bluuebunny/crossref_metadata_2025 huggingface dataset:
Amylase-binding proteins A (AbpA) and B (AbpB) differentially affect colonization of rats' teeth by Streptococcus gordonii
Authors: Tanzer J. M. , Grant L. , Thompson A. , Li L. , Rogers J. D. , Haase E. M. , Scannapieco F. A.
Published: August 2003

Streptococcus gordonii produces two α-amylase-binding proteins, AbpA and AbpB, that have been extensively studied in vitro. Little is known, however, about their significance in oral colonization and cariogenicity (virulence). To clarify these issues, weanling specific pathogen-free Osborne-Mendel rats, TAN : SPFOM(OM)BR, were inoculated either with wild-type strains FAS4-S or Challis-S or with strains having isogenic mutations of abpA, abpB, or both, to compare their colonization abilities and persistence on the teeth. Experiments were done with rats fed a sucrose-rich diet containing low amounts of starch or containing only starch. The mutants and wild-types were quantified in vivo and carious lesions were scored. In 11 experiments, S. gordonii was a prolific colonizer of the teeth when rats were fed the sucrose (with low starch)-supplemented diet, often dominating the flora. Sucrose-fed rats had several-fold higher recoveries of inoculants than those eating the sucrose-free, starch-supplemented diet, regardless of inoculant type. The strain defective in AbpB could not colonize teeth of starch-only-eating rats, but could colonize rats if sucrose was added to the diet. Strains defective in AbpA surprisingly colonized better than their wild-types. A double mutant deficient in both AbpA and AbpB (abpA/abpB) colonized like its wild-type. Wild-types FAS4-S and Challis-S had no more than marginal cariogenicity. Notably, in the absence of AbpA, cariogenicity was slightly augmented. Both the rescue of colonization by the AbpB− mutant and the augmentation of colonization by AbpA− mutant in the presence of dietary sucrose suggested additional amylase-binding protein interactions relevant to colonization. Glucosyltransferase activity was greater in mutants defective in abpA and modestly increased in the abpB mutant. It was concluded that AbpB is required for colonization of teeth of starch-eating rats and its deletion is partially masked if rats eat a sucrose-starch diet. AbpA appears to inhibit colonization of the plaque biofilm in vivo. This unexpected effect in vivo may be associated with interaction of AbpA with glucosyltransferase or with other colonization factors of these cells. These data illustrate that the complex nature of the oral environment may not be adequately modelled by in vitro systems.
N-Acetylglucosamine-inducible CaGAP1 encodes a general amino acid permease which co-ordinates external nitrogen source response and morphogenesis in Candida albicans
Authors: Biswas Subhrajit , Roy Monideepa , Datta Asis
Published: August 2003

Candida albicansis able to grow in a variety of reversible morphological forms (yeast, pseudohyphal and hyphal) in response to various environmental signals, noteworthy among them beingN-acetylglucosamine (GlcNAc). The geneCaGAP1, homologous toGAP1, which encodes the general amino acid permease fromSaccharomyces cerevisiae, was isolated on the basis of its induction by GlcNAc through differential screening of aC. albicansgenomic library. The gene could functionally complement anS. cerevisiae gap1mutant by rendering it susceptible to the toxic amino acid analogue mimosine in minimal proline media. As inS. cerevisiae, mutation of theCaGAP1gene had an effect on citrulline uptake inC. albicans. Northern analysis showed that GlcNAc-induced expression ofCaGAP1was further enhanced in synthetic minimal media supplemented with single amino acids (glutamate, proline and glutamine) or urea (without amino acids) but repressed in minimal ammonium media. Induction ofCaGAP1expression by GlcNAc was nullified inC. albicansdeleted for the transcription factorCPH1and the hyphal regulatorRAS1, indicating the involvement of Cph1p-dependent Ras1p signalling inCaGAP1expression. A homozygous mutant of this gene showed defective hyphal formation in solid hyphal-inducing media and exhibited less hyphal clumps when induced by GlcNAc. Alteration of morphology and short filamentation under nitrogen-starvation conditions in the heterozygous mutant suggested thatCaGAP1affects morphogenesis in a dose-dependent manner.
The Bacillus subtilis ywkA gene encodes a malic enzyme and its transcription is activated by the YufL/YufM two-component system in response to malate
Authors: Doan Thierry , Servant Pascale , Tojo Shigeo , Yamaguchi Hirotake , Lerondel Guillaume , Yoshida Ken-Ichi , Fujita Yasutaro , Aymerich Stéphane
Published: August 2003

A transcriptome comparison of a wild-typeBacillus subtilisstrain growing under glycolytic or gluconeogenic conditions was performed. In particular, it revealed that theywkAgene, one of the four paralogues putatively encoding a malic enzyme, was more transcribed during gluconeogenesis. Using alacZreporter fusion to theywkApromoter, it was shown thatywkAwas specifically induced by external malate and not subject to glucose catabolite repression. Northern analysis confirmed this expression pattern and demonstrated thatywkAis cotranscribed with the downstreamywkBgene. TheywkAgene product was purified and biochemical studies demonstrated its malic enzyme activity, which was 10-fold higher with NAD than with NADP (kcat/Km102 and 10 s−1 mM−1, respectively). However, physiological tests with single and multiple mutant strains affected inywkAand/or inywkAparalogues showed thatywkAdoes not contribute to efficient utilization of malate for growth. Transposon mutagenesis allowed the identification of the uncharacterized YufL/YufM two-component system as being responsible for the control ofywkAexpression. Genetic analysis andin vitrostudies with purified YufM protein showed that YufM binds just upstream ofywkApromoter and activatesywkAtranscription in response to the presence of malate in the extracellular medium, transmitted by YufL.ywkAandyufL/yufMcould thus be renamedmaeAformalicenzyme andmalK/malRformalatekinase sensor/malate responseregulator, respectively.
Induction of L-form-like cell shape change of Bacillus subtilis under microculture conditions
Authors: Shingaki Ryuji , Kasahara Yasuhiro , Iwano Megumi , Kuwano Masayoshi , Takatsuka Tomomasa , Inoue Tetsuyoshi , Kokeguchi Susumu , Fukui Kazuhiro
Published: August 2003

A remarkable cell shape change was observed inBacillus subtilisstrain 168 under microculture conditions on CI agar medium (Spizizen's minimal medium supplemented with a trace amount of yeast extract and Casamino acids). Cells cultured under a cover glass changed in form from rod-shaped to spherical, large and irregular shapes that closely resembled L-form cells. The cell shape change was observed only with CI medium, not with Spizizen's minimum medium alone or other rich media. The whole-cell protein profile of cells grown under cover glass and cells grown on CI agar plates differed in several respects. Tandem mass analysis of nine gel bands which differed in protein expression between the two conditions showed that proteins related to nitrate respiration and fermentation were expressed in the shape-changed cells grown under cover glass. The cell shape change of CI cultures was repressed when excess KNO3was added to the medium. Whole-cell protein analysis of the normal rod-shaped cells grown with 0·1 % KNO3and the shape-changed cells grown without KNO3revealed that the expression of the branched-chainα-keto acid dehydrogenase complex (coded by thebfmBgene locus) was elevated in the shape-changed cells. Inactivation of thebfmBlocus resulted in the repression of cell shape change, and cells in whichbfmBexpression was induced by IPTG did show changes in shape. Transmission electron microscopy of ultrathin sections demonstrated that the shape-changed cells had thin walls, and plasmolysis of cells fixed with a solution including 0·1 M sucrose was observed. Clarifying the mechanism of thinning of the cell wall may lead to the development of a new type of cell wall biosynthetic inhibitor.
A transposon encoding the complete 2,4-dichlorophenoxyacetic acid degradation pathway in the alkalitolerant strain Delftia acidovorans P4a
Authors: Hoffmann Doreen , Kleinsteuber Sabine , Müller Roland H. , Babel Wolfgang
Published: August 2003

The bacterial strainDelftia acidovoransP4a, isolated from an extreme environment (heavily contaminated with organochlorines, highly alkaline conditions in an aqueous environment), was found to mineralize 2,4-dichlorophenoxyacetic acid (2,4-D) and 2-methyl-4-chlorophenoxyacetic acid under alkaline conditions. Screening a genomic DNA library of the alkalitolerant strain for 2,4-D genes revealed the presence of the two 2,4-D gene clusterstfdCDEFandtfdCIIEIIBKA,tfdRgenes being located in the vicinity of eachtfdgene cluster. The results showed that the putative genes of the complete 2,4-D degradation pathway are organized in a single genomic unit. Sequence similarities to homologous gene clusters indicate that the individualtfdelements of strain P4a do not share a common origin, but were brought together by recombination events. The entire region is flanked by insertion elements of the IS1071and IS1380families, forming a transposon-like structure of about 30 kb, of which 28·4 kb were analysed. This element was shown to be located on the bacterial chromosome. The present study provides the first reported case of a chromosomally located catabolic transposon which carries the genes for the complete 2,4-D degradation pathway.
Multilocus sequence analysis of Porphyromonas gingivalis indicates frequent recombination
Authors: Koehler Andreas , Karch Helge , Beikler Thomas , Flemmig Thomas. F. , Suerbaum Sebastian , Schmidt Herbert
Published: August 2003

In this study, the genetic relationship of 19Porphyromonas gingivalisisolates from patients with periodontitis was investigated by multilocus sequence analysis. Internal 400–600 bp DNA fragments of the 10 chromosomal genesef-tu,ftsQ,hagB, gpdxJ,pepO,mcmA,dnaK,recA,pgaandnahwere amplified by PCR and sequenced. No two isolates were identical at all 10 loci. Phylogenetic analyses indicated a panmictic population structure ofP. gingivalis. Split decomposition analysis, calculation of homoplasy ratios and analyses of clustered polymorphisms all indicate that recombination plays a major role in creating the genetic heterogeneity ofP. gingivalis. A standardized index of association of 0·0898 indicates that theP. gingivalisgenes analysed are close to linkage equilibrium.
Calcium gradient dependence of Neurospora crassa hyphal growth
Authors: Silverman-Gavrila Lorelei B. , Lew Roger R.
Published: August 2003

A tip-high cytoplasmic calcium gradient has been identified as a requirement for hyphal growth in the fungusNeurospora crassa. The Ca2+gradient is less steep compared to wall vesicle, wall incorporation and vesicular Ca2+gradients, but this can be explained by Ca2+diffusion. Analysis of the relation between the rate of hyphal growth and the spatial distribution of tip-localized calcium indicates that hyphal growth rates depend upon the tip-localized calcium concentration. It is not the steepness of the calcium gradient, but tip-localized calcium and the difference in tip-localized calcium versus subapical calcium concentration which correlate closely with hyphal growth rate. A minimal concentration difference between the apex and subapical region of 30 nM is required for growth to occur. The calcium concentration dependence of growth may relate directly to biochemical functions of calcium in hyphal extension, such as vesicle fusion and enzyme activation during cellular expansion. Initiation of tip growth may rely upon random Ca2+motions causing localized regions of elevated calcium. Continued hyphal expansion may activate a stretch-activated phospholipase C which would increase tip-localized inositol 1,4,5-trisphosphate (IP3). Hyphal expansion, induced by mild hypoosmotic treatment, does increase diacylglycerol, the other product of phospholipase C activity. This is consistent with evidence that IP3-activated Ca2+channels generate and maintain the tip-high calcium gradient.
Aer and Tsr guide Escherichia coli in spatial gradients of oxidizable substrates
Authors: Greer-Phillips Suzanne E. , Alexandre Gladys , Taylor Barry L. , Zhulin Igor B.
Published: August 2003

The Aer and Tsr chemoreceptors in Escherichia coli govern tactic responses to oxygen and redox potential that are parts of an overall behaviour known as energy taxis. They are also proposed to mediate responses to rapidly utilized carbon sources, glycerol and succinate, via the energy taxis mechanism. In this study, the Aer and Tsr proteins were individually expressed in an ‘all-transducer-knockout’ strain of E. coli and taxis was analysed in gradients of various oxidizable carbon sources. In addition to the known response to glycerol and succinate, it was found that Aer directed taxis towards ribose, galactose, maltose, malate, proline and alanine as well as the phosphotransferase system (PTS) carbohydrates glucose, mannitol, mannose, sorbitol and fructose, but not to aspartate, glutamate, glycine and arabinose. Tsr directed taxis towards sugars (including those transported by the PTS), but not to organic acids or amino acids. When a mutated Aer protein unable to bind the FAD cofactor was expressed in the receptor-less strain, chemotaxis was not restored to any substrate. Aer appears to mediate responses to rapidly oxidizable substrates, whether or not they are effective growth substrates, whereas Tsr appears to mediate taxis to substrates that support maximal growth, whether or not they are rapidly oxidizable. This correlates with the hypothesis that Aer and Tsr sense redox and proton motive force, respectively. Taken together, the results demonstrate that Aer and Tsr mediate responses to a broad range of chemicals and their attractant repertoires overlap with those of specialized chemoreceptors, namely Trg (ribose, galactose) and Tar (maltose).
Streptomyces lividans contains a minimal functional signal recognition particle that is involved in protein secretion
Authors: Palacín Arantxa , de la Fuente Ricardo , Valle Inmaculada , Rivas Luis A. , Mellado Rafael P.
Published: August 2003

The bacterial version of the mammalian signal recognition particle (SRP) is well conserved and essential to all known bacteria. The genes for the Streptomyces lividans SRP components have been cloned and characterized. FtsY resembles the mammalian SRP receptor and the S. lividans SRP consists of Ffh, a homologue of the mammalian SRP54 protein, and scRNA, which is a small size RNA of 82 nt in length. Co-immunoprecipitation studies confirmed that Ffh and scRNA are probably the only components of the S. lividans SRP and that pre-agarase can co-immunoprecipitate with Ffh, suggesting that the SRP is involved in targeting secretory proteins.
Development of the Micromonospora carbonacea var. africana ATCC 39149 bacteriophage pMLP1 integrase for site-specific integration in Micromonospora spp.
Authors: Alexander Dylan C. , Devlin David J. , Hewitt Duane D. , Horan Ann C. , Hosted Thomas J.
Published: August 2003

Micromonospora carbonacea var. africana ATCC 39149 contains a temperate bacteriophage, pMLP1, that is present both as a replicative element and integrated into the chromosome. Sequence analysis of a 4·4 kb KpnI fragment revealed pMLP1 att/int functions consisting of an integrase, an excisionase and the phage attachment site (attP). Plasmids pSPRH840 and pSPRH910, containing the pMLP1 att/int region, were introduced into Micromonospora spp. by conjugation from Escherichia coli. Sequence analysis of DNA flanking the integration site confirmed site-specific integration into a tRNAHis gene in the chromosome. The pMLP1 attP element and chromosomal bacterial attachment (attB) site contain a 24 bp region of sequence identity located at the 3′ end of the tRNA. Integration of pMLP1-based plasmids in M. carbonacea var. africana caused a loss of the pMLP1 phage. Placement of an additional attB site into the chromosome allowed integration of pSPRH840 into the alternate attB site. Plasmids containing the site-specific att/int functions of pMLP1 can be used to integrate genes into the chromosome.
Dissection of homologous translocon operons reveals a distinct role for YopD in type III secretion by Yersinia pseudotuberculosis
Authors: Bröms Jeanette E. , Forslund Anna-Lena , Forsberg Åke , Francis Matthew S.
Published: August 2003

The homologouspcrGVHpopBDandlcrGVHyopBDtranslocase operons ofPseudomonas aeruginosaand pathogenicYersiniaspp., respectively, are responsible for the translocation of anti-host effectors into the cytosol of infected eukaryotic cells. InYersinia, this operon is also required foryop-regulatory control. To probe for key molecular interactions during the infection process, the functional interchangeability ofpopB/yopBandpopD/yopDwas investigated. Secretion of PopB producedin transin a ΔyopBnull mutant ofYersiniawas only observed when co-produced with its native chaperone PcrH, but this was sufficient to complement theyopBtranslocation defect. TheYersiniaΔyopDnull mutant synthesized and secreted PopD even in the absence of native PcrH, yet this did not restore YopD-dependentyop-regulatory control or effector translocation. Thus, this suggests that key residues in YopD, which are not conserved in PopD, are essential for functionalYersiniatype III secretion.
Glucose and trehalose PTS permeases of Spiroplasma citri probably share a single IIA domain, enabling the spiroplasma to adapt quickly to carbohydrate changes in its environment
Authors: André Aurélie , Maccheroni Walter , Doignon François , Garnier Monique , Renaudin Joël
Published: August 2003

Spiroplasma citri is a plant-pathogenic mollicute phylogenetically related to Gram-positive bacteria. Spiroplasma cells are restricted to the phloem sieve tubes and are transmitted from plant to plant by the leafhopper vector Circulifer haematoceps. In the plant sieve tubes, S. citri grows on glucose and fructose, whereas in the leafhopper haemolymph the spiroplasma must grow on trehalose, the major sugar in insects. Previous studies in this laboratory have shown that fructose utilization was a key factor of spiroplasmal pathogenicity. To further study the implication of sugar metabolism in the interactions of S. citri with its plant host and its leafhopper vector, genes encoding permease enzymes II (EIIGlc and EIITre) of the S. citri phosphoenolpyruvate : glucose and phosphoenolpyruvate : trehalose phosphotransferase systems (PTS) were characterized. Mapping studies revealed that the EIIGlc complex was split into two distinct polypeptides, IIAGlc and IICBGlc, encoded by two separate genes, crr and ptsG, respectively. As expected, S. citri polypeptides IIAGlc and IICBGlc were more phylogenetically related to their counterparts from Gram-positive than to those from Gram-negative bacteria. The trehalose operon consisted of three genes treR, treP and treA, encoding a transcriptional regulator, the PTS permease (EIITre) and the amylase, respectively. However, in contrast to the fructose-PTS permease, which is encoded as a single polypeptide (IIABCFru) containing the three domains A, B and C, the trehalose-PTS permease (IIBCTre) lacks its own IIA domain. No trehalose-specific IIA could be identified in the spiroplasmal genome, suggesting that the IIBCTre permease probably functions with the IIAGlc domain. In agreement with this statement, yeast two-hybrid system experiments revealed that the IIAGlc domain interacted not only with IIBGlc but also with the IIBTre domain. The results are discussed with respect to the ability of the spiroplasma to adapt from the phloem sap of the host plant to the haemolymph and salivary gland cells of the insect vector.
fusB is an allele of nadD, encoding nicotinate mononucleotide adenylyltransferase in Escherichia coli
Authors: Stancek Martin , Isaksson Leif A. , Rydén-Aulin Monica
Published: August 2003

Isolation of the temperature-sensitiveEscherichia colimutant 72c has been described previously. The mutant allele was namedfusBand causes a pleiotropic phenotype, the most striking features of which, besides temperature sensitivity, are the inability to grow on synthetic medium and supersensitivity to trimethoprim, an antibiotic that inhibits the C1metabolism. This work shows that thefusBmutation is a frameshift mutation in thenadDgene that encodes nicotinate mononucleotide adenylyltransferase. The frameshift leads to a change of the last 10 amino acids and an addition of 17 amino acids. This lesion, renamednadD72, leads to very little NAD+and NADPH synthesis at the permissive temperature and essentially no synthesis at the non-permissive temperature. As a comparison, a new mutation in thenadDgene, with an amino acid change in the ATP-binding site, has been isolated. Its NAD+synthesis is decreased at 30 °C but the level is still sufficient to support normal growth. At 42 °C, NAD+synthesis is reduced further, which leads to temperature sensitivity on minimal medium. This mutation was designatednadD74. Thus, a small decrease in NAD+levels affects ability to grow on minimal medium at 42 °C, while a large decrease leads to a more pleiotropic phenotype.
Shigella-induced necrosis and apoptosis of U937 cells and J774 macrophages
Authors: Nonaka Takashi , Kuwabara Taku , Mimuro Hitomi , Kuwae Asaomi , Imajoh-Ohmi Shinobu
Published: August 2003

It is currently unclear whetherShigellakills its phagocytic host cells by apoptosis or necrosis. This study shows that rapid necrosis ensues in macrophage-like cell lines (U937 cells differentiated by all-trans-retinoic acid and J774 cells) infected with theShigella flexneristrain YSH6000. The infected cells rapidly lose membrane integrity, a typical feature of necrosis, as indicated by the release of the cytoplasmic lactate dehydrogenase and the exposure of phosphatidylserine (PS) associated with the rapid uptake of propidium iodide (PI). The infected cells exhibit DNA fragmentation without nuclear condensation, and substantial involvement of either caspase-3/-7 or caspase-1 was not detected, which is also contrary to what is normally observed in apoptosis. Cytochalasin D potently inhibitedShigella-induced cell death, indicating that only internalizedShigellacan cause necrosis. Osmoprotectants such as polyethylene glycols could suppress cell death, suggesting that insertion of a pore byShigellainto the host cell membrane induces the necrosis. The pore was estimated to be 2·87±0·4 nm in diameter.Shigellawas also found to be able to induce apoptosis but only in one of the lines tested and under specific conditions, namely U937 cells differentiated with interferon-γ(U937IFN). Caspase-3/-7 but not caspase-1 activation was observed in these infected cells and the exposure of PS occurred without the uptake of PI. An avirulentShigellastrain, wild-typeShigellakilled with gentamicin, and evenEscherichia colistrain JM109, could also induce apoptosis in U937IFN cells, and cytochalasin D could not prevent apoptosis. It appears therefore thatShigella-induced apoptosis of U937IFN cells is unrelated toShigellapathogenicity and does not require bacterial internalization. Thus,Shigellacan induce rapid necrosis of macrophage-like cells in a virulence-related manner by forming pores in the host cell membrane while some cells can be killed through apoptosis in a virulence-independent fashion.
Spontaneous zygogenesis in Escherichia coli, a form of true sexuality in prokaryotes
Authors: Gratia Jean-Pierre , Thiry Marc
Published: August 2003

A new type of mating, differing from classic conjugation and previously observed in a certain strain ofEscherichia coliK-12, has also been found in strains derived from ordinary F−cells ofE. coliK-12 exposed to an exogenous factor originating in anE. coliclinical isolate. Immunofluorescence and electron microscopy after single and double labelling of DNA were used to produce evidence in favour of a novel mating mechanism by cell contact at the poles of the bacterial rod. These findings are supported by genetic analyses indicating complete genetic mixing. Unstable complementing diploids were formed, which throw off phenotypically haploid cells, of which some showed a parental phenotype and some were true genetic recombinants. Recombination was observed even when one parent was a UV-inactivated F−RecA−strain. The name ‘spontaneous zygogenesis' (Z-mating, for short) is proposed for this kind of mating.
Analysis of genes of tetrahydrofolate-dependent metabolism from cultivated spirochaetes and the gut community of the termite Zootermopsis angusticollis
Authors: Salmassi Tina M. , Leadbetter Jared R.
Published: August 2003

The hindguts of wood-feeding termites are the sites of intense, CO2-reductive acetogenesis. This activity profoundly influences host nutrition and methane emissions. Homoacetogens previously isolated from diverse termites comprised novel taxa belonging to two distinct bacterial phyla, Firmicutes and Spirochaetes. Little else is known about either the diversity or abundance of homoacetogenic species present in any given termite or the genetic details underlying CO2-reductive acetogenesis by Spirochaetes. A key enzyme of CO2-reductive acetogenesis is formyltetrahydrofolate synthetase (FTHFS). A previously designed primer set was used to amplify FTHFS genes from three isolated termite-gut spirochaetes. Sequencing DNA flanking the FTHFS gene of Treponema strain ZAS-2 revealed genes encoding two acetogenesis-related enzymes, methenyltetrahydrofolate cyclohydrolase and methylenetetrahydrofolate dehydrogenase. Although termite-gut spirochaetes are only distantly related to clostridia at the ribosomal level, their tetrahydrofolate-dependent enzymes appear to be closely related. In contrast, homologous proteins identified in the non-homoacetogenic oral spirochaete Treponema denticola were only distantly related to those from clostridia and the termite-gut treponemes. Having demonstrated their utility with spirochaete pure cultures, the FTHFS primers were used to construct a 91-clone library from the termite-gut community DNA. From this, 19 DNA and eight amino acid FTHFS types were identified. Over 75 % of the retrieved clones formed a novel, coherent cluster with the FTHFS homologues obtained from the termite-gut treponemes. Thus, FTHFS gene diversity in the gut of the termite Zootermopsis angusticollis appears to be dominated by spirochaetes. The homoacetogenic capacity of termite-gut spirochaetes may have been acquired via lateral gene transfer from clostridia.
An ordered collection of expressed sequences from Cryphonectria parasitica and evidence of genomic microsynteny with Neurospora crassa and Magnaporthe grisea
Authors: Dawe Angus L. , McMains Vanessa C. , Panglao Maria , Kasahara Shin , Chen Baoshan , Nuss Donald L.
Published: August 2003

Cryphonectria parasitica, the causative agent of chestnut blight, has proven to be a tractable experimental system for studying fungal pathogenesis. Moreover, the development of infectious cDNA clones ofC. parasiticahypoviruses, capable of attenuating fungal virulence, has provided the opportunity to examine molecular aspects of fungal plant pathogenesis in the context of biological control. In order to establish a genomic base for future studies ofC. parasitica, the authors have analysed a collection of expressed sequences. A mixed cDNA library was prepared from RNA isolated from wild-type (virus-free) and hypovirus-infectedC. parasiticastrains. Plasmid DNA was recovered from individual transformants and sequenced from the 5′ end of the insert. Contig analysis of the collected sequences revealed that they represented approximately 2200 individual ORFs. An assessment of functional diversity present in this collection was achieved by using theblastsoftware utilities and the NCBI protein database. Candidate genes were identified with significant potential relevance toC. parasiticagrowth, development, pathogenesis and vegetative incompatibility. Additional investigations of a 12·9 kbp genomic region revealed microsynteny betweenC. parasiticaand bothNeurospora crassa andMagnaporthegrisea, two closely related fungi. These data represent the largest collection of sequence information currently available forC. parasiticaand are now forming the basis of further studies using microarray analyses to determine global changes in transcription that occur in response to hypovirus infection.
Gene expression level influences amino acid usage, but not codon usage, in the tsetse fly endosymbiont Wigglesworthia
Authors: Herbeck Joshua T. , Wall Dennis P. , Wernegreen Jennifer J.
Published: August 2003

Wigglesworthia glossinidiabrevipalpis, the obligate bacterial endosymbiont of the tsetse flyGlossina brevipalpis, is characterized by extreme genome reduction and AT nucleotide composition bias. Here, multivariate statistical analyses are used to test the hypothesis that mutational bias and genetic drift shape synonymous codon usage and amino acid usage ofWigglesworthia. The results show that synonymous codon usage patterns vary little across the genome and do not distinguish genes of putative high and low expression levels, thus indicating a lack of translational selection. Extreme AT composition bias across the genome also drives relative amino acid usage, but predicted high-expression genes (ribosomal proteins and chaperonins) use GC-rich amino acids more frequently than do low-expression genes. The levels and configuration of amino acid differences betweenWigglesworthiaandEscherichia coliwere compared to test the hypothesis that the relatively GC-rich amino acid profiles of high-expression genes reflect greater amino acid conservation at these loci. This hypothesis is supported by reduced levels of protein divergence at predicted high-expressionWigglesworthiagenes and similar configurations of amino acid changes across expression categories. Combined, the results suggest that codon and amino acid usage in theWigglesworthiagenome reflect a strong AT mutational bias and elevated levels of genetic drift, consistent with expected effects of an endosymbiotic lifestyle and repeated population bottlenecks. However, these impacts of mutation and drift are apparently attenuated by selection on amino acid composition at high-expression genes.
The colicin G, H and X determinants encode microcins M and H47, which might utilize the catecholate siderophore receptors FepA, Cir, Fiu and IroN
Authors: Patzer S. I. , Baquero M. R. , Bravo D. , Moreno F. , Hantke K.
Published: August 2003

The colicin G producer Escherichia coli CA46, the colicin H producer E. coli CA58 and E. coli Nissle 1917 (DSM 6601) were shown to produce microcin H47 and the newly described microcin M. Both microcins were exported like colicin V by an RND-type export system, including TolC. The gene cluster encoding microcins H47 and M in strains CA46 and CA58 is nearly identical to that in strain DSM 6601, except that two additional genes are included. A Fur box identified in front of the microcin-encoding genes explained the observed iron regulation of microcin production. The catecholate siderophore receptors Fiu, Cir and FepA from E. coli and IroN, Cir and FepA from Salmonella were identified as receptors for microcins M, H47 and E492. IroN takes up the glucose-containing catecholate siderophore salmochelin, whose synthesis is encoded in the iro gene cluster found in Salmonella and certain, often uropathogenic, E. coli strains. A gene in this iro cluster, iroB, which encodes a putative glycosyltransferase, was also found in the microcin H47/M and microcin E492 gene clusters. These microcins could aid the producing strain in competing against enterobacteria that utilize catecholate siderophores.
The roles of SsrA–SsrB and OmpR–EnvZ in the regulation of genes encoding the Salmonella typhimurium SPI-2 type III secretion system
Authors: Garmendia Junkal , Beuzón Carmen R. , Ruiz-Albert Javier , Holden David W.
Published: August 2003

The type III secretion system (TTSS) encoded bySalmonella typhimuriumpathogenicity island 2 (SPI-2) is expressed after bacterial entry into host cells. The SPI-2 TTSS secretes the translocon components SseBCD, which translocate across the vacuolar membrane a number of effector proteins whose action is required for intracellular bacterial replication. Several of these effectors, including SifA and SifB, are encoded outside SPI-2. The two-component regulatory system SsrA–SsrB, encoded within SPI-2, controls the expression of components of the SPI-2 TTSS apparatus as well as its translocated effectors. The expression of SsrA–B is in turn regulated by the OmpR–EnvZ two-component system, by direct binding of OmpR to thessrABpromoter. Several environmental signals have been shown to inducein vitroexpression of genes regulated by the SsrA–B or OmpR–EnvZ systems. In this work, immunoblotting and flow cytometry were used to analyse the roles of SsrA–B and OmpR–EnvZ in coupling different environmental signals to changes in expression of a SPI-2 TTSS translocon component (SseB) and two effector genes (sifAandsifB). Using single and double mutant strains the relative contribution of each regulatory system to the response generated by low osmolarity, acidic pH or the absence of Ca2+was determined. SsrA–B was found to be essential for the induction of SPI-2 gene expression in response to each of these individual signals. OmpR–EnvZ was found to play a minor role in sensing these signals and to require a functional SsrA–B system to mediate their effect on SPI-2 TTSS gene expression.
Effect of enzyme I of the bacterial phosphoenolpyruvate : sugar phosphotransferase system (PTS) on virulence in a murine model
Authors: Kok Menno , Bron Guillaume , Erni Bernhard , Mukhija Seema
Published: August 2003

The phosphoenolpyruvate : sugar phosphotransferase system (PTS) catalyses translocation with concomitant phosphorylation of sugars and hexitols and it regulates metabolism in response to the availability of carbohydrates. The PTS forms an interface between energy and signal transduction and its inhibition is likely to have pleiotropic effects. It is present in about one-third of bacteria with fully sequenced genomes, including many common pathogens, but does not occur in eukaryotes. Enzyme I (ptsI) is the first component of the divergent protein phosphorylation cascade.ptsIdeletions were constructed inSalmonella typhimurium,Staphylococcus aureusandHaemophilus influenzaeand virulence of the mutants was characterized in an intraperitoneal mouse model. The log(attenuation) values were 2·3, 1·4 and 0·9 for theSal. typhimurium,Sta. aureusandH. influenzaeptsImutants, respectively. The degree of attenuation is correlated with the complexity of the respective PTS, which comprises approximately 40 components inSal. typhimurium, but only 5 inH. influenzae.
The complete genome sequence of the avian pathogen Mycoplasma gallisepticum strain Rlow
Authors: Papazisi Leka , Gorton Timothy S. , Kutish Gerald , Markham Philip F. , Browning Glenn F. , Nguyen Di Kim , Swartzell Steven , Madan Anup , Mahairas Greg , Geary Steven J.
Published: August 2003

The complete genome of Mycoplasma gallisepticum strain Rlow has been sequenced. The genome is composed of 996 422 bp with an overall G+C content of 31 mol%. It contains 742 putative coding DNA sequences (CDSs), representing a 91 % coding density. Function has been assigned to 469 of the CDSs, while 150 encode conserved hypothetical proteins and 123 remain as unique hypothetical proteins. The genome contains two copies of the rRNA genes and 33 tRNA genes. The origin of replication has been localized based on sequence analysis in the region of the dnaA gene. The vlhA family (previously termed pMGA) contains 43 genes distributed among five loci containing 8, 2, 9, 12 and 12 genes. This family of genes constitutes 10·4 % (103 kb) of the total genome. Two CDSs were identified immediately downstream of gapA and crmA encoding proteins that share homology to cytadhesins GapA and CrmA. Based on motif analysis it is predicted that 80 genes encode lipoproteins and 149 proteins contain multiple transmembrane domains. The authors have identified 75 proteins putatively involved in transport of biomolecules, 12 transposases, and a number of potential virulence factors. The completion of this sequence has spawned multiple projects directed at defining the biological basis of M. gallisepticum.
Klebsiella pneumoniae MrkD-mediated biofilm formation on extracellular matrix- and collagen-coated surfaces
Authors: Jagnow Jennifer , Clegg Steven
Published: August 2003

The type 3 fimbriae of Klebsiella pneumoniae are comprised of the major fimbrial subunit (MrkA) and the adhesin (MrkD) that has previously been shown to mediate binding to collagen. The ability of adhesive and non-adhesive derivatives of K. pneumoniae to form biofilms on collagen-coated surfaces in continuous-flow chambers was investigated. Unlike biofilm formation on abiotic plastic surfaces, the presence of the MrkD adhesin was necessary for growth on collagen-coated surfaces. Fimbriate strains lacking the MrkD adhesin did not efficiently adhere to and grow on these surfaces. Similarly, purified human extracellular matrix and the extracellular matrix formed by human bronchial epithelial cells grown in vitro provided a suitable substrate for MrkD-mediated biofilm formation, whereas direct binding to the respiratory cells was not observed. Type 3 fimbriae may therefore have two roles in the early stages of adherence and growth on in-dwelling devices such as endotracheal tubes. The MrkA polypeptide could facilitate adsorption to abiotic polymers of recently implanted devices and the MrkD adhesin could enable bacteria to adhere to and grow on polymers coated with host-derived proteins.
Membrane-bound hydrogenase and sulfur reductase of the hyperthermophilic and acidophilic archaeon Acidianus ambivalens
Authors: Laska Simone , Lottspeich Friedrich , Kletzin Arnulf
Published: August 2003

A sulfur reductase (SR) and a hydrogenase were purified from solubilized membrane fractions of anaerobically grown cells of the sulfur-dependent archaeon Acidianus
ambivalens and the corresponding genes were sequenced. The SR reduced elemental sulfur with hydrogen as electron donor [45 U (mg protein)−1] in the presence of hydrogenase and either 2,3-dimethylnaphthoquinone (DMN) or cytochrome c in the enzyme assay. The SR could not be separated from the hydrogenase during purification without loss of activity, whereas the hydrogenase could be separated from the SR. The specific activity of the hydrogenase was 170 U (mg protein)−1 with methyl viologen and 833 U (mg protein)−1 with DMN as electron acceptors. Both holoenzymes showed molecular masses of 250 kDa. In SDS gels of active fractions, protein bands with apparent masses of 110 (SreA), 66 (HynL), 41 (HynS) and 29 kDa were present. Enriched hydrogenase fractions contained 14 μmol Fe and 2 μmol Ni (g protein)−1; in addition, 2·5 μmol Mo (g protein)−1 was found in the membrane fraction. Two overlapping genomic cosmid clones were sequenced, encoding a five-gene SR cluster (sre) including the 110 kDa subunit gene (sreA), and a 12-gene hydrogenase cluster (hyn) including the large and small subunit genes and genes encoding proteins required for the maturation of NiFe hydrogenases. A phylogenetic analysis of the SR amino acid sequence revealed that the protein belonged to the DMSO reductase family of molybdoenzymes and that the family showed a novel clustering. A model of sulfur respiration in Acidianus developed from the biochemical results and the data of the amino acid sequence comparisons is discussed.
Gingival epithelial cell signalling and cytoskeletal responses to Porphyromonas gingivalis invasion
Authors: Yilmaz Özlem , Young Patrick A. , Lamont Richard J. , Kenny George E.
Published: August 2003

Porphyromonas gingivalis, an oral pathogen, can internalize within primary gingival epithelial cells (GECs) through an invasion mechanism mediated by interactions between P. gingivalis fimbriae and integrins on the surface of the GECs. Fimbriae–integrin-based signalling events were studied by fluorescence microscopy, and the subcellular localization of integrin-associated signalling molecules paxillin and focal adhesion kinase (FAK), and the architecture of the actin and microtubule cytoskeleton were examined. GECs infected with P. gingivalis for 30 min demonstrated significant redistribution of paxillin and FAK from the cytosol to cell peripheries and assembly into focal adhesion complexes. In contrast, a fimbriae-deficient mutant of P. gingivalis did not contribute substantially to activation of paxillin or FAK. After 24 h, the majority of paxillin and FAK had returned to the cytoplasm with significant co-localization with P. gingivalis in the perinuclear region. Wild-type P. gingivalis induced nucleation of actin filaments forming microspike-like protrusions and long stable microfilaments distributed throughout the cells. Fimbriae mutants promoted a rich cortical actin meshwork accompanied by membrane ruffling dispersed along the cell membrane. Remarkable disassembly and nucleation of the actin and microtubule filamentous network was observed following 24 h infection with either wild-type or fimbriae-deficient mutants of P. gingivalis. The results show that fimbriated P. gingivalis cells induce formation of integrin-associated focal adhesions with subsequent remodelling of the actin and tubulin cytoskeleton.
Anti-wurtzite reoriented
Authors: Hostettler M. , Flack H. D.
Published: August 2003

Anti-wurtzite and wurtzite are shown to be the same crystal structure despite the claims of a recent paper describing the crystal structure of the mineral rambergite, Mn1−x
Fe
x
S, x ≃ 0.05. The anti-wurtzite/wurtzite confusion is used as an illustration to help clarify the correct general approach to take in the treatment and presentation of achiral non-centrosymmetric crystal structures.
Intermolecular —CH3...O2N— contacts in two polymorphic modifications of (1E)-N'-[(E)-2-cyano-1-(dimethylamino)-2-nitrovinyl]-N,N-dimethylethanimidamide
Authors: Tafeenko V. A. , Chernyshev V. V. , Yatsenko A. V. , Makarov V. A. , Sonneveld E. J. , Peschar R. , Schenk H.
Published: August 2003

The title compound was synthesized and isolated in two crystal modifications. The structure of the orthorhombic modification was determined by the X-ray powder diffraction method and the structure of the monoclinic modification was determined using the X-ray single-crystal diffraction technique. The molecules in both polymorphs are E,E isomers. Intermolecular H3C...NO2 contacts and their role in the formation of the polymorphic modifications are analyzed.
Structural characterization of the n = 5 layered perovskite neodymium titanate using high-resolution transmission electron microscopy and image reconstruction
Authors: Sayagués Maria , Titmuss Katherine , Meyer Rudiger , Kirkland Angus , Sloan Jeremy , Hutchison John , Tilley Richard
Published: August 2003

The structure of Nd5Ti5O17 has been refined from a reconstruction of the specimen exit-plane wave restored from a series of incrementally defocused high-resolution transmission electron microscope (HRTEM) images. The phase of the exit-plane wave shows contrast attributable to the oxygen anion sublattice and coupled with simulations provides confirmation of the composition of the cation sites as a function of sample thickness. The enhanced resolution in the exit-plane wave additionally allows a direct measurement of the `skewing' of the perovskite slabs.
Structural characterization of YV4O8: simultaneous analysis of coexisting polytypes and simulation of diffuse scattering for a stacking disorder model
Authors: Onoda Mitsuko , Dhaussy Anne-Claire , Kanke Yasushi
Published: August 2003

The structure of a crystal of newly synthesized YV4O8 was refined on the assumption that two polytypes and their respective twin forms intergrow. The model was expressed as a commensurate composite crystal with two types of subsystem: one is a V4O8 framework with rather large tunnels and the other consists of Y ions. In the tunnels, Y ions and vacancies are located at every second site in an ordered manner that is characteristic of each polytype. Refinement was performed using a high-dimensional formalism and all reflections from all domains. Diffuse streaks observed in the X-ray and electron diffraction patterns were simulated using the matrix method that has been used for one-dimensional disorder such as stacking faults. The unusual diffraction phenomena that occur in a crystal of YV4O8 are explained as arising from a multiple-domain structure of coexisting polytypes.
Influence of pressure on the lengths of chemical bonds
Authors: Brown I. David , Klages Peter , Skowron Aniceta
Published: August 2003

An expression to describe the force that a chemical bond exerts on its terminal atoms is proposed, and is used to derive expressions for the bond force constant and bond compressibility. The unknown parameter in this model, the effective charge on the atoms that form the bond, is determined by comparing the derived force constants with those obtained spectroscopically. The resultant bond compressibilities are shown to generally agree well with those determined from high-pressure structure determinations and from the bulk moduli of high-symmetry structures. Bond valences can be corrected for pressure by recognizing that the bond-valence parameter, R
0, changes with pressure according to the equation{\rm d}R_0/{\rm d}P = 10^{-4} R_0^4/(1/B-2/R_0)\; \rm{\AA\,\,GPa}^{-1}
1,2-Dimethyl-4-nitro-5-morpholinoimidazole and its hydrate: a case of a centrosymmetric–noncentrosymmetric ambiguity
Authors: Kubicki Maciej , Borowiak Teresa , Dutkiewicz Grzegorz , Sobiak Stanisław , Weidlich Iwona
Published: August 2003

The compound studied is 1,2-dimethyl-4-nitro-5-morpholinoimidazole (1) in its anhydrous (1) and hydrated [(1)·H2O] crystal forms. In spite of the strong electron-withdrawing effect of the nitro group, the unsubstituted N atom of the imidazole moiety retains its basic character and acts as an acceptor for intermolecular hydrogen bonds: either weak C—H...N bonds in (1) or strong O—H...N bonds, with the water molecules, in (1)·H2O. The packing in (1) is determined by weak C—H...N and C—H...O hydrogen bonds, van der Waals interactions and the stacking of imidazole fragments. The crystal structure of (1)·H2O is determined by strong O—H(water)...N3(imidazole) and O—H(water)...O(water) hydrogen bonds. This structure consists of a centrosymmetric `matrix' of imidazole derivative molecules and locally noncentrosymmetric arrays of hydrogen-bonded water molecules. Each of these arrays is strictly homodromic, i.e. it runs only in one direction: ...H—O...H—O...H—O... or ...O—H...O—H...O—H.... These homodromic domains are statistically distributed within the crystal.
Structural trends and the electronic structure of the rare-earth oxomolybdates RMo5O8 (R = La, Ce, Pr, Nd, Sm, Eu and Gd) containing chains of bioctahedral Mo10 clusters
Authors: Gougeon P. , Gall P. , Halet J.-F. , Gautier R.
Published: August 2003

The crystal structures of the rare-earth members of the series RMo5O8 (R = Ce to Eu) have been investigated and compared with those of the La and Gd members previously published in order to understand the influences of the size and the charge of the cation on the different Mo—Mo bonds. The RMo5O8 compounds crystallize in the monoclinic space group P21/c. Their crystal structure is characterized by bioctahedral Mo10 clusters forming extended chains. The results of our single-crystal studies show that the modification of charge predominantly affects the Mo—Mo bonds between the Mo10 clusters and, to a lesser extent, the intra-cluster distances, while the cationic size induces only small variations. Theoretical investigations confirm this statement and allow the understanding of the bonding mode in these compounds.