bluuebunny/crossref_metadata_2025_split huggingface dataset:
Variations in the expression of pili: the effect on adherence of Neisseria meningitidis to human epithelial and endothelial cells
Authors: Virji Mumtaz , Alexandrescu Carol , Ferguson David J. P. , Saunders Jon R. , Moxon E. Richard 
Published: October 2006

SummaryThe effect of variations in Neisseria meningitidis pili on bacterial interactions with three epithelial cell lines as well as human umbilical vein endothelial cells was studied using a panel of seven strains expressing Class I or Class II pili. Comparison of adherence of piliated and pilus‐deficient variants of each strain to epithelial cells suggested that Class I pili may mediate bacterial adherence with all three epithelial cell lines. In contrast, Class II pili of the strains used did not increase bacterial adherence to Hep‐2 larynx carcinoma cells, although an increase in adherence to Chang conjunctival and A549 lung carcinoma epithelial cells was observed in the Class II pili‐expressing strains. In addition to these inter‐class functional variations, differences in adherence to epithelial cells were also observed among Class I and Class II strains. Functionally different pilin variants of one Class I strain, MC58, were obtained by single colony isolation. One piliated variant was identified which had concurrently lost the ability to adhere to both Chang and Hep‐2 cells (‘non‐adherent’ phenotype; adherence of < 2 bacteria per cell). In addition, several adherent pilin variants were isolated from non‐adherent Pil‐ and Pil‐ bacteria by selection on Chang cells (adherence of 10–25 bacteria per cell). In contrast to epithelial cells, al) variant pili, whether of Class I or Class II, adhered to endothelial cells in substantially larger numbers (<50 bacteria per cell) and therefore implied the existence of distinct mechanisms in pilus‐facilitated interactions of N. meningitidis with endothelial and epithelial cells.
Identification, genetic analysis and DNA sequence of a 7.8‐kb virulence region of the Salmonella typhimurium virulence plasmid
Authors: Gulig Paul A. , Caldwell Allison L. , Chiodo Vince A. 
Published: October 2006

SummaryThe 90‐kilobase (kb) virulence plasmid of Salmonella typhimurium is responsible for invasion from the intestines to mesenteric lymph nodes and spleens of orally inoculated mice. We used Tn5 and aminoglycoside phosphotransferase (aph) gene insertion mutagenesis and deletion mutagenesis of a previously identified 14‐kb virulence region to reduce this virulence region to 7.8kb. The 7.8‐kb virulence region subcloned into a low copy‐number vector conferred a wild‐type level of splenic infection to virulence plasmid‐cured S. typhimurium and conferred essentially a wild‐type oral LD50. Insertion mutagenesis identified five loci essential for virulence, and DNA sequence analysis of the virulence region identified six open reading frames. Expected protein products were identified from four of the six genes, with three of the proteins identified as doublet bands in Escherichia coli minicells. Three of the five mutated genes were able to be complemented by clones containing only the corresponding wild‐type gene. Only one of the five deduced amino acid sequences, that of the positive regulatory element, SpvR, possessed significant homology to other proteins. The codon usage for the virulence genes showed no codon bias, which is consistent with the low levels of expression observed for the corresponding proteins. Consensus promoters for several different sigma factors were identified upstream of several of the genes, whereas only consensus Rho‐dependent termination sequences were observed between certain of the genes. The operon structure of this virulence region therefore appears to be complex. The construction of the cloned 7.8‐kb virulence region and the determination of the DNA sequence will aid in the further genetic analysis of the five plasmid‐encoded virulence genes of S. typhimurium.
Carbon catabolite repression can account for the temporal pattern of expression of a penicillin biosynthetic gene in Aspergillus nidulans
Authors: Espeso Eduardo A. , Peñalva Miguel A. 
Published: October 2006

Summary Aspergillus nidulans synthesizes penicillins as secondary metabolites when grown under certain culture conditions. Broths containing carbon (C) sources that give rise to carbon catabolite repression (CCR) support a much lower antibiotic yield than broths with non‐repressing C sources. Steady‐state levels of the isopenicillin N synthetase (IPNS) gene transcript are considerably reduced in mycelia grown with repressing C sources and are derepressed in mycelia grown with sugars which do not cause CCR, indicating that penicillin biosynthesis is regulated by CCR through transcriptional control of structural genes. CCR is sufficient to explain the temporal window of expression of the IPNS gene during the growth cycle since (i) the transcript becomes derepressed as soon as the concentration of a repressing C source drops to non‐repressing levels and (ii) derepressing C sources sustain derepressed IPMS transcription at all tested moments of the growth cycle. Several tested hypofunctional mutations in creA (the negatively acting regulatory gene which mediates CCR in A. nidulans) do not cause full derepression of IPNS transcript in the presence of a repressing C source. The slight degree of IPNS derepression caused by some creAd (derepressed) alleles parallels the strength of the mutation (as determined by the morphological effect they elicit).
Starvation‐inducible loci of Salmonella typhimurium: regulation and roles in starvation‐survival
Authors: Spector Michael P. , Cubitt Christopher L. 
Published: October 2006

SummaryFour starvation‐inducible loci (stiA, stiB, stiC, and stiE) of Salmonella typhimurium have been extensively characterized as to their genetic and physiologic regulation, and their roles in survival during prolonged simultaneous phosphate (P)‐, carbon (C)‐and nitrogen (N)‐starvation (PCN‐starvation). Strains of S. typhimurium LT‐2, isogenic with the exception of lacking either the stiA, stiB or stiC locus, died off more quickly and survived at much reduced levels compared with their wild‐type parent. When certain sti mutations were combined in the same strain, we found that viability of these cultures declined even more rapidly, and starvation‐survival was affected to leveis over‐and‐above the additive effects of each individual mutation, indicating an epistatic relationship between these loci. All four sti loci were, directly or indirectly, under negative control by the crp gene product (cAMP receptor protein, CRP). With the exception of stiB, all were similarly regulated by the cya gene product (i.e., cAMP). This suggests that CRP acts alone, or with a signal molecule other than cAMP, to cause repression of the stiB locus. In addition, all four loci are under positive regulation by the relA gene product (i.e., ppGpp) during C‐ or N‐starvation, but not P‐starvation. Since not all relA‐depen‐dent sti loci are induced during both C‐ and N‐starvation, we propose that two separate ppGpp‐dependent pathways function during C‐starvation and N‐starvation, respectively. Possible models for separate P‐, C‐and N‐starvation‐induction pathways are discussed.
Insertional mutagenesis in the extreme thermophilic eubacteria Thermus thermophilus HB8
Authors: Lasa I. , Castón J. R. , Fernández‐Herrero L. A. , de Pedro M. A. , Berenguer J. 
Published: October 2006

SummaryThe transcription and translation signals of the S‐layer gene (slpA) from Thermus thermophilus HB8 have been used to express a thermostable kanamycin adenyl transferase gene in this organism. The chimaeric resistance gene was inserted in vitro into sIpA to produce different inactive forms of the gene, which were used to transform T. thermophilus HB8. After 48 hours of incubation at 70°C, only two constructions that contained the kat gene flanked by Thermus sequences from both sides of sIpA were able to produce protein layer (P100)‐defective mutants. The mutants obtained with both constructions showed identical protein patterns, in which a major 50 kDa protein and two other minor proteins were tentatively identified as P100 fragments, expressed from the extreme 5’end of slpA. They also exhibited important phenotypic defects, such as slow growth in liquid broth, a tendency to aggregate as rotund bodies, a twisted filamentous shape, and an extreme sensitivity to lysozyme, suggesting protective and shaping roles for the S‐layer in T. thermophilus HB8. These results also demonstrate for the first time the feasibility of using selective antibiotic‐resistance markers in extreme thermophiles.
Molecular analysis of the lac operon encoding the binding‐protein‐dependent lactose transport system and β‐galactosidase in Agrobacterium radiobacter
Authors: Williams S. G. , Greenwood J. A. , Jones C. W. 
Published: October 2006

SummaryThe genes coding for the binding‐protein‐dependent lactose transport system and β‐galactosidase in Agrobacterium radiobacter strain AR50 were cloned and partially sequenced. A novel lac operon was identified which contains genes coding for a lactose‐binding protein (lacE), two integral membrane proteins (lacF and lacG), an ATP‐binding protein (lacK) and β‐galactosidase (lacZ). The operon is transcribed in the order lacEFGZK, The operon is controlled by an upstream regulatory region containing putative ‐35 and ‐10 promoter sites, an operator site, a CRP‐binding site probably mediating catabolite repression by glucose and galactose, and a regulatory gene (lacl) encoding a repressor protein which mediates induction by lactose and other galactosides in wild‐type A. radiobacter (but not in strain AR50, thus allowing constitutive expression of the lac operon). The derived amino acid sequences of the gene products indicate marked similarities with other binding‐protein‐dependent transport systems in bacteria.
Clarification of the structural and functional features of the osmoregulated kdp operon of Escherichia coli
Authors: Sugiura A. , Nakashima K. , Tanaka K. , Mizuno T. 
Published: October 2006

SummaryExpression of the Escherichia coli kdpABC operon, which is responsible for a high‐affinity potassium‐uptake system, is regulated in response to a change in the medium osmolarity. In this study, we clarified the structure and function of the kdpABC promoter including its regulatory sequence at the molecular level. The canonical ‐35 and ‐10 regions determined for the promoter were not fully functional, i.e. in addition to them, a cis‐acting sequence located upstream of the ‐35 region was essential for full activation of the promoter. This upstream sequence was demonstrated to be the target site for the trans‐acting activator, KdpE.
Phosphotransfer signal transduction between two regulatory factors involved in the osmoregulated kdp operon in Escherichia coli
Authors: Nakashima K. , Sugiura A. , Momoi H. , Mizuno T. 
Published: October 2006

SummaryThe proteins KdpD and KdpE are regulatory factors critically involved in the osmotic regulation of the kdpABC operon that is responsible for a high‐affinity transport system in Escherichia coli. In this study, we obtained biochemical evidence supporting the view that the KdpD protein is a sensory protein kinase that exhibits autophosphorylation and KdpE‐phosphotransfer characteristics. During the course of such studies we established a procedure for purifying the KdpE protein in large quantities. We also developed a procedure for preparing cytoplasmic membrane enriched with the KdpD protein that exhibits in vitro ability with regard to phosphorylation of KdpE protein.
Interaction of the regulatory protein NicR1 with the promoter region of the pAO1‐encoded 6‐hydroxy‐D‐nicotine oxidase gene of Arthrobacter oxidans
Authors: Bernauer H. , Mauch L. , Brandsch R. 
Published: October 2006

SummaryThe d,l‐nicotine catabolism of the Gram‐positive soil bacterium Arthrobacter oxidans is linked to the presence within the cells of the 160 kb catabolic plasmid pAO1. pAO1‐cured cells lost the catabolic enzymes and reintroduction of pAO1 by electroporation into cured cells reestablished the nic+ phenotype. DNA band shift assays with extracts from cured and pAO1+ cells suggested that pAO1 encodes the regulatory protein NicR1. Footprint analysis revealed that two homologous palindromes (IR1 and IR2), present in the 5′‐regulatory region of the 6‐HDNO gene, were protected from DNase I digestion. Binding of NicR1 to the palindromes is symmetrical, co‐operative, and stronger to IR1 containing the 6‐HDNO gene promoter than to IR2. Site‐directed mutagenesis revealed that steric constraints and sequence requirements for NicR1 ‐binding are located exclusively in the palindromic sequences. Deletions and insertions in the interpalindromic region and in the 6‐HDNO promoter ‐10 sequence had no effect on the binding characteristics of NicR1 to the 6‐HDNO regulatory region. Acting as a repressor, NicR1 prevents binding of the E. coli RNA‐polymerase to the consensus σ70 promoter in vitro. However, the Interaction of NicR1 with the 6‐HDNO promoter region in extracts of nicotine‐induced cells from various growth stages did not differ from that observed with extracts of nicotine‐uninduced cells.
The POT1 gene for yeast peroxisomal thiolase is subject to three different mechanisms of regulation
Authors: Igual J. Carlos , González‐Bosch Carmen , Franco Luis , Pérez‐Ortin José E. 
Published: October 2006

SummarySummary The Saccharomyces cerevisiae POT1 gene is, as are other yeast peroxisomal protein genes, inducible by fatty acids and repressible by glucose. We have now found that it is also induced during the stationary phase of the culture. To investigate these three regulatory circuits, we have studied the mRNA levels of regulatory mutants as well as the changes in chromatin structure upon gene activation. We conclude that the regulation of transcriptional activity in glucose repression, oleate induction, and stationary phase induction follow different molecular mechanisms. We suggest that this multiplicity of regulatory mechanisms may represent a general rule for the yeast peroxisomal protein genes.
PuIO, a component of the pullulanase secretion pathway of Klebsiella oxytoca, correctly and efficiently processes gonococcal type IV prepilin in Escherichia coli
Authors: Dupuy Bruno , Taha Muhamed‐Kheir , Possot Odile , Marchal Christian , Pugsley Anthony P. 
Published: October 2006

SummaryThe PulO protein required for extracellular secretion of pullulanase by Klebsiella oxytoca is known to be highly homologous to two type IV prepilin peptidases, namely XcpA(PilD) (Pseudomonas aeruginosa) and TcpJ (Vibrio cholerae). The predicted prepilin peptidase activity of PulO was confirmed by showing that it could correctly process the product of the cloned pilE.1 type IV pilin structural gene from Neisseria gonorrhoeae in Escherichia coli. The P. aeruginosa prepilin peptidase and another putative prepilin peptidase, ComC from Bacillus subtilis, also processed prePilE. Subcellular fractionation showed that the pilE gene product that had been processed by PulO remained associated with the cytoplasmic membrane, as did the unprocessed precursor. PulO was also shown to process three of the four prePilE–PhoA hybrids tested. Southern hybridization experiments suggest that a PulO homologue is present in the N. gonorrhoeae chromosome.
Pneumocystis carinii shows DNA homology with the ustomycetous red yeast fungi
Authors: Wakefield Ann E. , Peters Sarah E. , Banerji Suneale , Bridge Paul D. , Hall Geoffrey S. , Hawksworth David L. , Guiver Lynden A. , Allen Andrew G. , Hopkin Julian M. 
Published: October 2006

Summary Pneumocystis carinii causes life‐threatening pneumonia in T‐lymphocyte‐immunodeficient subjects in transplant and oncology units or with acquired immune deficiency syndrome (AIDS). Recent DNA homology studies show P. carinii to be a fungus. To investigate the biology and epidemiology of this parasite further, we elected to determine for it a more precise taxonomic assignment within the fungal kingdom. We screened a wide range of organisms representing the major orders of fungi using DNA amplification and subsequently sequenced a portion of the mitochondrial gene encoding the large subunit ribosomal RNA. Our data show that the opportunistic pulmonary pathogen P. carinii is closely related to the ustomycetous red yeast fungi, a group which includes organisms that are extensively distributed throughout the environment and which release many widely dispersed airborne spores.
Negative transcriptional control of iron transport in Erwinia chrysanthemi involves an iron‐responsive two‐factor system
Authors: Expert Dominique , Sauvage Chrystèle , Neilands J. B. 
Published: October 2006

SummarySystemic virulence of the phytopathogen Erwinia chrysanthemi 3937 requires a functional iron assimilation system which, in this enterobacterium, is mediated by the siderophore chrysobactin and the outer membrane transport protein Fct. We investigated the regulation of this system by iron. No direct similarity with the Escherichia coli fur gene was found. Insertional mutagenesis allowed isolation of a regulatory mutant which expressed chrysobactin and two other high‐affinity iron transport systems previously characterized in strain 3937, regardless of the iron level. RNA/DNA hybridization analysis established that regulation of chrysobactin by iron occurs at the transcriptional level. From a wild‐type gene library, a recombinant cosmid able to restore normal regulation in the mutant strain was isolated. By generating a series of subclones and mini‐Mulac insertions, we identified a regulatory locus (cbr) extending beyond c. 2.5 kb which encodes two polypeptides, Cbr A and CbrB, with molecular weights of 34000 and 55000 respectively. Functional analysis of the locus suggests that the cognate genes cbrA and cbrB are clustered within an operon. Their expression was studied through chromosomal lac gene fusions, in the presence of plasmid‐borne wild‐type constructions, under high‐ and low‐iron conditions. In summary, the data show that in the presence of iron, cbr negatively regulates the chrysobactin biosynthetic and transport genes, while under conditions of depletion, cbr is subject to negative autogeneous regulation.
Localization of an immunodominant 64kDa lipoprotein (LP 64) in the membrane of Mycoplasma gallisepticum and its role in cytadherence
Authors: Forsyth Mark H. , Tourtellotte Mark E. , Geary Steven J. 
Published: October 2006

SummaryA64kDa lipoprotein (LP 64) haemagglutinin (pI 4.9–5.0) was isolated from the membrane of Mycoplasma gallisepticum. Triton X‐114 phase partitioning has demonstrated that the hydrophobic nature of this haemagglutinin is due to a lipid portion of the molecule. Autoradiography of [3H]‐palmitate‐labelled M. gallisepticum revealed the presence of several additional lipoproteins. Immunoelectron microscopy demonstrated the localization of LP 64 to the base of the terminal structure. Densitometric scans of stained polyacrylamide gels of M. gallisepticum showed that LP 64 constitutes 1.7% of the total protein. Scans of immunoblots of M. gallisepticum indicate that LP 64 is highly immunogenic in chickens, accounting for 7.4% of the total serum IgG response at four weeks post‐infection. A quantitative value for the IgG response to LP 64, relative to the percentage of total protein (the Relative Immunogenicity Index) was 4.4. LP 64 is conserved among several strains of M. gallisepticum, but its presence could not be detected in Mycoplasma synoviae. Antiserum raised to electroeluted LP 64 reacted specifically with this lipoprotein when assessed on either one‐or two‐dimensional immunoblots of M. gallisepticum. This antiserum, as well as Fab fragments, inhibited haemagglutination of chicken erythrocytes and inhibited the attachment of 14Clabelled M. gallisepticum to chicken tracheal epithelium in vitro by 62%.
Variation in length and sequence of porin (ompP2) alleles of non‐capsulate Haemophilus influenzae
Authors: Forbes Kenneth J. , Bruce Kenneth D. , Ball Alison , Pennington T. Hugh 
Published: October 2006

SummaryLength variations of Haemophilus influenzae outer membrane porin protein P2 were found at the DNA and protein levels, notably in non‐capsulate strains. Protein length, measured by SDS‐polyacrylamide gel electrophoresis, was found to correlate with the length of the gene, measured by polymerase chain reaction amplification, and ranged from 35–42 kDa and 970–1090 nucleotides, respectively. This represents a length variation of some 15%. The genetic location of these variations was studied by restriction enzyme mapping 10 of the non‐capsulate strains revealing further polymorphisms at the DNA level. All 10 strains were distinct and differed from a type b strain. The conservation and assortment of the different restriction sites in the alleles is discussed in relation to the very great diversity previously described for this protein and of the whole genome itself in non‐capsulate strains. The roles of selection, horizontal gene transfer, and transformation in generating this diversity are discussed.
Cloning and characterization of fxp, the flexible pilin gene of Aeromonas hydrophila
Authors: Ho Alice S. Y. , Sohel Indira , Schoolnik Gary K. 
Published: October 2006

SummaryThe flexible pilus of Aeromonas hydrophila is a morphologically and biochemically unique organelle which binds eukaryotic cell surfaces and whose expression is induced by specific physiochemical conditions, fxp, the structural gene coding for the flexible pilus subunit, was localized on a 7.6 kb plasmid of A. hydrophila strain AH26. A putative Shine‐Dalgarno sequence and ‐10 and ‐35 regions were identified, a signal peptide sequence delineated, and the coding sequence compared with other bacterial sequences and found to be unique. Plasmid and chromosomal DNA was prepared from 66 other Aeromonas strains and 12 strains from other bacterial genera and examined by Southern blot hybridization using a labelled fxp oligonucleotide and the 7.6 kb plasmid as probes. No hybridizing sequences were identified except in the original strain, AH26. It is proposed that fxp codes for a highly evolved organelle, possibly widely distributed in nature, but that it is carried on a genetic element that is rapidly lost from most strains upon in vitro cultivation and storage.
Transcriptional control, translation and function of the products of the five open reading frames of the Escherichia coli nir operon
Authors: Harborne Nerina R. , Griffiths Lesley , Busby Stephen J. W. , Cole Jeffrey A. 
Published: October 2006

SummaryFive open reading frames designated nirB, nirD, nirE, nirC and cysG have been identified from the DNA sequence of the Escherichia coli nir operon. Complementation experiments established that the NirB, NirD and CysG polypeptides are essential and sufficient for NADH‐dependent nitrite reductase activity (EC 1.6.6.4). A series of plasmids has been constructed in which each of the open reading frames has been fused in‐phase with the p‐galactosidase gene, lacZ. Rates of β‐galactosidase synthesis during growth in different media revealed that nirB, ‐D, ‐E and ‐C are transcribed from the FNR‐dependent promoter, p‐nirB, located just upstream of the nirB gene: expression is co‐ordinately repressed by oxygen and induced during anaerobic growth. Although the nirB, ‐D and ‐C open reading frames are translated into protein, no translation of nirE mRNA was detected. The cysG gene product is expressed from both p‐nirB and a second, FNR‐independent promoter, p‐cysG, located within the nirC gene.No NADH‐dependent nitrite reductase activity was detected in extracts from bacteria lacking either NirB or NirD, but a mixture of the two was as active as an extract from wild‐type bacteria. Reconstitution of enzyme activity in vitro required stoichiometric quantities of NirB and NirD and was rapid and independent of the temperature during mixing. NirD remained associated with NirB during the initial stages of purification of the active enzyme, suggesting that NirD is a second structural subunit of the enzyme.