that one function of GlnK is to prevent AmtB‐mediated antagonism of PII signalling after nitrogen starvation. Other roles of GlnK in controlling Ntr gene expression and maintaining viability during nitrogen starvation were unaffected by AmtB. Expression of AmtB from a constitutive promoter under nitrogen‐rich conditions induced full expression of glnALG and elevated expression of glnK in wild‐type and glnK cells; thus, the ability of AmtB to raise Ntr gene expression did not require a factor found only in nitrogen‐starved cells. Experiments with intact cells showed that AmtB acted downstream of a uridylyl transferase uridylyl‐removing enzyme (UTase/UR) and upstream of NRII, suggesting that the target was PII. AmtB also slowed the deuridylylation of PII∼UMP upon ammonia addition, showing that multiple PII interactions were affected by AmtB. Our data are consistent with a hypothesis that AmtB interacts with PII and GlnK, and that co‐transcription of glnK and amtB prevents titration of PII when AmtB is highly expressed.
Medicinal genetics approach towards identifying the molecular target of a novel inhibitor of fungal cell wall assembly
Authors: Tsukahara Kappei , Hata Katsura , Nakamoto Kazutaka , Sagane Koji , Watanabe Nao‐aki , Kuromitsu Junro , Kai Junko , Tsuchiya Mamiko , Ohba Fuminori , Jigami Yoshifumi , Yoshimatsu Kentaro , Nagasu Takeshi
Published: May 2003

Glycosylphosphatidylinositol (GPI)‐anchored cell wall mannoproteins are required for the adhesion of pathogenic fungi, such as Candida albicans, to human epithelium. Small molecular inhibitors of the cell surface presentation of GPI‐anchored mannoproteins would be promising candidate drugs to block the establishment of fungal infections. Here, we describe a medicinal genetics approach to identifying the gene encoding a novel target protein that is required for the localization of GPI‐anchored cell wall mannoproteins. By means of a yeast cell‐based screening procedure, we discovered a compound, 1‐[4‐butylbenzyl]isoquinoline (BIQ), that inhibits cell wall localization of GPI‐anchored mannoproteins in Saccharomyces cerevisiae. Treatment of C. albicans cells with this compound resulted in reduced adherence to a rat intestine epithelial cell monolayer. A previously uncharacterized gene YJL091c, named GWT1, was cloned as a dosage‐dependent suppressor of the BIQ‐induced phenotypes. GWT1 knock‐out cells showed similar phenotypes to BIQ‐treated wild‐type cells in terms of cell wall structure and transcriptional profiles. Two different mutants resistant to BIQ each contained a single missense mutation in the coding region of the GWT1 gene. These results all suggest that the GWT1 gene product is the primary target of the compound.
Identification of a 5‐nucleotide sequence that controls expression of the ica locus in Staphylococcus aureus and characterization of the DNA‐binding properties of IcaR
Authors: Jefferson Kimberly K. , Cramton Sarah E. , Götz Friedrich , Pier Gerald B.
Published: May 2003

Biofilm formation is an important aspect of the pathogenesis of staphylococcal infections. A β‐1,6‐linked N‐acetyl glucosamine polysaccharide is critical to biofilm elaboration and is synthesized by proteins encoded by the intercellular adhesion (ica) locus. These studies were undertaken to characterize the mechanism by which transcription of the ica locus in S. aureus is regulated using isogenic S. aureus MN8 and MN8 mucoid (MN8m) strains, the latter of which constitutively overproduces biofilm. Transformation of the ica locus from MN8m to the ica knock‐out mutants of two strains, MN8 and NCTC 10833, conferred a strong biofilm‐producing phenotype. Sequence analysis revealed a 5‐nucleotide deletion within the promoter region of the ica locus in MN8m compared with the sequence in the wild‐type locus. Deletion or substitution of these 5 nucleotides within the wild‐type ica locus augmented transcription of the ica locus and induced the strong biofilm‐producing phenotype. Gel shift analysis demonstrated that a protein(s) within cell‐free lysates from strain MN8 bind(s) specifically to oligonucleotides representative of the wild‐type ica promoter sequence and that this binding is greatly diminished by the deletion or substitution of the 5 nucleotides. DNase I footprint analysis revealed that purified IcaR, thought to be a regulator of ica transcription, also binds to the ica promoter sequence just upstream of the ica start codon, but its affinity for the ica promoter is unaffected by deletion of the 5‐nucleotide motif. These findings identify a 5‐nucleotide motif within the ica promoter region that has a functional role in transcriptional regulation of the ica locus that is independent of IcaR, and also show that IcaR binds to the promoter region of the S. aureus ica locus.
Sequence‐specific recognition but position‐dependent cleavage of two distinct telomeres by the Borrelia burgdorferi telomere resolvase, ResT
Authors: Tourand Yvonne , Kobryn Kerri , Chaconas George
Published: May 2003

An unusual feature of bacteria in the genus Borrelia (causative agents of Lyme disease and relapsing fever) is a segmented genome consisting of multiple linear DNA molecules with covalently closed hairpin ends, known as telomeres. The hairpin telomeres are generated by a DNA breakage and reunion process (telomere resolution) promoted by ResT, an enzyme using an active site related to that of tyrosine recombinases and type IB topoisomerases. In this study, we define the minimal sequence requirements for a functional telomere and identify specific basepairs that appear to be important for telomere resolution. In addition, we show that the two naturally occurring and distinct telomere spacings found in B. burgdorferi can both be efficiently processed by ResT. This flexibility for substrate utilization by ResT supports the argument for a single telomere resolvase in Borrelia. Furthermore, although telomere recognition requires sequence specificity in part of the substrate, DNA cleavage is instead position dependent and occurs at a fixed distance from the axis of symmetry and the conserved sequence of box 3 in the different replicated telomere substrates. This positional dependence for DNA cleavage has not been observed previously for a tyrosine recombinase.
Substrate specificity of type III flagellar protein export in Salmonella is controlled by subdomain interactions in FlhB
Authors: Fraser Gillian M. , Hirano Takanori , Ferris Hedda U. , Devgan Lara L. , Kihara May , Macnab Robert M.
Published: May 2003

FlhB, an integral membrane protein, gates the type III flagellar export pathway of Salmonella. It permits export of rod/hook‐type proteins before hook completion, whereupon it switches specificity to recognize filament‐type proteins. The cytoplasmic C‐terminal domain of FlhB (FlhBC) is cleaved between Asn‐269 and Pro‐270, defining two subdomains: FlhBCN and FlhBCC. Here, we show that subdomain interactions and cleavage within FlhB are central to substrate‐specificity switching. We found that deletions between residues 216 and 240 of FlhBCN permitted FlhB cleavage but abolished function, whereas a deletion spanning Asn‐269 and Pro‐270 abolished both. The mutation N269A prevented cleavage at the FlhBCN–FlhBCC boundary. Cells producing FlhB(N269A) exported the same amounts of hook‐capping protein as cells producing wild‐type FlhB. However, they exported no flagellin, even when the fliC gene was being expressed from a foreign promoter to circumvent regulation of expression by FlgM, which is itself a filament‐type substrate. Electron microscopy revealed that these cells assembled polyhook structures lacking filaments. Thus, FlhB(N269A) is locked in a conformation specific for rod/hook‐type substrates. With FlhB(P270A), cleavage was reduced but not abolished, and cells producing this protein were weakly motile, exported reduced amounts of flagellin and assembled polyhook filaments.
A LuxR‐type regulator from Agrobacterium tumefaciens elevates Ti plasmid copy number by activating transcription of plasmid replication genes
Authors: Pappas Katherine M. , Winans Stephen C.
Published: May 2003

TraR, a LuxR‐type quorum‐sensing transcription factor in Agrobacterium tumefaciens, activates genes required for conjugal transfer of the Ti plasmid and also enhances the copy number of a nopaline‐type Ti plasmid. Here, we show that TraR increases the copy number of an octopine‐type Ti plasmid up to eightfold and that TraR activates transcription of the repABC operon up to 25‐fold. The ability of TraR to increase copy number was strictly dependent on several TraR‐activated promoters of this operon, indicating that TraR affects copy number solely at the level of transcription. Promoter resections and mRNA transcript analysis revealed the presence of three TraR‐dependent promoters. Two TraR‐dependent transcription start sites are located 45.5 and 65.5 nucleotides downstream of a site called tra box II, whereas the third start site lies 42.5 nucleotides downstream of a site called tra box III. Purified TraR bound to both tra boxes with comparable affinities, causing moderate DNA bending. TraR bound and bent these two sites independently rather than synergistically. Alteration of tra box III to match the consensus sequence dramatically increased TraR‐dependent expression of repABC and plasmid copy number. TraR‐dependent elevation of Ti plasmid copy number caused a three‐ to fourfold increase in plant tumorigenesis.
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

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

The 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

We 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

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

The 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

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

Intra‐ 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

In 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

Expression 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

Cyclin‐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

The 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

Transgene 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.
Emergence of Chenopodium album and Stellaria media of different origins under different climatic conditions
Authors: Grundy A C , Peters N C B , Rasmussen I A , Hartmann K M , Sattin M , Andersson L , Mead A , Murdoch A J , Forcella F
Published: May 2003

Summary The emergence behaviour of weed species in relation to cultural and meteorological events was studied. Dissimilarities between populations in dormancy and germination ecology, between‐year maturation conditions and seed quality and burial site climate all contribute to potentially unpredictable variability. Therefore, a weed emergence data set was produced for weed seeds of Stellaria media and Chenopodium album matured and collected from three populations (Italy, Sweden and UK). The seeds were collected in two consecutive seasons (1999 and 2000) and subsequently buried in the autumn of the same year of maturation in eight contrasting climatic locations throughout Europe and the USA. The experiment sought to explore and explain differences between the three populations in their emergence behaviour. Evidence was demonstrated of synchrony in the timing of the emergence of different populations of a species at a given burial site. The relative magnitudes of emergence from the three populations at a given burial site in a given year were generally similar across all the burial sites in the study. The resulting data set was also used to construct a simple weed emergence model, which was tested for its application to the range of different burial environments and populations. The study demonstrated the possibility of using a simple thermal time‐based model to describe part of the emergence behaviour across different burial sites, seed populations and seasons, and a simple winter chilling relationship to adjust for the magnitude of the flush of emergence at a given burial site. This study demonstrates the possibility of developing robust generic models for simple predictions of emergence timing across populations.
Effect of Modified Eros Centre on Some Reproductive Parameters of the Sow
Authors: Molnar GB , Bilkei G
Published: May 2003

Contents

The 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.