bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of cpsD, a gene essential for type III capsule expression in group B streptococci
Authors: Rubens Craig E. , Heggen Laura M. , Haft Rachel F. , Wessels Michael R. 
Published: October 2006

SummaryWe showed previously that a mutant strain of group B Streptococcus (GBS) defective in capsule production was avirulent. This study describes the derivation of an unencapsulated mutant from a highly encapsulated wild‐type strain of type III GBS, COH1, by transposon mutagenesis with Tn916ΔE. The mutant, COH1‐13, was sensitive to phagocytic killing by human leukocytes in vitro and was relatively avirulent in a neonatal rat sepsis model compared with the wild‐type strain. No capsular polysaccharide was evident in the cytoplasm or on the cell surface of the mutant strain. The Tn916ΔE insertion site in COH1‐13 was mapped to the same chromosomal location as the Tn916 insertion site in the unencapsulated type III mutant COH31‐15 reported previously. Nucleotide sequencing of DNA flanking the insertion site in COH1‐13 revealed an open reading frame, designated cpsD, with significant homology to the rfbP gene of Salmonella typhimurium. RfbP encodes a galactosyl transferase enzyme that catalyses the transfer of galactose to undecaprenol phosphate, the initial step in O‐polysaccharide synthesis. A particulate fraction of a lysate of wild‐type strain GBS COH1 mediated the transfer of galactose from UDP‐galactose to an endogenous acceptor. The galactose–acceptor complex partitioned into organic solvents, suggesting it is lipid in nature or membrane‐associated. Galactosyl transferase activity was significantly reduced in the unencapsulated mutant strain COH1‐13. These results, together with the similarity in deduced amino acid sequence between cpsD and rfbP suggest that cpsD encodes a galactosyl transferase essential for assembly of the GBS type III capsular polysaccharide.
Proteins essential for expression of the Hms+ phenotype of Yersinia pestis
Authors: Pendrak Michael L. , Perry Robert D. 
Published: October 2006

SummaryOne characteristic of pigmented (Pgm+) cells of Yersinia pestis is the adsorption of sufficient quantities of exogenous haemin during growth at 26°C to form dark‐brown colonies. Carriage of the cloned haemin‐storage (hms) locus in pHMS1 restores this phenotype to spontaneous Pgm− chromosomal deletion mutants of Y. pestis. We have mapped the location of the structural genes for four proteins encoded on pHMS1 using minicell, in vitro transcription/translation, and complementation analysis. The hmsH and hmsF genes encode 90kDa and 72 kDa protein precursors processed to surface‐exposed, outer membrane proteins of 86kDa and 70kDa, respectively. Beta‐galactosidase positive MudII1734 insertions in hmsR suggest that it encodes a protein that is also essential for haemin storage. Finally, the structural gene for a 41 kDa protein lies distal to the hmsH gene but, unlike hmsH, hmsF, and hmsR, its expression is not essential for the Hms+ phenotype in Y. pestis.
Synthesis of the Escherichia coli K‐12 nucleoid‐associated DNA‐binding protein H‐NS is subjected to growth‐phase control and autoregulation
Authors: Dersch Petra , Schmidt Keratin , Bremer Erhard 
Published: October 2006

SummaryMutations in the structural gene (hns) for the Escherichia coli nucleoid‐associated DNA‐binding protein H‐NS cause highly pleiotropic effects on gene expression, site‐specific recombination, transposition of phage Mu, the stability of the genetic material and the topological state of the DNA. We have investigated the regulation of hns expression and found that hns transcription is subjected to stationary phase induction and negative autoregulation. A set of hns–lacZ protein and operon fusions was constructed in vitro and integrated in single copy into the attB site of the bacterial genome. Quantification of β‐galactosidase activity along the bacterial growth curve showed that hns expression increases approximately 10‐fold in stationary phase compared with exponentially growing cells. Immunological detection of the H‐NS protein in growing and stationary phase cells supported the genetic data and showed that H‐NS synthesis varies with growth phase. In addition, primer extension experiments demonstrated that the amount of hns mRNA is elevated in stationary phase cultures and that hns transcription is directed by a unique promoter functioning in both log and stationary phase. Disruption of the hns gene by an insertion mutation led to the derepression (approximately fourfold) of the expression of an hns–lacZ operon fusion integrated at the attB site, showing that hns transcription is subjected to negative regulation by its own gene product. Autoregulation of hns expression is particularly pronounced in log phase. Both stationary phase control and autoregulation of hns transcription are associated with a 130 bp fragment that contains the hns promoter. In order to study the interaction of H‐NS with its own regulatory region, we developed an efficient overproduction procedure and a simple purification scheme for H‐NS. DNA gel retardation assays showed that the H‐NS protein can preferentially interact with a restriction fragment carrying the hnspromoter. This restriction fragment showed features of curved DNA as judged by two‐dimensional polyacrylamide gel electrophoresis performed at 4°C and 60°C.
The role of galE in the biosynthesis and function of gonococcal lipopolysaccharide
Authors: Robertson Brian D. , Frosch Matthias , van Putten Jos P. M. 
Published: October 2006

SummaryLipopolysaccharide is an essential component of the outer membrane of Gram‐negative bacteria and an important virulence factor of many pathogens, such as Neisseria gonorrhoeae. We have cloned the gonococcal galE gene which was found to be located in the gonococcal homologue of the meningococcal capsule gene complex region D. Sequence alignment indicated extensive homology with the Escherichia coli and Salmonella GalE proteins. Mutants with insertions in the galE gene were used as a tool to characterize the structure and function of gonococcal lipopolysaccharide. They displayed deep rough phenotypes, and chemical analysis confirmed the loss of galactose from the mutant lipopolysaccharide. Functional analysis indicated that the terminal oligosaccharides contain galactose and that these are lost in galE mutants. The importance of these oligosaccharides in gonococcal biology is clear from the fact that they contain the epitopes that are the targets for killing by normal human serum, and the acceptor site for sialic acid, which acts to protect the gonococcus from this killing. Furthermore, infection experiments in vitro indicate that the galE mutants exhibit unaltered intergonococcal adhesion as well as adhesion to, and invasion of, epithelial cells.
Sequence, genetic, and lacZ fusion analyses of a nifR3–ntrB–ntrC operon in Rhodobacter capsulatus
Authors: Foster‐Hartnett Dawn , Cullen Paul J. , Gabbert Karen K. , Kranz Robert G. 
Published: October 2006

SummaryTranscription of Rhodobacter capsulatus genes encoding the nitrogenase polypeptides (nifHDK) is repressed by fixed nitrogen and oxygen. Regulatory genes required to sense and relay the nitrogen status of the cell are gInB, ntrB (nifR2), and ntrC (nifR1). R. capsulatus nifA1 and nifA2 require ntrC for activation when fixed nitrogen is limiting. The polypeptides encoded by nifA1 and nif42 along with the alternate Sigma factor RpoN activate nifHDK and the remaining nif genes in the absence of both fixed nitrogen and oxygen. In this study we report the sequence and genetic analysis of the previously Identified nifR3–ntrB–ntrC regulatory locus. nifR3 is predicted to encode a 324‐amino‐acid protein with significant homology to an upstream open reading frame cotranscribed with the Escherichia coli fregulatory gene, fis. Analysis of ntrC–lacZ fusions and complementation data indicate that nifR3ntrBC constitute a single operon. nifR3–lacZfusions are expressed only when lacZ is in the proper reading frame with the predicted nifR3 gene product. Tn5, a kanamycin‐resistance cassette, and miniMu insertions in nifR3 are polar on nfrBC (required for nif transcription). This gene organization suggests that the nifR3gene product may be involved in nitrogen regulation, although nifR3 is not stringently required for nitrogen fixation when ntrBC are present on a multicopy plasmid. In addition, a R. capsulatus strain with a 22‐nucleotide insert in the chromosomal nifR3 gene was constructed. This nifR3 strain is able to fix nitrogen and activate nifA1 and nifA2 genes, again supporting the hypothesis that nifR3 is not stringently required for nfrC‐dependent gene activation in R. capsulatus.
Formation of a putative relaxation intermediate during T‐DNA processing directed by the Agrobacterium tumefaciens VirD1, D2 endonuclease
Authors: Filichkin Sergei A. , Gelvin Stanton B. 
Published: October 2006

SummaryDuring the initial stages of crown gall tumorigenesis, the T‐DNA region of the Agrobacterium tumefaciens Ti‐plasmid is processed, resulting in the production of T‐DNA molecules that are subsequently transferred to the plant cell. Processing of the T‐DNA in the bacterium involves the nicking of T‐DNA border sequences by an endonuclease encoded by the virD locus, and the subsequent tight (possibly covalent) association of the VirD2 protein with the 5′ end of the processed single‐stranded or double‐stranded T‐DNA molecule. To investigate the interaction of the VirD1,D2 endonuclease with a right T‐DNA border, a set of plasmids containing both the border and virD sequences on the same high‐copy‐number replicon has been constructed and introduced into Escherichia coli. In this model system a tight nucleoprotein complex is formed between the relaxed double‐stranded substrate plasmid and the VirD2 protein. This putative T‐DNA processing complex may be analogous to the covalent relaxation complex formed between the pilot protein and plasmid DNA during bacterial conjugation. VirD2 attachment to the relaxed substrate plasmid was resistant to denaturing agents but sensitive to S1 nuclease digestion, indicating a single‐stranded region near the site of protein attachment. We speculate that this structure may be an intermediate formed prior to T‐strand unwinding from the substrate plasmid in a host bacterium.
Molecular cloning of a gene involved in glucose sensing in the yeast Saccharomyces cerevisiae
Authors: Van Aelst Linda , Hohmann Stefan , Bulaya Botchaka , de Koning Wim , Sierkstra Laurens , Neves Maria José , Luyten Kattie , Alijo Rafael , Ramos José , Coccetti Paola , Martegani Enzo , de Magalhães‐Rocha Neuza Maria , Brandão Rogelio Lopes , Van Dijck Patrick , Vanhalewyn Mieke , Durnez Peter , Arnold W. H. Jans  , Thevelein Johan M. 
Published: October 2006

SummaryCells of the yeast Saccharomyces cerevisiae display a wide range of glucose‐induced regulatory phenomena, including glucose‐induced activation of the RAS‐adenylate cyclase pathway and phosphatidylinositol turnover, rapid post‐translational effects on the activity of different enzymes as well as long‐term effects at the transcriptional level. A gene called GGS1 (for General Glucose Sensor) that is apparently required for the glucose‐induced regulatory effects and several ggs1 alleles (fdp1, byp1 and cif1) has been cloned and characterized. A GGS1 homologue is present in Methanobacterium thermoautotrophicum. Yeast ggs1 mutants are unable to grow on glucose or related readily fermentable sugars, apparently owing to unrestricted influx of sugar into glycolysis, resulting in its rapid deregulation. Levels of intracellular free glucose and metabolites measured over a period of a few minutes after addition of glucose to cells of a ggsi1Δ strain are consistent with our previous suggestion of a functional interaction between a sugar transporter, a sugar kinase and the GGS1 gene product. Such a glucose‐sensing system might both restrict the influx of glucose and activate several signal transduction pathways, leading to the wide range of glucose‐induced regulatory phenomena. Deregulation of these pathways in ggs1 mutants might explain phenotypic defects observed in the absence of glucose, e.g. the inability of ggs1 diploids to sporulate.
The importance of morphological events and intercellular interactions in the regulation of prespore‐specific gene expression during sporulation in Bacillus subtilis
Authors: Partridge S. R. , Errington J. 
Published: October 2006

SummaryWe have established a time course for the early morphological events of sporulation in Bacillus subtilis and related this to changes in gene expression, particularly those occurring in the prespore compartment. We have also systematically studied the effects of mutations in various regulatory (spo) genes on prespore‐specific gene expression. On the basis of these results, and those of other laboratories, at least four distinct temporal classes of prespore‐specific gene expression can now be distinguished. The first class begins within 15min of the formation of the spore septum, and requires the σ;F form of RNA polymerase. The second class, also directed by RNA polymerase containing σ;F, begins soon after the completion of prespore engulfment, and depends on an intercellular signal from the mother cell. This transcription results in synthesis of σ;G. However, σ;G activity, directing the third class of gene expression, appears only about 30min later and is dependent on the completion of prespore engulfment and on further interactions with the mother cell. The fourth class of gene expression has been described. The results demonstrate that the prespore programme of gene expression incorporates a series of control points modulated by information from the mother cell and on progress through the morphogenetic process.
Linear chromosomal physical and genetic map of Borrelia burgdorferi, the Lyme disease agent
Authors: Casjens Sherwood , Huang Wai Mun 
Published: October 2006

SummaryA physical map of the 952kbp chromosome of Borrelia burgdorferi Sh‐2‐82 has been constructed. Eighty‐three intervals on the chromosome, defined by the cleavage sites of 15 restriction enzymes, are delineated. The intervals vary in size from 96kbp to a few hundred bp, with an average size of 11.5 kbp. A striking feature of the map is its linearity; no other bacterial groups are known to have linear chromosomes. The two ends of the chromosome do not hybridize with one another, indicating that there are no large common terminal regions. The chromosome of this strain was found to be stable in culture; passage 6, 165 and 320 cultures have identical chromosomal restriction maps. We have positioned all previously known Borrelia burgdorferi chromosomal genes and several newly identified ones on this map. These include the gyrA/gyrB/dnaA/dnaN gene cluster, the rRNA gene cluster, fla, flgE, groEL (hsp60), recA, the rho/hip cluster, the dnaK (hsp70)/dnaJ/grpE cluster, the pheT/pheS cluster, and the genes which encode the potent immunogen proteins p22A, p39 and p83. Our electrophoretic analysis detects five linear and at least two circular plasmids in B. burgdorferi Sh‐2‐82. We have constructed a physical map of the 53 kbp linear plasmid and located the operon that encodes the two major outer surface proteins ospA and ospB on this plasmid. Because of the absence of functional genetic tools for this organism, these maps will serve as a basis for future mapping, cloning and sequencing studies of B. burgdorferi.
Nucleotide substitutions and small‐scale insertion produce size and antigenic variation in group A streptococcal M1 protein
Authors: Harbaugh M. P. , Podbielski A. , Hügl S. , Cleary P. P. 
Published: October 2006

SummaryThe presence of M protein on the surface of group A streptococci (GAS) confers the ability of the cell to resist phagocytosis in the absence of type‐specific antibodies. It undergoes antigenic variation with more than 80 different serotypes having been defined. We have sequenced the M protein gene (emm1.1) from strain CS190 and present evidence that individual nucleotide substitutions are responsible for sequence variation in the N‐terminal non‐repeat region of emm1.1 and these substitutions have altered antibody recognition of opsonic epitopes. The N‐terminal non‐repeat domains of two other closely related strains, 71‐155 and 76‐088, were found to have sequence identical to emm1.1 with the addition of a 21 bp insert. This study provides the first evidence that nucleotide substitutions and small insertions are responsible for size and antigenic variation in the N terminal non‐repeat domain of the M protein of GAS.
Cloning and nucleotide sequence of anaerobically induced porin protein E1 (OprE) of Pseudomonas aeruginosa PAO1
Authors: Yamano Yoshinori , Nishikawa Tohru , Komatsu Yoshihide 
Published: October 2006

SummaryThe porin oprE gene of Pseudomonas aeruginosa PAO1 was isolated. Its nucleotide sequence indicated that the structural gene of 1383 nucleotide residues encodes a precursor consisting of 460 amino acid residues with a signal peptide of 29 amino acid residues, which was confirmed by the N‐terminal 23‐amino‐acid sequence and the reaction with anti‐OprE polyclonal antiserum. Anaerobiosis induced OprE production at the transcription level. The transcription start site was determined to be 40 nucleotides upstream from the ATG initiation codon. The control region contained an appropriately situated Eσ54 recognition site and the putative second half of an ANR box. The amino acid sequence of OprE had some clusters of sequence homologous with that of OprD of P. aeruginosa, which might be responsible for the outer membrane permeability of imipenem and basic amino acids.
Regulation by Ca2+ in the Yersinia low‐Ca2+ response
Authors: Straley Susan C. , Plano Gregory V. , Skrzypek Elźbieta , Haddix Pryce L. , Fields Kenneth A. 
Published: October 2006

SummaryThe Yersinia low‐Ca2+ response (LCR) is a regulatory response in which a set of plasmid‐borne operons is transcriptionally regulated at 37°C in response to the presence or absence of mM concentrations of Ca2+. LCR‐regulated operons encode secreted proteins with regulatory and virulence roles as well as non‐secreted regulatory proteins and components of the secretion machinery. Downregulation by Ca2+ is imposed by a signalling cascade that includes secreted proteins and possibly also components of the secretion system and is hypothesized to act on membrane‐bound inductive components. An important rote in LCR induction is played by LcrD, an inner‐membrane protein with homologues in several virulence‐associated and flagella assembly‐related systems in diverse bacterial species. The mechanism of signal transduction in response to Ca2+ is not known, and the proteins that bind DNA to downregulate transcription have not been identified.
Multicopy plasmid instability: the dimer catastrophe hypothesis
Authors: Summers David K. , Beton Christopher W. H. , Withers Helen L. 
Published: October 2006

SummaryMultimer formation reduces plasmid copy number and is an established cause of segregational instability. Nevertheless, it is difficult to rationalize observations that low levels of dimers can cause severe instability, if we assume they are distributed evenly in cell populations. We report here that dimer distribution is in fact heterogeneous in recombination‐proficient strains. Most cells in the population contain only monomers; dimers are confined to a small sub‐population from which plasmid‐free daughters arise at high frequency. In a rec+ culture where 4% of pBR322 molecules are dimers, more than half are in dimer‐only cells. We show that this situation is inevitable because dimers replicate at twice the rate of monomers. Runaway multimerization is avoided because dimer‐containing cells grow more slowly than their monomer‐containing counterparts. A computer simulation is used to show how dimers proliferate after formation by homologous recombination. The equilibrium concentration of dimers is proportional to the inter‐plasmid recombination rate and is essentially independent of the rate at which homologous recombination converts dimers to monomers.
The alkane oxidation system of Pseudomonas oleovorans: induction of the alk genes in Escherichia coli W3110(pGEc47) affects membrane biogenesis and results in overexpression of alkane hydroxylase in a distinct cytoplasmic membrane subtraction
Authors: Nieboer Maarten , Kingma Jaap , Witholt Bernard 
Published: October 2006

SummaryThe alkane hydroxylase system of Pseudomonas oleovorans, which catalyses the initial oxidation of aliphatic substrates, is encoded by three genes. One of the gene products, the alkane hydroxyiase AlkB, is an integral cytoplasmic membrane protein. Induction leads to the synthesis of 1.5–2% AlkB relative to the total cell protein, both in P. oleovorans and in recombinant Escherichia coli DH1. We present a study on the Induction and localization of the alkane hydroxylase in E. coli W3110, which appears to be an interesting host strain because it permits expression levels of AlkB of up to 10–15% of the total cell protein. This expression level had negative effects on cell growth. The phospholipid content of such cells was about threefold higher than that of wild‐type W3110. Freeze‐fracture electron microscopy showed that induction of the alk genes led to the appearance of membrane vesicles in the cytoplasm; these occurred much more frequently in cells expressing alkB than in the negative control, which contained all of the alk genes except for alkB. Isolation and separation of the membranes of cells expressing alkB by density gradient centrifugation showed the customary cytoplasmic and outer membranes, as well as a low‐density membrane fraction. This additional fraction was highly enriched in AlkB, as shown both by SDS‐PAGE and enzyme activity measurements. A typical cytoplasmic membrane protein, NADH oxidase, was absent from the low‐density membrane fraction, alkB expression in W3110 changed the composition of the phospholipid headgroup in the membrane, as well as the fatty acid composition of the membrane. The major changes occurred in the unsaturated fatty acids: C16:1 and C18:1 increased at the expense of C17:0cyc and C19:0cyc*
Genetic stability and xanthan gum production in Xanthomonas campestris pv. campestris NRRL B1459
Authors: Martínez‐Salazar Jaime M. , Palacios Alma Nirvana , Sánchez Rosalba , Caro Alma Delia , Soberón‐Chavez Gloria 
Published: October 2006

SummaryA transposon (Tn5‐SC) was constructed that can be used to quantify genetic deletions or amplifications. This transposon was used to evaluate the genomic stability of Xanthomonas campestris pv. campestris NRRL B1459 and we found that the genome of this bacterium is as stable as other Gram‐negative bacteria or even more stable. Homologous recombination between plasmid sequences was determined in strain NRRL B1459 and was found to occur at a similar level to that reported for other Gram‐negative bacteria. We report here that in X.c.c. NRRL B1459 there is no straightforward correlation between the occurrence of genetic rearrangements and frequency of homologous recombination. These data are discussed with respect to the reported instability of strain NRRL B1459 for xanthan gum production.