bluuebunny/crossref_metadata_2025 huggingface dataset:
Are some putative glycogen accumulating organisms (GAO) in anaerobic : aerobic activated sludge systems members of the α-Proteobacteria?
Authors: Beer Michael , Kong Yun H. , Seviour Robert J.
Published: July 2004

Activated sludge plants designed to remove phosphorus microbiologically often perform unreliably. One suggestion is that the polyphosphate-accumulating organisms (PAO) are out-competed for substrates by another group of bacteria, the glycogen-accumulating organisms (GAO) in the anaerobic zones of these processes. This study used fluorescence in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE) to analyse the communities from laboratory-scale anaerobic : aerobic sequencing batch reactors. Members of the genus Sphingomonas in the α-Proteobacteria were present in large numbers in communities with poor phosphorus removal capacity where the biomass had a high glycogen content. Their ability to store poly-β-hydroxyalkanoates anaerobically, but not aerobically, and not accumulate polyphosphate aerobically is consistent with these organisms behaving as GAO there. No evidence was found to support an important role for the γ-Proteobacteria as possible GAO in these communities, although these bacterial populations have been considered in other studies to act as possible competitors for the PAO.
DNA-containing membrane vesicles of Pseudomonas aeruginosa PAO1 and their genetic transformation potential
Authors: Renelli Marika , Matias Valério , Lo Reggie Y. , Beveridge Terry J.
Published: July 2004

Natural membrane vesicles (n-MVs) produced byPseudomonas aeruginosaPAO1 and PAO1 carrying plasmid pAK1900 (p-MVs) were purified and analysed for DNA content. The MVs were isolated by a procedure designed to ensure no cellular contamination from the parent MV-producing cells. Fluorometry analysis revealed that p-MVs were associated with 7·80 ng DNA (20 μg MV protein)−1. PCR analysis using specific primers for pAK1900 sequences and a chromosomal target,oprL, indicated that only plasmid DNA was contained within the lumen of p-MVs after exogenous DNA was digested by DNase. MVs have previously been shown to be capable of fusing into the outer membrane (OM) of PAO1 andEscherichia coliDH5α. Accordingly, p-MVs should deliver the plasmid into the periplasm, where it would only have to by-pass the plasma membrane (PM) for effective transformation. It was speculated that p-MVs should increase transformation efficiency but the data suggested otherwise. p-MVs did not transform PAO1 nor DH5αunder a variety of transforming conditions. To characterize p-MVs and to ensure that membrane-encapsulated pAK1900 was not derived from a small proportion of lysed cells within the culture and bound by PM instead of OM, which typically forms n-MVs, the physical and ultrastructural differences between n- and p-MVs were determined. Cryo-transmission electron microscopy (cryo-TEM) revealed that n-MVs and p-MVs closely resembled isolated OM. Buoyant density measurements using isopycnic sucrose gradients on isolated PM, OM, n- and p-MVs demonstrated that isolated OM and n-MVs both fractionated into two bands (ρ=1·240 and 1·260 g ml−1). p-MVs also produced two bands but at two different densities (ρ=1·250 and 1·265 g ml−1) which may be attributed to the presence of DNA. SDS-PAGE showed that p-MVs possessed most major OM proteins and also contained 43·70 nmol 3-deoxy-d-manno-octulosonic acid (KDO) (mg protein)−1as an LPS marker. The amount of NADH oxidase activity, a PM enzyme, in the p-MVs was barely detectable. These data strongly suggest that p-MVs are OM-based, with little if any PM material associated with them. The possibility of whether exogenous plasmid DNA could enter n-MVs once the vesicles had departed from cells was also tested; surprisingly, a small amount of DNA could. Accordingly, the data suggest that DNA can be taken up by MVs using two separate routes: (1) via a periplasmic route and (2) via an extracellular, exogenous route.
Identification and characterization of two α-1,6-mannosyltransferases, Anl1p and Och1p, in the yeast Yarrowia lipolytica
Authors: Barnay-Verdier Stéphanie , Boisramé Anita , Beckerich Jean-Marie
Published: July 2004

In this study, the identification and characterization of theYarrowia lipolyticahomologues ofSaccharomyces cerevisiaeα-1,6-mannosyltransferases Anp1p and Och1p, designated YlAnl1p and YlOch1p, are described. In order to confirm the function of theY. lipolyticaproteins, including the previously isolated YlMnn9p, in theN-glycosylation pathway, a phenotypic analysis of the disrupted strains ΔYlmnn9, ΔYlanl1, ΔYloch1, ΔYlanl1ΔYlmnn9and ΔYlmnn9ΔYloch1was performed. Disruption of theYlMNN9,YlANL1andYlOCH1genes caused an increased sensitivity to SDS, compatible with a glycosylation defect, and to Calcofluor White, characteristic of cell-wall defects. Moreover, Western-blot analysis of a heterologous glycosylated protein confirmed a direct role of YlMnn9p and YlAnl1p in theN-glycosylation process. These mutant strains, ΔYlmnn9, ΔYlanl1, ΔYloch1, ΔYlanl1ΔYlmnn9and ΔYlmnn9ΔYloch1may thus be used to establish a model for theY. lipolyticaN-linked glycosylation pathway.
Extended phenotype of an mreB-like mutant in Azospirillum brasilense
Authors: Biondi Emanuele G. , Marini Francesca , Altieri Fabio , Bonzi Laura , Bazzicalupo Marco , del Gallo Maddalena
Published: July 2004

Tn5mutagenesis was used to generate anAzospirillum brasilenseSPF94 mutant. Genetic analysis of this mutant revealed that a homologue of themreBgene, which controls cell shape inBacillus subtilisandEscherichia coli, was inactivated. The cell-surface properties of the mutant were different from those of the parental strain. The mutant colonies were highly fluorescent when grown on plates containing Calcofluor White. Light and electron microscopy revealed that the mutant cells were round and had thicker capsules than the spiral parental strain. The mutants contained up to ten times more capsule protein than the parental strain, but lacked a 40 kDa protein that is abundant in the parental strain. The phenotype of the isolated mutant resembled that of the cyst-like differentiated forms ofAzospirillum, suggesting that themreBhomologue could be involved in differentiation.
Three genes, lgtF, lic2C and lpsA, have a primary role in determining the pattern of oligosaccharide extension from the inner core of Haemophilus influenzae LPS
Authors: Hood Derek W. , Deadman Mary E. , Cox Andrew D. , Makepeace Katherine , Martin Adele , Richards James C. , Moxon E. Richard
Published: July 2004

Lipopolysaccharide (LPS) is a virulence determinant of Haemophilus influenzae and exhibits substantial heterogeneity in structure within and between strains. Key factors contributing to this heterogeneity are the genes required to add the first glycose to each of the three heptose residues of the LPS inner core. In each case this addition can facilitate further oligosaccharide extension. lgtF is invariably present in strains and the product has a function in adding the glucose to the first heptose. lic2C is present in half the strains and was found to add a glucose to the second heptose. Insertion of lic2C into a strain that does not naturally contain it resulted in hexose incorporation from the second heptose of the LPS. The product of the lpsA gene can add a glucose or galactose to the third heptose. By allelic replacement of lpsA between strains it is shown that the sequence of the gene can be the sole determinant of this specificity. Thus, lgtF, lic2C and lpsA make significant but very distinct contributions to the conservation and variable patterns of oligosaccharide extensions seen in H. influenzae LPS.
Genetic analysis of the Bacillus subtilis sigG promoter, which controls the sporulation-specific transcription factor σ G
Authors: Evans Louise , Feucht Andrea , Errington Jeff
Published: July 2004

At the onset of sporulation inBacillus subtilis, an asymmetric cell division gives rise to two unequal-sized compartments with distinct developmental fates. The smaller compartment, or prespore, becomes the spore, whilst the larger compartment, or mother cell, eventually lyses after contributing to spore maturation. The fate of each compartment is determined by differential gene expression, controlled by the activation of four compartment-specificσ-factors. The expression and activity of all fourσ-factors are tightly regulated to ensure the correct sequence of morphological events. Prespore-specific genes are transcribed by twoσ-factors,σFfollowed byσG. The gene encodingσG(sigG) is transcribed byσF, but also requires the activity of one of the mother-cell-specificσ-factors,σE, for its expression. The minimal promoter required for dependence onσEwas found to stretch to just upstream of the −35 site. Analysis of mutantsigGpromoters generated by site-directed mutagenesis andsigGpromoters from other species suggests the presence of a binding site for a transcriptional repressor within thesigGpromoter region. Replacement of the wild-type promoter withσE-independent promoters resulted in impairment of sporulation. These data support the idea thatσEactivity is required for the transcription ofsigG.
ALS3 and ALS8 represent a single locus that encodes a Candida albicans adhesin; functional comparisons between Als3p and Als1p
Authors: Zhao Xiaomin , Oh Soon-Hwan , Cheng Georgina , Green Clayton B. , Nuessen Jennifer A. , Yeater Kathleen , Leng Roger P. , Brown Alistair J. P. , Hoyer Lois L.
Published: July 2004

The ALS (agglutinin-likesequence) gene family ofCandida albicansencodes eight cell-surface glycoproteins, some of which are involved in adherence to host surfaces. A mutational analysis of each ALS gene is currently being performed to deduce the functions of the encoded proteins and to better understand the role of these proteins inC. albicansbiology and pathogenesis. This paper describes construction of anals3/als3mutant and comparison of its phenotype to anals1/als1strain. Efforts to disruptALS3indicated that the gene could be deleted in two transformation steps, suggesting that the gene is encoded by a single locus and that theALS3-like locus,ALS8, does not exist. Strains lackingALS3orALS1did not exhibit a defect in germ tube formation when grown in RPMI 1640 medium, but theals1/als1mutant formed significantly fewer germ tubes in Lee medium. Analysis ofALS3andALS1promoter activity using green fluorescent protein (GFP) reporter strains and flow cytometry showed that when cells are placed into medium that promotes germ tube formation,ALS1is transcribed prior toALS3. Comparison of the mutant strains in adhesion assays showed that theals3/als3strain was defective in adhesion to both human umbilical vein endothelial cells (HUVEC) and buccal epithelial cells (BEC), but not to fibronectin-coated plastic plates. In contrast, theals1/als1strain showed decreased adherence to HUVEC, but adherence to BEC and fibronectin were the same as wild-type controls. Inoculation of the buccal reconstituted human epithelium (RHE) model of oral candidiasis with the mutant strains showed nearly a total lack of adhesion and epithelial destruction by theals3/als3mutant while theals1/als1strain showed only a slightly reduced degree of epithelial destruction compared to the wild-type control. Adhesion data presented here suggest that, in the assays performed, loss of Als3p affectsC. albicansadhesion more than loss of Als1p. Collectively, these results demonstrate functional similarities and differences between Als1p and Als3p, and suggest the potential for more complex interrelationships between the ALS genes and their encoded proteins.
Communications blackout? Do N-acylhomoserine-lactone-degrading enzymes have any role in quorum sensing?
Authors: Roche Declan M. , Byers Joseph T. , Smith Debra S. , Glansdorp Freija G. , Spring David R. , Welch Martin
Published: July 2004

A number of bacteria, including some significant pathogens, utilize N-acylhomoserine lactones (AHLs) as quorum sensing signals. There is considerable interest in the therapeutic potential of disrupting quorum sensing. Recently, a number of bacteria have been identified which are capable of enzymic inactivation of AHLs. These enzymes show considerable promise as ‘quenchers' of quorum sensing. However, the assumption that the natural function of these enzymes is to disrupt or modulate quorum sensing has yet to be established. This review surveys the progress made to date in this field and examines what implications these findings have for our understanding of the role played by these enzymes in vivo.
Structure–function relationships of UMP kinases from pyrH mutants of Gram-negative bacteria
Authors: Sakamoto Hiroshi , Landais Stéphanie , Evrin Cécile , Laurent-Winter Christine , Bârzu Octavian , Kelln Rod A.
Published: July 2004

Bacterial uridine monophosphate (UMP) kinases are essential enzymes encoded bypyrHgenes, and conditional-lethal or otherpyrHmutants were analysed with respect to structure–function relationships. A set of thermosensitivepyrHmutants fromEscherichia coliwas generated and studied, along with already describedpyrHmutants fromSalmonella entericaserovar Typhimurium. It is shown that Arg-11 and Gly-232 are key residues for thermodynamic stability of the enzyme, and that Asp-201 is important for both catalysis and allosteric regulation. A comparison of the amino acid sequence of UMP kinases from several prokaryotes showed that these were conserved residues. Discussion on the enzyme activity level in relation to bacterial viability is also presented.
α-Aminoadipate aminotransferase from an extremely thermophilic bacterium, Thermus thermophilus
Authors: Miyazaki Takashi , Miyazaki Junichi , Yamane Hisakazu , Nishiyama Makoto
Published: July 2004

The extremely thermophilic bacteriumThermus thermophilusHB27 synthesizes lysine throughα-aminoadipate (AAA). In this study, aT. thermophilusgene encoding the enzyme that catalyses transamination of AAA was cloned as a mammalian kynurenine/AAA aminotransferase (Kat2) gene homologue. AT. thermophilusmutant with disruption of theKat2homologue required a longer lag phase for growth and showed slower growth in minimal medium. Furthermore, addition of AAA or lysine shortened the lag phase and improved the growth rate. TheKat2homologue was therefore termedlysN. LysN recognizes not only 2-oxoadipate, an intermediate of lysine biosynthesis, but also 2-oxoisocaproate, 2-oxoisovalerate and 2-oxo-3-methylvalerate, intermediates of leucine, valine and isoleucine biosyntheses, respectively, along with oxaloacetate, a compound in the TCA cycle, as an amino acceptor. These results suggest multiple roles of LysN in several cellular metabolic pathways including lysine and branched-chain amino acid biosyntheses.
Comparative analysis of eukaryotic-type protein phosphatases in two streptomycete genomes
Authors: Shi Liang , Zhang Weiwen
Published: July 2004

Inspection of the genomes ofStreptomyces coelicolorA3(2) andStreptomyces avermitilisreveals that each contains 55 putative eukaryotic-type protein phosphatases (PPs), the largest number ever identified from any single prokaryotic organism. Unlike most other prokaryotic genomes that have only one or two superfamilies of eukaryotic-type PPs, the streptomycete genomes possess the eukaryotic-type PPs that belong to four superfamilies: 2 phosphoprotein phosphatases and 2 low-molecular-mass protein tyrosine phosphatases in each species, 49 Mg2+- or Mn2+-dependent protein phosphatases (PPMs) and 2 conventional protein tyrosine phosphatases (CPTPs) inS. coelicolorA3(2), and 48 PPMs and 3 CPTPs inS. avermitilis. Sixty-four percent of the PPs found inS. coelicolorA3(2) have orthologues inS. avermitilis, indicating that they originated from a common ancestor and might be involved in the regulation of more conserved metabolic activities. The genes of eukaryotic-type PP unique to each surveyed streptomycete genome are mainly located in two arms of the linear chromosomes and their evolution might be involved in gene acquisition or duplication to adapt to the extremely variable soil environments where these organisms live. In addition, 56 % of the PPs fromS. coelicolorA3(2) and 65 % of the PPs fromS. avermitilispossess at least one additional domain having a putative biological function. These include the domains involved in the detection of redox potential, the binding of cyclic nucleotides, mRNA, DNA and ATP, and the catalysis of phosphorylation reactions. Because they contained multiple functional domains, most of them were assigned functions other than PPs in previous annotations. Although few studies have been conducted on the physiological functions of the PPs in streptomycetes, the existence of large numbers of putative PPs in these two streptomycete genomes strongly suggests that eukaryotic-type PPs play important regulatory roles in primary or secondary metabolic pathways. The identification and analysis of such a large number of putative eukaryotic-type PPs fromS. coelicolorA3(2) andS. avermitilisconstitute a basis for further exploration of the signal transduction pathways mediated by these phosphatases in industrially important strains of streptomycetes.
Enzyme system of Clostridium stercorarium for hydrolysis of arabinoxylan: reconstitution of the in vivo system from recombinant enzymes
Authors: Adelsberger Helmuth , Hertel Christian , Glawischnig Erich , Zverlov Vladimir V. , Schwarz Wolfgang H.
Published: July 2004

Four extracellular enzymes of the thermophilic bacteriumClostridium stercorariumare involved in the depolymerization of de-esterified arabinoxylan: Xyn11A, Xyn10C, Bxl3B, and Arf51B. They were identified in a collection of eight clones producing enzymes hydrolysing xylan (xynA,xynB,xynC),β-xyloside (bxlA,bxlB,bglZ) andα-arabinofuranoside (arfA,arfB). The modular enzymes Xyn11A and Xyn10C represent the major xylanases in the culture supernatant ofC. stercorarium. Both hydrolyse arabinoxylan in an endo-type mode, but differ in the pattern of the oligosaccharides produced. Of the glycosidases, Bxl3B degrades xylobiose and xylooligosaccharides to xylose, and Arf51B is able to release arabinose residues from de-esterified arabinoxylan and from the oligosaccharides generated. The other glycosidases either did not attack or only marginally attacked these oligosaccharides. Significantly more xylanase and xylosidase activity was produced during growth on xylose and xylan. This is believed to be the first time that, in a single thermophilic micro-organism, the complete set of enzymes (as well as the respective genes) to completely hydrolyse de-esterified arabinoxylan to its monomeric sugar constituents, xylose and arabinose, has been identified and the enzymes producedin vivo. The active enzyme system was reconstitutedin vitrofrom recombinant enzymes.
FixJ-regulated genes evolved through promoter duplication in Sinorhizobium meliloti
Authors: Ferrières Lionel , Francez-Charlot Anne , Gouzy Jérôme , Rouillé Stéphane , Kahn Daniel
Published: July 2004

The FixLJ two-component system ofSinorhizobium melilotiis a global regulator, turning on nitrogen-fixation genes in microaerobiosis. Up to now,nifAandfixKwere the only genes known to be directly regulated by FixJ. We used a genomic SELEX approach in order to isolate new FixJ targets in the genome. This led to the identification of 22 FixJ binding sites, including the known sites in thefixK1andfixK2promoters. FixJ binding sites are unevenly distributed among the three replicons constituting theS. melilotigenome: a majority are carried either by pSymA or by a short chromosomal region of non-chromosomal origin. Thus FixJ binding sites appear to be preferentially associated with the pSymA replicon, which carries thefixJgene. Functional analysis of FixJ targets led to the discovery of two new FixJ-regulated genes,smc03253andproB2. This FixJ-dependent regulation appears to be mediated by a duplication of the wholefixKpromoter region, including the beginning of thefixKgene. Similar duplications were previously reported for thenifHpromoter. By systematic comparison of all promoter regions we found 17 such duplications throughout the genome, indicating that promoter duplication is a common mechanism for the evolution of regulatory pathways inS. meliloti.
Positive effects of multiple pch genes on expression of the locus of enterocyte effacement genes and adherence of enterohaemorrhagic Escherichia coli O157 : H7 to HEp-2 cells
Authors: Iyoda Sunao , Watanabe Haruo
Published: July 2004

Enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC, respectively) genomes contain a pathogenicity island, termed the locus of enterocyte effacement (LEE), which encodes genes involved in the formation of attaching and effacing lesions on epithelial cells. To elucidate the regulatory mechanism of the LEE genes in EHEC, an EHEC O157 genomic library was screened for clones which modulated expression of the LEE genes. From more than 5000 clones, a DNA fragment was obtained containing a perC homologue as a positive regulator for the LEE genes. In EPEC, perC is known to be part of the per operon, along with perA and perB, located on the EPEC adherence factor plasmid, which is not found in EHEC. However, the complete genome sequence of EHEC O157 Sakai strain reveals that there are five perC-like sequences, but no perA and perB, on the chromosome. These five perC homologues were characterized, and it was found that three of the homologues (renamed
perC

homologue pchA, pchB and pchC) encoded 104 aa proteins, and when expressed on a multicopy plasmid enhanced the expression of LEE genes. In contrast, perC homologues encoding proteins of 89 and 90 aa, renamed pchD and pchE, respectively, had no significant effect. Deletion mutants of the pch genes were constructed, and the effect on the expression of LEE-encoded type III effector proteins, such as EspA, B and D, and adhesion phenotype to HEp-2 cells was examined. Deletion of pchA or pchB, but not pchC, decreased the expression of Esp proteins and adhesion to HEp-2 cells. Such effects were more apparent with mutants carrying double deletions of pchA/pchB or pchA/pchC, suggesting that pchA/B/C are all necessary for full expression of the LEE genes and adhesion to HEp-2 cells. Further study demonstrated that the positive effect of pchA/B/C was caused by enhanced transcription of the LEE-encoded regulatory gene, ler. Introduction of a multicopy plasmid carrying each pchA/B/C gene significantly induced microcolony formation by EHEC O157 on HEp-2 cells. These results suggest that the pchABC genes are necessary for full virulence of EHEC O157.
Biochemical and molecular characterization of Lactobacillus reuteri 121 reuteransucrase
Authors: Kralj S. , van Geel-Schutten G. H. , van der Maarel M. J. E. C. , Dijkhuizen L.
Published: July 2004

Lactobacillus reuteristrain 121 uses sucrose for synthesis of a unique, soluble glucan (‘reuteran’) with mainlyα-(1→4) glucosidic linkages. The gene (gtfA) encoding this glucansucrase enzyme had previously been characterized. Here, a detailed biochemical and molecular analysis of the GTFA enzyme is presented. This is believed to be the first report describing reuteransucrase enzyme kinetics and the oligosaccharides synthesized with various acceptors. Alignments of the GTFA sequence with glucansucrases fromStreptococcusandLeuconostocidentified conserved amino-acid residues in the catalytic core critical for enzyme activity. Mutants Asp1024Asn, Glu1061Gln and Asp1133Asn displayed 300- to 1000-fold-reduced specific activities. To investigate the role of the relatively large N-terminal variable domain (702 amino acids) and the relatively short C-terminal putative glucan-binding domain (267 amino acids, with 11 YG repeats), various truncated derivatives of GTFA (1781 amino acids) were constructed and characterized. Deletion of the complete N-terminal variable domain of GTFA (GTFA-ΔN) had little effect on reuteran characteristics (size, distribution of glycosidic linkages), but the initial transferase activity of the mutant enzyme increased drastically. Sequential C-terminal deletions (up to six YG repeats) in GTFA-ΔN also had little effect on reuteran characteristics. However, enzyme kinetics drastically changed. Deletion of 7, 8 or 11 YG repeats resulted in dramatic loss of total enzyme activity (43-, 63- and 1000-fold-reduced specific activities, respectively). Characterization of sequential C-terminal deletion mutants of GTFA-ΔN revealed that the C-terminal domain of reuteransucrase has an important role in glucan binding.
rexAB mutants in Streptococcus pneumoniae
Authors: Halpern David , Gruss Alexandra , Claverys Jean-Pierre , Karoui Meriem El
Published: July 2004

Streptococcus pneumoniae is a human pathogen that is naturally transformable. In this study a major component of the homologous recombination pathway, the RexAB exonuclease/helicase, was characterized. rexA and rexB insertional mutants were constructed using mariner mutagenesis and found to have identical phenotypes. Both rexAB mutants displayed poor cell viability, reduced double-strand exonuclease activity, UV sensitivity and a reduced level of gene conversion compared to the wild-type strain. No effect was observed on plasmid and chromosomal transformation efficiencies. These results indicate that in S. pneumoniae, RexAB is required for DNA repair, but not for chromosomal transformation and plasmid establishment.
A two-plasmid system for stable, selective-pressure-independent expression of multiple extracellular proteins in mycobacteria
Authors: Harth Günter , Masleša-Galić Saša , Horwitz Marcus A.
Published: July 2004

Recombinant mycobacteria expressing Mycobacterium tuberculosis extracellular proteins are leading candidates for new vaccines against tuberculosis and other mycobacterial diseases, and important tools both in antimycobacterial drug development and basic research in mycobacterial pathogenesis. Recombinant mycobacteria that stably overexpress and secrete major extracellular proteins of M. tuberculosis in native form on plasmids pSMT3 and pNBV1 were previously constructed by the authors. To enhance the versatility of this plasmid-based approach for mycobacterial protein expression, the Escherichia coli/mycobacteria shuttle plasmid pGB9 was modified to accommodate mycobacterial genes expressed from their endogenous promoters. Previous studies showed that the modified plasmid, designated pGB9.2, derived from the cryptic Mycobacterium fortuitum plasmid pMF1, was present at a low copy number in both E. coli and mycobacteria, and expression of recombinant M. tuberculosis proteins was found to be at levels paralleling its copy number, that is, approximating their endogenous levels. Plasmid pGB9.2 was compatible with the shuttle vectors pSMT3 and pNBV1 and in combination with them it simultaneously expressed the M. tuberculosis 30 kDa extracellular protein FbpB. Plasmid pGB9.2 was stably maintained in the absence of selective pressure in three mycobacterial species: Mycobacterium bovis BCG, M. tuberculosis and M. smegmatis. Plasmid pGB9.2 was found to be self-transmissible between both fast- and slow-growing mycobacteria, but not from mycobacteria to E. coli or between E. coli strains. The combination of two compatible plasmids in one BCG strain allows expression of recombinant mycobacterial proteins at different levels, a potentially important factor in optimizing vaccine potency.
Symbionts of the gut flagellate Staurojoenina sp. from Neotermes cubanus represent a novel, termite-associated lineage of Bacteroidales: description of ‘Candidatus Vestibaculum illigatum’
Authors: Stingl Ulrich , Maass Annelie , Radek Renate , Brune Andreas
Published: July 2004

The symbioses between cellulose-degrading flagellates and bacteria are one of the most fascinating phenomena in the complex micro-ecosystem found in the hindgut of lower termites. However, little is known about the identity of the symbionts. One example is the epibiotic bacteria colonizing the surface of hypermastigote protists of the genusStaurojoenina. By using scanning electron microscopy, it was shown that the whole surface ofStaurojoeninasp. from the termiteNeotermes cubanusis densely covered with long rod-shaped bacteria of uniform size and morphology. PCR amplification of 16S rRNA genes from isolated protozoa and subsequent cloning yielded a uniform collection of clones with virtually identical sequences. Phylogenetic analysis placed them as a new lineage among theBacteroidales, only distantly related to other uncultivated bacteria in the hindgut of other termites, including an epibiont of the flagellateMixotricha paradoxa. The closest cultivated relative wasTannerella forsythensis(<85 % sequence identity). Fluorescencein situhybridization with a newly designed clone-specific oligonucleotide probe confirmed that these sequences belong to the rod-shaped epibionts ofStaurojoeninasp. Transmission electron microscopy confirmed the presence of a Gram-negative cell wall and revealed special attachment sites for the symbionts on the cell envelope of the flagellate host. Based on the isolated phylogenetic position and the specific association with the surface ofStaurojoeninasp., we propose to classify this new taxon ofBacteroidalesunder the provisional name ‘CandidatusVestibaculum illigatum’.
HthA, a putative DNA-binding protein, and HthB are important for fruiting body morphogenesis in Myxococcus xanthus
Authors: Nielsen Mette , Rasmussen Anders Aa. , Ellehauge Eva , Treuner-Lange Anke , Søgaard-Andersen Lotte
Published: July 2004

In response to starvation,Myxococcus xanthusinitiates a developmental programme that results in the formation of spore-filled multicellular fruiting bodies. Fruiting body formation depends on the temporal and spatial coordination of aggregation and sporulation and involves temporally and spatially coordinated changes in gene expression. This paper reports the identification of two genes,hthAandhthB, that are important for fruiting body formation.hthAandhthBare co-transcribed, and transcription of the two genes decreases strongly during development. Loss of HthA and HthB function results in delayed aggregation, a reduction in the level of sporulation, and abnormal developmental gene expression. Extracellular complementation experiments showed that the developmental defects caused by loss of HthA and HthB function are not due to the inability to synthesize an intercellular signal required for fruiting body formation. HthA, independent of HthB, is required for aggregation. HthB, alone or in combination with HthA, is required for sporulation. HthA is predicted to contain a C-terminal helix–turn–helix DNA-binding domain. Intriguingly, the N-terminal part of HthA does not exhibit significant amino acid similarity to proteins in the databases. The HthB protein lacks homologues in the databases. The results suggest that HthA is a novel DNA-binding protein, which regulates transcription of genes important for aggregation, and that HthB, alone or in combination with HthA, stimulates sporulation.
A global role for Fis in the transcriptional control of metabolism and type III secretion in Salmonella enterica serovar Typhimurium
Authors: Kelly Arlene , Goldberg Martin D. , Carroll Ronan K. , Danino Vittoria , Hinton Jay C. D. , Dorman Charles J.
Published: July 2004

Fis is a key DNA-binding protein involved in nucleoid organization and modulation of many DNA transactions, including transcription in enteric bacteria. The regulon of genes whose expression is influenced by Fis inSalmonella entericaserovar Typhimurium (S. typhimurium) has been defined by DNA microarray analysis. These data suggest that Fis plays a central role in coordinating the expression of both metabolic and type III secretion factors. The genes that were most strongly up-regulated by Fis were those involved in virulence and located in the pathogenicity islands SPI-1, SPI-2, SPI-3 and SPI-5. Similarly, motility and flagellar genes required Fis for full expression. This was shown to be a direct effect as purified Fis protein bound to the promoter regions of representative flagella and SPI-2 genes. Genes contributing to aspects of metabolism known to assist the bacterium during survival in the mammalian gut were also Fis-regulated, usually negatively. This category included components of metabolic pathways for propanediol utilization, biotin synthesis, vitamin B12transport, fatty acids and acetate metabolism, as well as genes for the glyoxylate bypass of the tricarboxylic acid cycle. Genes found to be positively regulated by Fis included those for ethanolamine utilization. The data reported reveal the central role played by Fis in coordinating the expression of both housekeeping and virulence factors required byS. typhimuriumduring life in the gut lumen or during systemic infection of host cells.
A signaling mucin at the head of the Cdc42- and MAPK-dependent filamentous growth pathway in yeast
Authors: Cullen Paul J. , Sabbagh Walid , Graham Ellie , Irick Molly M. , van Olden Erin K. , Neal Cassandra , Delrow Jeffrey , Bardwell Lee , Sprague George F.
Published: July 2004

Signaling molecules such as Cdc42 and mitogen-activated protein kinases (MAPKs) can function in multiple pathways in the same cell. Here, we propose one mechanism by which such factors may be directed to function in a particular pathway such that a specific response is elicited. Using genomic approaches, we identify a new component of the Cdc42- and MAPK-dependent signaling pathway that regulates filamentous growth (FG) in yeast. This factor, called Msb2, is a FG-pathway-specific factor that promotes differential activation of the MAPK for the FG pathway, Kss1. Msb2 is localized to polarized sites on the cell surface and interacts with Cdc42 and with the osmosensor for the high osmolarity glycerol response (HOG) pathway, Sho1. Msb2 is glycosylated and is a member of the mucin family, proteins that in mammalian cells promote disease resistance and contribute to metastasis in cancer cells. Remarkably, loss of the mucin domain of Msb2 causes hyperactivity of the FG pathway, demonstrating an inhibitory role for mucin domains in MAPK pathway activation. Taken together, our data suggest that Msb2 is a signaling mucin that interacts with general components, such as Cdc42 and Sho1, to promote their function in the FG pathway.
Differential gene regulation by the SRC family of coactivators
Authors: Zhang Hua , Yi Xia , Sun Xiaojing , Yin Na , Shi Bin , Wu Huijian , Wang Dan , Wu Ge , Shang Yongfeng
Published: July 2004

SRCs (steroid receptor coactivators) are required for nuclear receptor-mediated transcription and are also implicated in the transcription initiation by other transcription factors, such as STATs and NFκB. Despite phenotypic manifestations in gene knockout mice for SRC-1, GRIP1, and AIB1 of the SRC (Steroid Receptor Coactivator) family indicating their differential roles in animal physiology, there is no clear evidence, at the molecular level, to support a functional specificity for these proteins. We demonstrated in this report that two species of SRC coactivators, either as AIB1:GRIP1 or as AIB1:SRC-1 are recruited, possibly through heterodimerization, on the promoter of genes that contain a classical hormone responsive element (HRE). In contrast, on non-HRE-containing gene promoters, on which steroid receptors bind indirectly, either GRIP1 or SRC-1 is recruited as a monomer, depending on the cellular abundance of the protein. Typically, non-HRE-containing genes are early genes activated by steroid receptors, whereas HRE-containing genes are activated later. Our results also showed that SRC proteins contribute to the temporal regulation of gene transcription. In addition, our experiments revealed a positive correlation between AIB1/c-myc overexpression in ER+ breast carcinoma samples, suggesting a possible mechanism for AIB1 in breast cancer carcinogenesis.
Dm-myb mutant lethality in Drosophila is dependent upon mip130: positive and negative regulation of DNA replication
Authors: Beall Eileen L. , Bell Maren , Georlette Daphne , Botchan Michael R.
Published: July 2004

Gene amplification at the chorion loci in Drosophila ovarian follicle cells is a model for the developmental regulation of DNA replication. Previously, we showed that the Drosophila homolog of the Myb oncoprotein family (DmMyb) is tightly associated with four additional proteins and that DmMyb is required for this replication-mediated amplification. Here we used targeted mutagenesis to generate a mutant in the largest subunit of the DmMyb complex, the Aly and Lin-9 family member, Myb-interacting protein 130 (Mip130). We found that mip130 mutant females are sterile and display inappropriate bromodeoxyuridine (BrdU) incorporation throughout the follicle cell nuclei at stages undergoing gene amplification. Whereas mutations in Dm-myb are lethal, mutations in mip130 are viable. Surprisingly, Dm-myb mip130 double mutants are also viable and display the same phenotypes as mip130 mutants alone. This suggests that Mip130 activity without DmMyb counteraction may be responsible for the Dm-myb mutant lethality. RNA interference (RNAi) to selectively remove each DmMyb complex member revealed that DmMyb protein levels are dependent upon the presence of several of the complex members. Together, these data support a model in which DmMyb activates a repressive complex containing Mip130 into a complex competent to support replication at specific loci in a temporally and developmentally proscribed manner.
Topological domain structure of the Escherichia coli chromosome
Authors: Postow Lisa , Hardy Christine D. , Arsuaga Javier , Cozzarelli Nicholas R.
Published: July 2004

The circular chromosome of Escherichia coli is organized into independently supercoiled loops, or topological domains. We investigated the organization and size of these domains in vivo and in vitro. Using the expression of >300 supercoiling-sensitive genes to gauge local chromosomal supercoiling, we quantitatively measured the spread of relaxation from double-strand breaks generated in vivo and thereby calculated the distance to the nearest domain boundary. In a complementary approach, we gently isolated chromosomes and examined the lengths of individual supercoiled loops by electron microscopy. The results from these two very different methods agree remarkably well. By comparing our results to Monte Carlo simulations of domain organization models, we conclude that domain barriers are not placed stably at fixed sites on the chromosome but instead are effectively randomly distributed. We find that domains are much smaller than previously reported, ∼10 kb on average. We discuss the implications of these findings and present models for how domain barriers may be generated and displaced during the cell cycle in a stochastic fashion.
Tal, a Tsg101-specific E3 ubiquitin ligase, regulates receptor endocytosis and retrovirus budding
Authors: Amit Ido , Yakir Liat , Katz Menachem , Zwang Yaara , Marmor Mina D. , Citri Ami , Shtiegman Keren , Alroy Iris , Tuvia Shmuel , Reiss Yuval , Roubini Eli , Cohen Maya , Wides Ron , Bacharach Eran , Schubert Ullrich , Yarden Yosef
Published: July 2004

The tumor suppressor gene 101 (tsg101) regulates vesicular trafficking processes in yeast and mammals. We report a novel protein, Tal (Tsg101-associated ligase), whose RING finger is necessary for multiple monoubiquitylation of Tsg101. Bivalent binding of Tsg101 to a tandem tetrapeptide motif (PTAP) and to a central region of Tal is essential for Tal-mediated ubiquitylation of Tsg101. By studying endocytosis of the epidermal growth factor receptor and egress of the human immunodeficiency virus, we conclude that Tal regulates a Tsg101-associated complex responsible for the sorting of cargo into cytoplasm-containing vesicles that bud at the multivesicular body and at the plasma membrane.
Cdk1-Clb4 controls the interaction of astral microtubule plus ends with subdomains of the daughter cell cortex
Authors: Maekawa Hiromi , Schiebel Elmar
Published: July 2004

As in many polarized cells, spindle alignment in yeast is essential and cell cycle regulated. A key step that governs spindle alignment is the selective binding of the Kar9 protein to only one of the two spindle pole bodies (SPBs). It has been suggested that cyclin-dependent kinase Cdc28, in complex with cyclin Clb4, associates only with the SPB in the mother cell and so prevents Kar9 binding to this SPB. However, here we show that the nonoverexpressed Clb4 associates with the budward-directed SPB through Kar9. Cdc28-Clb4 then uses Kar9 as a carrier to move from this SPB to the plus ends of astral microtubules, where Cdc28-Clb4 regulates the interactions between microtubule ends and subdomains of the bud cortex. In the absence of Cdc28-Clb4 activity (G1/S phase), astral microtubules interact with the bud tip in a manner dependent on actin, Myo2, and Kar9. Coincidentally with reaching the bud cortex in S phase, Cdc28-Clb4 facilitates the dissociation of the microtubule bud tip interaction and their capture by the bud neck. This transition prevents the preanaphase spindle from becoming prematurely pulled into the bud. Thus, Cdc28-Clb4 facilitates spindle alignment by regulating the interaction of astral microtubules with subdomains of the bud cortex.
Role ofAplysiaCell Adhesion Molecules During 5-HT-Induced Long-Term Functional and Structural Changes
Authors: Han Jin-Hee , Lim Chae-Seok , Lee Yong-Seok , Kandel Eric R. , Kaang Bong-Kiun
Published: July 2004

We previously reported that five repeated pulses of 5-HT lead to down-regulation of the TM-apCAM isoform at the surface ofAplysiasensory neurons (SNs). We here examined whether apCAM down-regulation is required for 5-HT-induced long-term facilitation. We also analyzed the role of the cytoplasmic and extracellular domains by overexpressing various apCAM mutants by DNA microinjection. When TM-apCAM was up-regulated in SNs by DNA microinjection, five pulses of 5-HT failed to produce either synaptic facilitation or an enhancement of synaptic growth, suggesting that down-regulation of apCAM is required for 5-HT-induced EPSP enhancement and new varicosity formation. However, disrupting the extracellular domain function of overexpressed apCAM with a specific antibody restored 5-HT-induced excitatory postsynaptic potential increase but not synaptic growth. The overexpression of the MAP Kinase mutant of TM-apCAM, which is not internalized by 5-HT, inhibited new varicosity formation, but did not inhibit excitatory postsynaptic potential increase. Deletion mutants containing only the cytoplasmic portion of apCAM blocked 5-HT-induced synaptic growth but not excitatory postsynaptic potential increase. Thus, our data suggest that TM-apCAM may act as a suppressor of both synaptic-strength enhancement in pre-existing synapses and of new synaptic varicosity formation in the nonsynaptic region, via different mechanisms.
Intra-Amygdala Muscimol Injections Impair Freezing and Place Avoidance in Aversive Contextual Conditioning
Authors: Holahan Matthew R. , White Norman M.
Published: July 2004

Rats were trained by shocking them in a closed compartment. When subsequently tested in the same closed compartment with no shock, normal rats showed an increased tendency to freeze. They also showed an increased tendency to actively avoid the compartment when given access to an adjacent neutral compartment for the first time. Amygdala inactivation with bilateral muscimol injections before training attenuated freezing and eliminated avoidance during the test. Rats trained in a normal state and given intra-amygdala muscimol injections before the test did not freeze or avoid the shock-paired compartment. This pattern of effects suggests that amygdala inactivation during training impaired acquisition of a conditioned response (CR) due either to inactivation of a neural substrate essential for its storage or to elimination of a memory modulation effect that facilitates its storage in some other brain region(s). The elimination of both freezing and active avoidance by amygdala inactivation during testing suggests that neither of these behaviors is the CR. The possibility that the CR is a set of internal responses that produces both freezing and avoidance as well as other behavioral effects is discussed.
The Hippocampus and Memory for “What,” “Where,” and “When”
Authors: Ergorul Ceren , Eichenbaum Howard
Published: July 2004

Previous studies have indicated that nonhuman animals might have a capacity for episodic-like recall reflected in memory for “what” events that happened “where” and “when”. These studies did not identify the brain structures that are critical to this capacity. Here we trained rats to remember single training episodes, each composed of a series of odors presented in different places on an open field. Additional assessments examined the individual contributions of odor and spatial cues to judgments about the order of events. The results indicated that normal rats used a combination of spatial (“where”) and olfactory (“what”) cues to distinguish “when” events occurred. Rats with lesions of the hippocampus failed in using combinations of spatial and olfactory cues, even as evidence from probe tests and initial sampling behavior indicated spared capacities for perception of spatial and odor cues, as well as some form of memory for those individual cues. These findings indicate that rats integrate “what,” “where,” and “when” information in memory for single experiences, and that the hippocampus is critical to this capacity.
Synaptic Plasticity and Translation Initiation
Authors: Klann Eric , Antion Marcia D. , Banko Jessica L. , Hou Lingfei
Published: July 2004

It is widely accepted that protein synthesis, including local protein
synthesis at synapses, is required for several forms of synaptic plasticity.
Local protein synthesis enables synapses to control synaptic strength
independent of the cell body via rapid protein production from pre-existing
mRNA. Therefore, regulation of translation initiation is likely to be
intimately involved in modulating synaptic strength. Our understanding of the
translation-initiation process has expanded greatly in recent years. In this
review, we discuss various aspects of translation initiation, as well as
signaling pathways that might be involved in coupling neurotransmitter and
neurotrophin receptors to the translation machinery during various forms of
synaptic plasticity.
Determination of the optimal allocation of parameters for gauge repeatability and reproducibility study
Authors: Pan Jeh‐Nan
Published: July 2004

Recently, gauge repeatability and reproducibility (GR&R) study has been highly regarded by the quality practitioners when QS9000 and D19000 become fashionable requirements for manufacturing industries. Measurement plays a significant role in helping organizations improve their product quality. Good quality of products is the key factor towards business success. Therefore, how to ensure the quality of measurement becomes an important task for quality practitioners. In performing the GR&R study, several parameters, such as the appropriate sample size of parts (n), number of inspectors (p) and replicate measurements (k) are frequently asked by quality personnel in industries. The adequacy of current way of (n, p, k) selection is very questionable. A statistical method using the shortest confidence interval and its associated computer programming algorithm are presented in this paper for evaluating the optimal allocation among sample size of parts (n), number of inspectors (p) and replicate measurements (k). Hopefully, it can provide a useful reference for quality practitioners in industries.