bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of HasB, a Serratia marcescens TonB‐like protein specifically involved in the haemophore‐dependent haem acquisition system
Authors: Paquelin Annick , Ghigo Jean Marc , Bertin Stephane , Wandersman Cécile
Published: March 2003

In Gram‐negative bacteria, the TonB–ExbB–ExbD inner membrane multiprotein complex is required for active transport of diverse molecules through the outer membrane. We present evidence that Serratia marcescens, like several other Gram‐negative bacteria, has two TonB proteins: the previously characterized TonBSM, and also HasB, a newly identified component of the has operon that encodes a haemophore‐dependent haem acquisition system. This system involves a soluble extracellular protein (the HasA haemophore) that acquires free or haemoprotein‐bound haem and presents it to a specific outer membrane haemophore receptor (HasR). TonBSM and HasB are significantly similar and can replace each other for haem acquisition. However, TonBSM, but not HasB, mediates iron acquisition from iron sources other than haem and haemoproteins, showing that HasB and TonBSM only display partial redundancy. The reconstitution in Escherichia coli of the S. marcescens Has system demonstrated that haem uptake is dependent on the E. coli ExbB, ExbD and TonB proteins and that HasB is non‐functional in E. coli. Nevertheless, a mutation in the HasB transmembrane anchor domain allows it to replace TonBEC for haem acquisition. As the change affects a domain involved in specific TonBEC–ExbBEC interactions, HasB may be unable to interact with ExbBEC, and the HasB mutation may allow this interaction. In E. coli, the HasB mutant protein was functional for haem uptake but could not complement the other TonBEC‐dependent functions, such as iron siderophore acquisition, and phage DNA and colicin uptake. Our findings support the emerging hypothesis that TonB homologues are widespread in bacteria, where they may have specific functions in receptor–ligand uptake systems.
Efficient uptake of Yersinia pseudotuberculosis via integrin receptors involves a Rac1–Arp 2/3 pathway that bypasses N‐WASP function
Authors: Alrutz Michael A. , Srivastava Amit , Wong Ka‐Wing , D'Souza‐Schorey Crislyn , Tang May , Ch'Ng Lian‐Ee , Snapper Scott B. , Isberg Ralph R.
Published: March 2003

Efficient uptake of Yersinia pseudotuberculosis into cultured mammalian cells is the result of high‐affinity binding of invasin to β1 chain integrins. We demonstrate here that uptake requires Rac1 and Arp 2/3 function. Bacterial uptake was stimulated by GTPγS, but was inhibited in mammalian cells transfected with the interfering Rac1‐N17 derivative. Rac1 was found to be activated in response to integrin engagement by invasin, whereas Rac1 and Arp 2/3 were found to be intensely localized around phagosomes bearing bacteria, indicating a specific role for Rac1 signalling from the nascent phagosome to downstream effectors. To determine whether the Arp 2/3 complex was a component of this proposed pathway, cells overproducing various derivatives of Scar1/WAVE1, an Arp 2/3‐binding protein, were analysed. Sequestration of Arp 2/3 away from the phagocytic cup as a result of Scar1/WAVE1 overproduction dramatically inhibited uptake. To determine whether signalling from Rac1 to Arp 2/3 occurred via N‐WASP, uptake was analysed in a cell line lacking expression of WASP and N‐WASP. Uptake was unaffected by the absence of these proteins, indicating that β1 integrin signalling from Rac1 to Arp 2/3 can occur in the absence of N‐WASP function.
Systematic mutational analysis of the amino‐terminal domain of the Listeria monocytogenes ActA protein reveals novel functions in actin‐based motility
Authors: Lauer Peter , Theriot Julie A. , Skoble Justin , Welch Matthew D. , Portnoy Daniel A.
Published: March 2003

The Listeria monocytogenes ActA protein acts as a scaffold to assemble and activate host cell actin cytoskeletal factors at the bacterial surface, resulting in directional actin polymerization and propulsion of the bacterium through the cytoplasm. We have constructed 20 clustered charged‐to‐alanine mutations in the NH2‐terminal domain of ActA and replaced the endogenous actA gene with these molecular variants. These 20 clones were evaluated in several biological assays for phenotypes associated with particular amino acid changes. Additionally, each protein variant was purified and tested for stimulation of the Arp2/3 complex, and a subset was tested for actin monomer binding. These specific mutations refined the two regions involved in Arp2/3 activation and suggest that the actin‐binding sequence of ActA spans 40 amino acids. We also identified a ‘motility rate and cloud‐to‐tail transition’ region in which nine contiguous mutations spanning amino acids 165–260 caused motility rate defects and changed the ratio of intracellular bacteria associated with actin clouds and comet tails without affecting Arp2/3 activation. Several unusual motility phenotypes were associated with amino acid changes in this region, including altered paths through the cytoplasm, discontinuous actin tails in host cells and the tendency to ‘skid’ or dramatically change direction while moving. These unusual phenotypes illustrate the complexity of ActA functions that control the actin‐based motility of L. monocytogenes.
Cryptic plasmids of Mycobacterium avium: Tn552 to the rescue
Authors: Kirby Carolyn , Waring Al , Griffin Thomas J. , Falkinham Joseph O. , Grindley Nigel D. F. , Derbyshire Keith M.
Published: March 2003

Plasmids have been described in almost all bacterial species analysed and have proven to be essential genetic tools. In many bacteria these extrachro‐mosomal DNAs are cryptic with no known markers or function, which makes their characterization and genetic exploitation extremely difficult. Here we describe a system that will allow the rescue of any circular DNA (plasmid or phage) using an in vitro
transposition system to deliver both a selectable marker (kanamycin) and an Escherichia
coli plasmid origin of replication. In this study, we demonstrate the rescue
of four cryptic plasmids from the opportunistic pathogen Mycobacterium avium.
To evaluate the host range of the rescued plasmids, we have examined their ability
to be propagated in Mycobacterium smegmatis and Mycobacterium bovis BCG, and their compatibility with other mycobacterial plasmids. In addition, we use a library of transposon insertions to sequence one plasmid, pVT2, and to begin a genetic analysis of plasmid genes. Using this approach, we identified a putative conjugative relaxase, suggesting this myco‐bacterial plasmid is transferable, and three genes required for plasmid establishment and replication.
Functional complementation reveals the importance of intermolecular monomer interactions for Helicobacter pylori VacA vacuolating activity
Authors: Ye Dan , Blanke Steven R.
Published: March 2003

The Helicobacter pylori vacuolating cytotoxin (VacA) induces degenerative

vacuolation of sensitive mammalian cell lines. Although evidence is accumulating

that VacA enters cells and functions from an intracellular site of action, the biochemical

mechanism by which VacA mediates cellular vacuolation has not been established. In

this study, we used functional complementation and biochemical approaches to probe

the structure of VacA. VacA consists of two discrete fragments, p37 and p58, that

are both required for vacuolating activity. Using a transient transfection system,

we expressed genetically modified forms of VacA and identified mutations in either

p37 or p58 that inactivated the toxin. VacA with an inactivating single‐residue substitution

in the p37 domain [VacA (P9A)] functionally complemented a second mutant form of

VacA with an inactivating two‐residue deletion in the p58 domain [VacA Δ(346–347)].

VacA (P9A) and VacA Δ(346–347) also co‐immunoprecipitated from

vacuolated monolayers, supporting the hypothesis that these two inactive mutants

associate directly to function in trans. p37 and p58 interact directly when expressed as separate fragments within HeLa cells, suggesting that p37–p58 inter‐actions facilitate VacA monomer associations. Collectively, these results support a model in which the active form of VacA requires assembly into a complex of two or more monomers to elaborate toxin function.
The Sinorhizobium meliloti stringent response affects multiple aspects of symbiosis
Authors: Wells Derek H. , Long Sharon R.
Published: March 2003

Sinorhizobium meliloti and host legumes enter into a nitrogen‐fixing, symbiotic

relationship triggered by an exchange of signals between bacteria and plant. S.

meliloti produces Nod factor, which elicits the formation of nodules on plant

roots, and succinoglycan, an exopolysaccharide that allows for bacterial invasion

and colonization of the host. The biosynthesis of these molecules is well defined,

but the specific regu‐lation of these compounds is not completely understood. Bacteria

control complex regulatory networks by the production of ppGpp, the effector molecule

of the stringent response, which induces physiological change in response to adverse

growth conditions and can also control bacterial development and virulence. Through

detailed analysis of an S. meliloti mutant incapable of producing ppGpp, we

show that the stringent response is required for nodule formation and regulates the

production of succinoglycan. Although it remains unknown whether these phenotypes

are connected, we have isolated suppressor strains that restore both defects and

potentially identify key downstream regulatory genes. These results indicate that

the S. meliloti stringent response has roles in both succinoglycan production and nodule formation and, more importantly, that control of bacterial physiology in response to the plant and surrounding environment is critical to the establishment of a successful symbiosis.
Endonuclease cleavage of messenger RNA in Bacillus subtilis
Authors: Drider Djamel , DiChiara Jeanne M. , Wei Jin , Sharp Josh S. , Bechhofer David H.
Published: March 2003

A deletion derivative of the ermC gene was constructed that expresses a

254‐nucleotide mRNA. The small size of this mRNA facilitated the detection of processing

products that did not differ greatly in size from the full‐length transcript. In

the presence of erythromycin, which induces ribosome stalling near the 5′

end of ermC mRNA, the 254‐nucleotide mRNA was cleaved endonucleolytically

at the site of ribosome stalling. Only the downstream product of this cleavage was

detectable; the upstream product was apparently too unstable to be detected. The

downstream cleavage product accumulated at times after rifampicin addition, suggesting

that the stalled ribosome at the 5′ end conferred stability to this

RNA fragment. Neither Bs‐RNase III nor RNase M5, the two known narrow‐specificity

endoribonucleases of Bacillus subtilis, was responsible for this cleavage.

These results indicate the presence in B. subtilis of another specific endoribonuclease, which may be ribosome associated.
HSF and Msn2/4p can exclusively or cooperatively activate the yeast HSP104 gene
Authors: Grably Melanie R. , Stanhill Ariel , Tell Osnat , Engelberg David
Published: March 2003

In an effort to understand how an accurate level of stress‐specific expression is obtained, we studied the promoter of the yeast HSP104 gene. Through 5′ deletions, we defined a 334 bp fragment upstream of the first coding AUG as sufficient and essential for maximal basal activity and a 260 bp fragment as sufficient and essential for heat shock responsiveness. These sequences contain heat shock elements (HSEs) and stress response elements (STREs) that cooperate to achieve maximal inducible expression. However, in the absence of one set of factors (e.g. in msn2Δmsn4Δ cells) proper induction is obtained exclusively through HSEs. We also show that HSP104 is constitutively derepressed in ras2Δ cells. This derepression is achieved exclusively through activation of STREs, with no role for HSEs. Strikingly, in ras2Δmsn2Δmsn4Δ cells the HSP104 promoter is also derepressed, but in this strain derepression is mediated through HSEs, showing the flexibility and adaptation of the promoter. Thus, appropriate transcription of HSP104 is usually obtained through cooperation between the Msn2/4/STRE and the HSF/ HSE systems, but each factor could activate the promoter alone, backing up the other. Transcription control of HSP104 is adaptive and robust, ensuring proper expression under extreme conditions and in various mutants.
Expression, mutagenesis and kinetic analysis of recombinant K1E endosialidase to define the site of proteolytic processing and requirements for catalysis
Authors: Leggate Daniel R. , Bryant J. Mark , Redpath Maria B. , Head Denise , Taylor Peter W. , Luzio J. Paul
Published: March 2003

Catalytically active, recombinant fusion proteins of bacteriophage E endosialidase were expressed and purified fromEscherichia coli. Constructs with different fusion partners added to the amino terminus of the endosialidase were enzymatically active. A post‐translational proteolytic cleavage was shown to occur between serine 706 and aspartate 707 to generate the 76 kDa mature enzyme from the 90 kDa translation product. Endosialidase truncated at the C‐terminus from aspartate 707 was observed to have the same 76 kDa molecular weight as wild‐type enzyme using denaturing SDS–PAGE but, under native PAGE conditions, was not observed to form the≈250 kDa trimeric wild‐type enzyme, implying that the C‐terminus of the enzyme may be required for correct assembly of active trimer, rather than as part of the active site as has been previously suggested. Mutagenesis of aspartate 138 to alanine greatly reduced enzyme activity whereas conversion of other selected aspartate residues to alanine had less effect, consistent with similarities between the structure and cata‐lytic mechanism of bacteriophage E endosialidase and those of exosialidases.
Pyruvate kinase fromChlamydia trachomatisis activated by fructose‐2,6‐bisphosphate
Authors: Iliffe‐Lee Emma R. , McClarty Grant
Published: March 2003

Pyruvate kinase is the final regulatory point in the catabolic Embden–Meyerhoff–Parnas pathway, which controls the carbon flux of glycolytic intermediates and regulates the level of ATP in the cell. In a previous study, we identified, cloned and sequenced pyruvate kinase from the obligate intracellular bacteriumChlamydia trachomatisand demonstrated that the enzyme was active in crude extract. Here, we report the kinetic properties of highly purifiedC. tracho‐matispyruvate kinase. The results indicate thatC. trachomatispyruvate kinase is 53.5 kDa with a pH optima of 7.3. Kinetic studies show thatC. trachomatispyruvate kinase requires both K+and Mg2+ions for activity, exhibits sigmoidal kinetics with respect to phosphoenolpyruvate and Michaelis–Menten kinetics with respect to ADP. In addition,C. trachomatispyruvate kinase is able to use alternative nucleoside diphosphates as phosphate acceptors, although it shows the greatest activity with ADP. In contrast to other bacterial pyruvate kinases that are activated by AMP, our data show that AMP, in addition to ATP and GTP, inhibitsC. trachomatispyruvate kinase. Surprisingly, unlike any other known bacterial pyruvate kinase,C. trachomatispyruvate kinase was alloste‐rically activated by fructose‐2,6‐bisphosphate, an important regulatory metabolite that has only been reported in eukaryotes.
Bacterial conjugative transfer: visualization of successful mating pairs and plasmid establishment in live Escherichia coli
Authors: Lawley Trevor D. , Gordon G. Scott , Wright Andrew , Taylor Diane E.
Published: March 2003

We used the LacO/GFP–LacI system to label and visualize the IncPβ plasmid R751 fluorescently during conjugative transfer between live donor and recipient bacteria. Comparisons of R751 in conjugative and non‐conjugative conditions have allowed us to identify key localizations and movements associated with the initiation of conjugative transfer in the donor and the establishment of R751 in the recipient. A survey of successful mating pairs demonstrates that close physical contact between donor and recipient bacteria is required for DNA transfer and that regions of intimate contact can occur at any location on the donor or recipient cell membrane. The transferred DNA is positioned at the characteristic centre or quarter‐cell position after conversion to a double‐stranded molecule in the recipient cell. Initial duplication of plasmids often results in an asymmetric distribution of plasmid foci. Symmetric localization (either at centre or at 1/4 and 3/4 cell lengths) occurs only after a significant lag, presumably reflecting the time required to synthesize the plasmid‐encoded partitioning proteins.
Structural characterization of lipo‐oligosaccharide (LOS) from Yersinia pestis: regulation of LOS structure by the PhoPQ system
Authors: Hitchen Paul G. , Prior Joann L. , Oyston Petra C. F. , Panico Maria , Wren Brendan W. , Titball Richard W. , Morris Howard R. , Dell Anne
Published: March 2003

The two‐component regulatory system PhoPQ has been shown to regulate the expression of virulence factors in a number of bacterial species. For one such virulence factor, lipopolysaccharide (LPS), the PhoPQ system has been shown to regulate structural modifications in Salmonella enterica var Typhi‐murium. In Yersinia pestis,
which expresses lipo‐oligosaccharide (LOS), a PhoPQ regulatory system has been identified
and an isogenic mutant constructed. To investigate potential modifications to LOS
from Y. pestis, which to date has not been fully characterized, purified LOS
from wild‐type plague and the phoP defective mutant were analysed by mass
spectrometry. Here we report the structural characterization of LOS from Y. pestis
and the direct comparison of LOS from a phoP mutant. Structural modifications
to lipid A, the host signalling portion of LOS, were not detected but analysis of
the core revealed the expression of two distinct molecular species in wild‐type LOS,
differing in terminal galactose or heptose. The phoP mutant was restricted
to the expression of a single molecular species, containing terminal heptose. The
minimum inhibitory concentration of cationic antimicrobial peptides for the two strains
was determined and compared with the wild‐type: the phoP mutant was highly
sensitive to polymyxin. Thus, LOS modification is under the control of the PhoPQ
regulatory system and the ability to alter LOS structure may be required for survival
of Y. pestis within the mammalian and/or flea host.
‘The glideosome’: a dynamic complex powering gliding motion and host cell invasion by Toxoplasma gondii
Authors: Opitz Corinna , Soldati Dominique
Published: March 2003

Motion is an intrinsic property of all living organisms, and each cell displays a variety of shapes and modes of locomotion. How structural proteins support cellular movement and how cytoskeletal dynamics and motor proteins are harnessed to generate order and movement are among the fundamental and not fully resolved questions in biology today. Protozoan parasites belonging to the Apicomplexa are of enormous medical and veterinary significance, being responsible for a wide variety of diseases in human and animals, including malaria, toxoplasmosis, coccidiosis and cryptosporidiosis. These obligate intracellular parasites exhibit a unique form of actin‐based gliding motility, which is essential for host cell invasion and spreading of parasites throughout the in‐fected hosts. A motor complex composed of a small myosin of class XIV associated with a myosin light chain and a plasma membrane‐docking protein is present beneath the parasite's plasma membrane. According to the capping model, this complex is connected directly or indirectly to transmembrane adhesin complexes, which are delivered to the parasite surface upon microneme secretion. Together with F‐actin and as yet unknown bridging molecules and proteases, these complexes are among the structural and functional components of the ‘glideosome’.
Disruption of msl3 abolishes the synthesis of mycolipanoic and mycolipenic acids required for polyacyltrehalose synthesis in Mycobacterium tuberculosis H37Rv and causes cell aggregation
Authors: Dubey Vinod S. , Sirakova Tatiana D. , Kolattukudy P. E.
Published: March 2003

Cell wall lipids of Mycobacterium tuberculosis containing multiple methylbranched fatty acids play critical roles in pathogenesis and thus offer targets for new antimycobacterial drugs. Mycocerosic acid synthase gene (mas) encodes the enzyme that produces one class of such acids. Seven mas‐like genes (msls) were identified in the genome. One of them, msl3, originally annotated as two separate genes, pks 3 and pks 4, is now shown to constitute a single open reading frame, which encodes a 220.3 kDa protein. Msl3 was disrupted using a phage mediated delivery system and the gene replacement in the mutant was confirmed by polymerase chain reaction analysis of the flanking regions of the introduced disrupted gene and by Southern analysis. Biochemical analysis showed that the msl3 mutant does not produce mycolipanoic acids and mycolipenic (phthienoic) acids, the major constituents of polyacyl trehaloses and thus lacks this cell wall lipid, but synthesizes all of the other classes of lipids. The absence of the major acyl chains that anchor the surface‐exposed acyltrehaloses causes a novel growth morphology; the cells stick to each other, most probably via the intercellular interaction between the exposed hydrophobic cell surfaces, manifesting a bead‐like growth morphology without affecting the overall growth rate.
Activation of a p44 pseudogene in Anaplasma phagocytophila by bacterial RNA splicing: a novel mechanism for post‐transcriptional regulation of a multigene family encoding immunodominant major outer membrane proteins
Authors: Zhi Ning , Ohashi Norio , Rikihisa Yasuko
Published: March 2003

Immunodominant 44 kDa major outer membrane proteins of Anaplasma phagocytophila (human granulocytic ehrlichiosis agent) are encoded by the p44 multigene family. One of the paralogues, p44–18 is predominantly expressed by A. phagocytophila in mammalian hosts, but is downregulated in the arthropod vector. The expression of p44–18 was upregulated in A. phagocytophila cultivated in HL‐60 cells at 37°C compared with 24°C. However, the molecular mechanism of such gene expression was unclear, as p44–18 has a pseudogene‐like structure, i.e. it lacks an AUG start codon and is out of frame with an upstream overlapping paralogue, p44–1. In the present study, we found that an amplicon detected by reverse transciption‐polymerase chain reaction (RT‐PCR) [808 basepair (bp)] for the p44–1/p44–18 gene locus was smaller than that detected by PCR with the genomic DNA (1652 bp) in the A. phagocytophila‐infected HL‐60 cells cultured at 37°C. A circularized RNA molecule corresponding to the 844 bp region missing from the locus in the RT‐PCR product was detected by inverse RT‐PCR, indicating that this is an intron (designated p44–1 intervening sequence, p44–1 IVS). The splicing event of p44–1 IVS was also observed when the p44–1 IVS‐carrying plasmid was introduced into Escherichia coli, suggesting that the splicing is sequence‐dependent. Structural analysis and in vitro splicing experiments of p44–1 IVS suggested that this is likely to represent a new class of introns in eubacteria. The primer extension analysis showed the presence of a putative σ32‐type promoter in region upstream from p44–1. Collectively, the novel RNA splicing and the temperature‐dependent transcription may account for the dominant p44–18 expression in mammals.
J‐binding protein increases the level and retention of the unusual base J in trypanosome DNA
Authors: Cross Mike , Kieft Rudo , Sabatini Robert , Dirks‐Mulder Anita , Chaves Inês , Borst Piet
Published: March 2003

The nuclear DNA of Trypanosoma brucei and other kinetoplastid flagellates contains the unusual base β‐d‐glucosyl‐hydroxymethyluracil, called J, replacing part of the thymine in repetitive sequences. We have described a 100 kDa protein that specifically binds to J in duplex DNA. We have now disrupted the genes for this J‐binding protein (JBP) in T. brucei. The disruption does not affect growth, gene expression or the stability of some repetitive DNA sequences. Unexpectedly, however, the JBP KO trypanosomes contain only about 5% of the wild‐type level of J in their DNA. Excess J, randomly introduced into T. brucei DNA by growing the cells in the presence of the J precursor 5‐hydroxymethyldeoxyuridine, is lost by simple dilution as the KO trypanosomes multiply, showing that JBP does not protect J against removal. In contrast, cells containing JBP lose excess J only sluggishly. We conclude that JBP is able to activate the thymine modification enzymes to introduce additional J in regions of DNA already containing a basal level of J. We propose that JBP is a novel DNA modification maintenance protein.
Generation of conditional lethal Candida albicans mutants by inducible deletion of essential genes
Authors: Michel Sonja , Ushinsky Sophia , Klebl Bert , Leberer Ekkehard , Thomas David , Whiteway Malcolm , Morschhäuser Joachim
Published: March 2003

The yeast Candida albicans is the most important fungal pathogen of humans and a model organism for studying fungal virulence. Sequencing of the C. albicans genome will soon be completed, allowing systematic approaches to analyse gene function. However, techniques to define and characterize essential genes in this permanently diploid yeast are limited. We have developed an efficient method to create conditional lethal C. albicans null mutants by inducible, FLP‐mediated gene deletion. Both wild‐type alleles of the CDC42 or the BEM1 gene were deleted in strains that carried an additional copy of the respective gene that could be excised from the genome by the site‐specific recombinase FLP. Expression of a C. albicans‐adapted FLP gene under the control of an inducible promoter generated cell populations consisting of ≥99.9% null mutants. Upon plating, these cells were unable to form colonies, demonstrating that CDC42 and BEM1 are essential genes in C. albicans. The cdc42 null mutants failed to produce buds and hyphae and grew as large, round cells instead, suggesting that they lacked the ability to produce polarized cell growth. However, the cells still responded to hyphal inducing signals by ag‐gregating and expressing hypha‐specific genes, behaviours typical of the mycelial growth form of C. albicans. Budding cells and germ tubes of bem1 null mutants exhibited morphological abnormalities, demonstrating that BEM1 is essential for normal growth of both yeast and hyphae. Inducible, FLP‐mediated gene deletion provides a powerful approach to generate conditional lethal C. albicans mutants and allows the functional analysis of essential genes.
Ralstonia solanacearum requires type 4 pili to adhere to multiple surfaces and for natural transformation and virulence
Authors: Kang Yaowei , Liu Huanli , Genin Stéphane , Schell Mark A. , Denny Timothy P.
Published: March 2003

As reported previously for Ralstonia solanacearum strain GMI1000, wild‐type strains AW1 and K60 were shown to produce Hrp pili. AW1 and K60 mutants lacking Hrp pili still exhibited twitching motility, which requires type 4 pili (Tfp), and electron microscopy revealed that they still made flexuous polar pili. Twitching‐positive cells had an extracellular 17 kDa protein that was associated with piliation, and an internal 43‐amino‐acid sequence of this protein was typical of type 4 pilins. This amino acid sequence is encoded by an open reading frame, designated pilA, in the genomic sequence of GMI1000. PilA is 46% identical to a Pseudomonas aeruginosa type 4 pilin over its entire length and has all the conserved residues and motifs characteristic of type 4 group A pilins. pilA mutants did not make the 17 kDa PilA protein and did not exhibit twitching motility. When compared with its parent, an AW1 pilA mutant was reduced in virulence on tomato plants and in autoaggregation and biofilm formation in broth culture. Unlike AW1, a pilA mutant did not exhibit polar attachment to tobacco suspension culture cells or to tomato roots; it was also not naturally competent for transformation. We reported previously that twitching motility ceases in maturing AW1 colonies and that inactivation of PhcA, a global transcriptional regulator, results in colonies that continue to exhibit twitching motility. Similarly, in broth culture, expression of a pilA::lacZ fusion in AW1 decreased 10‐fold at high cell density, but expression remained high in a phcA mutant. In addition, pilA::lacZ expression was positively regulated 10‐fold by PehR, a response regulator that is known to be repressed by PhcA. This signal cascade is sufficient to explain why pilA expression, and thus twitching motility, decreases at high cell densities.
Molecular analysis of fusidic acid resistance in Staphylococcus aureus
Authors: Besier Silke , Ludwig Albrecht , Brade Volker , Wichelhaus Thomas A.
Published: March 2003

Fusidic acid is a potent antibiotic against severe Gram‐positive infections that interferes with the function of elongation factor G (EF‐G), thereby leading to the inhibition of bacterial protein synthesis. In this study, we demonstrate that fusidic acid resistance in Staphylococcus aureus results from point mutations within the chromosomal fusA gene encoding EF‐G. Sequence analysis of fusA revealed mutational changes that cause amino acid substitutions in 10 fusidic acid‐resistant clinical S. aureus strains as well as in 10 fusidic acid‐resistant S. aureus mutants isolated under fusidic acid selective pressure in vitro. Fourteen different amino acid exchanges were identified that were restricted to 13 amino acid residues within EF‐G. To confirm the importance of observed amino acid exchanges in EF‐G for the generation of fusidic acid resistance in S. aureus, three mutant fusA alleles encoding EF‐G derivatives with the exchanges P406L, H457Y and L461K were constructed by site‐directed mutagenesis. In each case, introduction of the mutant fusA alleles on plasmids into the fusidic acid‐susceptible S. aureus strain RN4220 caused a fusidic acid‐resistant phenotype. The elevated minimal inhibitory concentrations of fusidic acid determined for the recombinant bacteria were analogous to those observed for the fusidic acid‐resistant clinical S. aureus isolates and the in vitro mutants containing the same chromosomal mutations. Thus, the data presented provide evidence for the crucial importance of individual amino acid exchanges within EF‐G for the generation of fusidic acid resistance in S. aureus.
Induction of apoptosis in macrophages by Pseudomonas aeruginosa azurin: tumour‐suppressor protein p53 and reactive oxygen species, but not redox activity, as critical elements in cytotoxicity
Authors: Goto Masatoshi , Yamada Tohru , Kimbara Kazuhide , Horner John , Newcomb Martin , Gupta T. K. Das , Chakrabarty A. M.
Published: March 2003

Azurin is a copper‐containing protein involved in electron transfer during denitrification. We reported recently that purified azurin demonstrates cytotoxicity to macrophages by forming a complex with the tumour‐suppressor protein p53, thereby stabilizing it and enhancing its function as an inducer of proapoptotic activity (Yamada, T., Goto, M., Punj, V., Zaborina, O., Kimbara, K., Das Gupta, T. K., and Chakrabarty, A. M. 2002, Infect Immun70: 7054–7062). It is, however, not known whether the oxidoreductase (redox) activity of azurin or the involvement of copper is important for its cytotoxicity. We have isolated apo‐azurin devoid of copper and site‐directed mutants that are redox negative because of either replacement of a cysteine residue (Cys‐112) involved in co‐ordination with copper or mutational replacement of two methionine residues (Met‐44 and Met‐64) that are present in the hydrophobic patch of azurin and allow interaction of azurin with its redox partner cytochrome c551. We demonstrate that, although the wild type (wt) and the Cys‐112 Asp mutant azurin can form complexes with the tumour‐suppressor protein p53 and generate high levels of reactive oxygen species (ROS), the redox‐negative Met‐44LysMet‐64Glu mu‐tant azurin is defective in complex formation with p53, generates low levels of ROS and lacks appreciable cytotoxicity towards macrophages. Thus, complex formation with p53 and ROS generation, rather than azurin redox activity, are important in the cytotoxic action of azurin towards macrophages.
Characterization of potassium transport in wild‐type and isogenic yeast strains carrying all combinations of trk1, trk2 and tok1 null mutations
Authors: Bertl Adam , Ramos José , Ludwig Jost , Lichtenberg‐Fraté Hella , Reid John , Bihler Hermann , Calero Fernando , Martínez Paula , Ljungdahl Per O.
Published: March 2003

Saccharomyces cerevisiae
cells express three defined potassium‐specific transport systems en‐coded by
TRK1
,
TRK2
and
TOK1
. To gain a more complete understanding of the physiological function of these transport proteins, we have constructed a set of isogenic yeast strains carrying all combinations of
trk1
Δ,
trk2
Δ and
tok1
Δ null mutations. The
in vivo
K
+
transport characteristics of each strain have been documented using growth‐based assays, and the
in vitro
biochemical and electrophysiological properties associated with K
+
transport have been determined. As has been reported previously, Trk1p and Trk2p facilitate high‐affinity potassium uptake and appear to be functionally redundant under a wide range of environmental conditions. In the absence of
TRK1
and
TRK2
, strains lack the ability specifically to take up K
+
, and
trk1
Δ
trk2
Δ double mutant cells depend upon poorly understood non‐specific cation uptake mechanisms for growth. Under conditions that impair the activity of the non‐specific uptake system, termed NSC1, we have found that the presence of functional Tok1p renders cells sensitive to Cs
+
. Based on this finding, we have established a growth‐based assay that monitors the
in vivo activity of Tok1p.
Translocation of YopE and YopN into eukaryotic cells by Yersinia pestis yopN, tyeA, sycN, yscB and lcrG deletion mutants measured using a phosphorylatable peptide tag and phosphospecific antibodies
Authors: Day James B. , Ferracci Franco , Plano Gregory V.
Published: March 2003

Yersinia pestis
, the causative agent of plague, exports a set of virulence proteins called Yops upon contact with eukaryotic cells. A subset of these Yops is translocated directly into the cytosol of host cells. In this study, a novel protein tag‐based reporter system is used to measure the translocation of Yops into cultured eukaryotic cells. The reporter system uses a small bipartite phosphorylatable peptide tag, termed the Elk tag. Translocation of an Elk‐tagged protein into eukaryotic cells results in host cell protein kinase‐dependent phosphorylation of the tag at a specific serine residue, which can subsequently be detected with phosphospecific antibodies. The YopN, TyeA, SycN, YscB and LcrG proteins function to prevent Yop secretion before host cell contact. The role of these proteins was investigated in the translocation of Elk‐tagged YopE (YopE
129
–Elk) and YopN (YopN
293
–Elk) into HeLa cells.
Y. pestis yopN
,
tyeA
,
sycN
and
yscB
deletion mutants showed reduced levels of YopE
129
–Elk phosphorylation compared with the parent strain, indicating that these mutants translocate reduced amounts of YopE. We also demonstrate that YopN
293
–Elk is translocated into HeLa cells and that this process is more efficient in a
Yersinia yop
polymutant strain lacking the six translocated effector Yops.
Y. pestis sycN
and
yscB
mutants translocated reduced amounts of YopN
293
–Elk; however,
tyeA
and
lcrG
mutants translocated higher amounts of YopN
293 –Elk compared with the parent strain. These data suggest that TyeA and LcrG function to suppress the secretion of YopN before host cell contact, whereas SycN and YscB facilitate YopN secretion and subsequent translocation.
Characterization of the vls antigenic variation loci of the Lyme disease spirochaetes Borrelia garinii Ip90 and Borrelia afzelii ACAI
Authors: Wang Dachun , Botkin Douglas J. , Norris Steven J.
Published: March 2003

The vls locus of Borrelia burgdorferi B31 consists of 15 silent cassettes and one expression site (vlsE), and the presence of the encoding plasmid lp28‐1 correlates with high infectivity. Recombination between the expression cassette and silent cassettes occurs in vivo, and this process may enable B. burgdorferi to evade the immune response. To determine the characteristics of the vls loci in other Borrelia strains, we have cloned and characterized the vls silent cassette loci of Borrelia garinii Ip90 and Borrelia afzelii ACAI, consisting of 11 vls silent cassettes and 14 vls silent cassettes respectively. The silent cassettes share 90–97% nucleotide sequence identity with one another within the Ip90 vls locus and 84–91% within the ACAI vls locus. In both organisms, the silent cassettes resemble the B31 Vls sequences in overall amino acid similarity (50–65%) and in the presence of six variable regions interspersed between six relatively invariant regions. The vlsE expression sites of these two strains have not been isolated, but transcripts of vlsE were detected by reverse transcriptase‐polymerase chain reaction for both Ip90 and ACAI. In addition, the occurrence of sequence variation within the vlsE cassette region of these transcripts was verified. This study indicates that the vls loci present in B. garinii Ip90 and B. afzelii ACAI have characteristics similar to those found in B. burgdorferi B31.
The Leopoldina international symposium on parasitism, commensalism and symbiosis – common themes, different outcome
Authors: Gross Roy , Hacker Jörg , Goebel Werner
Published: March 2003

The development of new methods, including genomics, which can even be applied to unculturable microorganisms, has significantly increased our knowledge about bacterial pathogenesis and symbiosis and, in consequence, is profoundly modifying our views on the evolution and the genetic and physiological basis of bacteria–host interactions. The presentations at this symposium revealed conceptual links between bacterial pathogenesis and symbiosis. The close co‐operation of experts in both fields will result in significant synergy and new insights into basic mechanisms of bacteria–host interactions and their evolution. The meeting provided fascinating news about the genetic and metabolic consequences that the change in their lifestyle had for bacteria that developed from free‐living to permanent host‐associated organisms exemplified by intracellular pathogens or symbionts. In addition, surprising similarities but also striking differences between the strategies involved in the establishment of a symbiotic versus a parasitic lifestyle can be noted. In the long run, the characterization of such differences might lead to lifestyle prediction or to an evaluation of the pathogenic potential of newly isolated bacteria via the definition of genetic and/or metabolic signatures characteristic for pathogenic or symbiotic organisms. Moreover, it is expected that these investigations will lead to new strategies for the treatment or prevention of bacterial infections, or the avoidance of pathogen transmission.
Gastric myoelectrical and antroduodenal motor activity in patients with achalasia
Authors: Verhagen M. A. M. T. , Samsom M. , Smout A. J. P. M.
Published: March 2003

Achalasia is a primary motor disorder of the oesophagus, in which the myenteric plexus is involved. However, abnormalities in other parts of the digestive tract have also been described in achalasia. Whether gastric myoelectrical and duodenal motor activity in these patients is also affected is unknown. Therefore, interdigestive and postprandial gastric myoelectrical and antroduodenal motor activity were studied in 11 patients with achalasia, using electrogastrography (EGG) and stationary antroduodenal manometry.

Electrogastrographically, no differences were found in the gastric frequency, incidence of dysrhythmias and postprandial/fasting power ratio. In the interdigestive state a lower propagation velocity of phase III episodes was found in the achalasia patients, but other parameters were unaltered. Postprandially, no differences were found in the number of pressure waves, in the amplitude of pressure waves or in antro‐duodenal coordination.

We conclude that gastric myoelectrical activity and antral motor activity in patients with achalasia is normal, suggesting an intact extrinsic and intrinsic neural innervation of the distal stomach. Although postprandial duodenal motility is normal, a lower propagation velocity of phase III suggests involvement of the small intestine in achalasia.
Postprandial peristalsis in the human duodenum
Authors: Castedal M. , Björnsson E. , Abrahamsson H.
Published: March 2003

MMC‐related retroperistalsis is a cyclical phenomenon in the duodenum linked to phase III. The aim of this study was to elucidate the direction of propagation of juxtapyloric duodenal pressure waves in the postprandial state in healthy humans and to compare with the contractions in the interdigestive phase II. Antroduodenal manometry was performed in 11 healthy subjects. Individual pressure waves propagating along a 6‐cm duodenal segment were analysed with respect to the proportions of antegrade and retrograde propagation in the four duodenal subsegments (D1–D2) to (D4–D5), each subsegment being 15 mm. A test meal was given 30 min after a phase III had passed and motility recording continued for 60 min after the meal. During both the first and the second 30‐min period of postprandial recording the proportion of retrograde pressure waves was larger just distal to the pylorus, (D1–D2), 40% (23–68) and 50% (23–68), respectively, compared to the distal part, (D4–D5), of the duodenal segment, 29% (12–30) and 10%(10–24), respectively (P < 0.05 and 0.01). In contrast, during late phase II of the interdigestive state antegrade pressure waves predominated in all four duodenal subsegments. We conclude that in the postprandial state a high proportion of the duodenal pressure waves (40–50%) is retrograde in the immediate juxtapyloric area while antegrade contractions predominate at a distance 5–6 cm distal to the pylorus. These manometric data together with recent observations of postprandial transpyloric liquid flow, indicate that retrograde duodenogastric propelling of contents may be an important determinant for the gastric emptying rate.
Perirectal graciloplasty: a novel surgical technique for the treatment of the neurogenic rectum and rectal inertia
Authors: SHAFIK
Published: March 2003

A novel technique for evacuation of the denervated rectum was carried out on 11 mongrel dogs (mean weight 15.8 ± 4.6 kg). The gracilis muscle was mobilized from the thigh, pedicled on its origin from the pubic ramus, and was introduced into the ischiorectal fossa through a para‐anal incision. The abdomen was opened and the rectum mobilized and wrapped with the gracilis muscle which was pulled through the ischiorectal fossa into the pelvis. Rectal denervation was induced by bilateral pelvic ganglionectomy; cystostomy was performed owing to the resultant vesical denervation. Two electrodes were sutured to the gracilis muscle and connected to a myostimulator. The effect of electrostimulation of the gracilis muscle on the rectal and rectal neck (anal canal) pressures and on balloon expulsion was determined. Gracilis muscle stimulation with a burst of 30 Hz during 300 msec with a stimulus of 0.5 msec and an intensity just suprathreshold effected a rectal pressure rise (P < 0.001) with no rectal neck pressure change (P > 0.05); balloon expulsion occurred at a mean balloon filling of 14.8 ± 3.2 mL. Gracilis muscle transposition could be a suitable experimental model for evacuation of the neurogenic rectum. The technique might be considered for the treatment of the neurogenic rectum, or rectal inertia.
Discrimination of irritable bowel syndrome by non‐linear analysis of 24‐h jejunal motility
Authors: WACKERBAUER  , SCHMIDT  , WIDMER  , PFEIFFER  , MORFILL  , KAESS
Published: March 2003

Conventional analysis of ambulatory long‐term manometry of the small intestine has revealed abnormalities in patients with the irritable bowel syndrome (IBS). The aim was to use methods from non‐linear dynamics, in particular the concepts of symbolic dynamics and entropy, in order to discriminate motility in IBS from healthy subjects. 24‐h jejunal motility was recorded in 30 IBS patients and 30 healthy subjects. Computerized analysis of contraction amplitudes and intercontractile intervals was performed. Both time series were analysed by symbolic dynamics and entropy to quantify the degree of randomness inherent in the signal. During phase II IBS patients exhibited significantly increased entropies for the intercontractile intervals (IBS: 0.978 ± 0.016, C: 0.965 ± 0.020, P = 0.004) and for the contraction amplitudes (IBS: 0.978 ± 0.008, C: 0.970 ± 0.026, P = 0.05) compared with healthy subjects. In addition, a significantly increased mean contraction amplitude (mmHg) was found in patients (IBS: 25.2 ± 4.8, C: 22.2 ± 3.1, P = 0.003). Considering a combination of both parameters, a diagnostic accuracy of 82% was achieved. We conclude that phase II motility of the small intestine is more random in IBS than in health. Symbolic dynamics seems to be a promising new concept for the analysis of long‐term gastrointestinal motility recordings.
Structural relationships between immune cells and longitudinal muscle during a Trichinella spiralis infection in the rat intestine
Authors: BEREZIN  , HUIZINGA  , COLLINS
Published: March 2003

A Trichinella spiralis infection produces an acute inflammatory reaction and tissue damage in the mucosa, and, in addition, functional changes occur in the external muscle layers. The aim of the present study was to characterize structural changes in the musculature that occur during early infection, and to identify relationships between immune cells and muscle cells, as part of an ongoing investigation into the immune modulation of motor function in the gut. Rats were infected with T. spiralis larvae and the gut fixed at 12 h, 24 h, 48 h and 6 days post‐infection for electron microscopy of the longitudinal muscle. Macrophages and lymphocytes penetrated the longitudinal musculature 12–24 h post‐infection. Distinct contacts were observed between specific cell types; cellular protrusions from macrophages or lymphocytes made close apposition contacts with smooth muscle cells. Resident macrophages in the subserosal space, fibroblast‐like cells as well as smooth muscle cells showed marked activation during inflammation. Fibroblast‐like cells were frequently seen intercalated between lymphocytes and smooth muscle cells, hence they may mediate communication between immune cells and the musculature.
Effect of an o‐raffinose cross‐linked haemoglobin product on oesophageal and lower oesophageal sphincter motor function
Authors: Xue  , Paterson  , Valdez  , Miller  , Christoff  , Wong  , Diamant
Published: March 2003

The present experiments evaluate the effects on oesophageal motility of an o‐raffinose cross‐linked haemoglobin‐based oxygen carrier (HBOC) purified from outdated donated human blood cells (HemolinkTM), with attention to dose–response (0.6–2.4 g kg–1), oxygenation status and low molecular weight components (4.4–36.4% 64 kDa or less). In ketamine‐anaesthetized cats, lower oesophageal sphincter (LES) function and oesophageal peristalsis were monitored 0.5 h before, during and up to 3.5 h after HBOC infusion, and in some cats at 24 h. (1) All products significantly inhibited LES relaxation and increased peristaltic velocity in the distal smooth muscle oesophagus, without consistently altering resting LES pressure. (2) Effects on peristaltic velocity reached a maximum at the smallest dose, whereas the effects on LES relaxation had a maximum effect at 1.2 g kg–1. (3) Effects were not significantly altered by the haemoglobin oxygenation status or presence of low molecular weight components. (4) Repetitive oesophageal contractions occurred.

In the cat, an o‐raffinose cross‐linked human haemoglobin product produces changes in oesophageal body and LES function, which are independent of the HBOC oxygenation status and composition of the low molecular weight components tested. Changes may persist for at least 24 h. These motility changes are likely due to scavenging of nitric oxide by the haemoglobin.