bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

SummaryThe 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

SummaryMotion 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

SummaryCell 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

SummaryImmunodominant 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

SummaryThe 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

SummaryThe 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

SummaryAs 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

SummaryFusidic 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

SummaryAzurin 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

Summary 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

Summary 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

SummaryThe 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

SummaryThe 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.