bluuebunny/crossref_metadata_2025_split huggingface dataset:
Targeting and assembly of an oligomeric bacterial enterotoxoid in the endoplasmic reticulum of Saccharomyces cerevisiae
Authors: Schonberger O. , Hirst T. R. , Pines O. 
Published: October 2006

SummaryA hybrid protein consisting of the Escherichia coli lipoprotein signal sequence attached to the mature sequence of the B subunit of heat‐labile enterotoxrn (Lipo‐EtxB) was expressed in yeast and E. coli Analyses of cell lysates from Saccharomyces cerevisiae and E. coli expressing the protein revealed that both organisms were able to assemble Lipo‐EtxB into oligomers that were (i) stable in the presence of sodium dodecyl sulphate, (ii) resistant to proteinase K degradation, and (iii) able to bind to GM1‐ganglio‐side receptors. Each of these properties are characteristic of the wild‐type B subunit pentamer produced in E. coli. Assembly of Lipo‐EtxB was found to be unaffected in a sec18 mutant of S. cerevisiae, which possesses a temperature‐sensitive defect in protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus, but was found not to assemble in a sec53 mutant, which causes the misfolding of proteins targeted to the ER. A kar2‐1 mutation with a defect in the yeast homologue of BiP caused an 18‐fold reduction in Lipo‐EtxB assembly at the non‐permissive temperature in S. cerevisiae. However, introduction of the wild‐type KAR2gene on a plasmid into the kar2‐1 mutant completely suppressed the inhibition of Lipo‐EtxB assembly. This provides the first evidence that KAR2 facilitates the assembly of an oligomeric protein in yeast and thus implicates KAR2 as a‘molecular chaperone'. The possible mechanisms of enterotoxoid assembly In E. coli and S. cerevisiae are discussed.
Identification of a nifA‐like regulatory gene of Azospirillum brasilense Sp7 expressed under conditions of nitrogen fixation and in the presence of air and ammonia
Authors: Liang Y. Y. , Kaminski P. A. , Elmerich C. 
Published: October 2006

SummaryA gene bank of Azospirillum lipoferum Br17 constructed in the vector λGEM11 was screened with a Bradyrhizobium japonicum nifA gene probe. A 7.3 kb EcoRI fragment carrying a nifA‐like gene was thereby isolated and subsequently used to screen a gene bank of Azospirillum brasilense Sp7 constructed in pUC18. Two EcoRI fragments of 5.6 kb and 3.6 kb covering the nifA‐homology region were found. Mutants with Nif‐phenotype were obtained by site‐directed Tn5 mutagenesis of the 5.6 kb fragment and subsequent recombination into the A. brasilense Sp7 genome. The mutations were clustered into two loci located at each extremity of the fragment. One of these loci corresponded to nifA and the other to nifB. The nucleotide sequence of nifA of A. brasilense Sp7 was determined. Comparison of the deduced amino acid sequences of NifA of A. brasilense Sp7 and NifA of B. Japonicum, Rhizobium leguminosarum biovar trifolii and Klebsiella pneumoniae confirmed that it was a nifA‐like gene. Construction of a nifA‐lacZ fusion and mapping of the RNA transcriptional start site showed that the nifA‐like gene was expressed from an unidentified promoter, under conditions of nitrogen fixation and in the presence of oxygen and ammonia.
New approaches in genome analysis by pulsed‐field gel electrophoresis: application to the analysis of Pseudomonas species
Authors: Grothuesi D. , Tümmler B. 
Published: October 2006

SummarySummary A general method for the evaluation of macrorestriction fragment patterns is presented and its applicability to the taxonomy of bacteria is demonstrated for 32 Pseudomonas species. Strains were differentiated at the species and subspecies level by genome size and macrorestriction fragment fingerprints of the chromosome that had been separated on pulsed‐field gels. The relatedness of bacteria was ascertained from the similarity of Asnl, Dral, Spel, Sspl or Xbal fragment patterns. In general, the dendrograms calculated from the genome fingerprints corresponded with the phylogenetic classification obtained from phenotypic marker or nucleic acid hybridization analysis, but several exceptions were noted. The techniques and algorithms presented herein are generally applicable to the genome analysis of bacteria, lower eukaryotes, and DNA fragments cloned in yeast artificial chromosomes.
The tonB gene of Serratia marcescens: sequence, activity and partial complementation of Escherichia coli tonB mutants
Authors: Gaisser S. , Braun V. 
Published: October 2006

SummaryThe TonB protein plays a key role in the energy‐coupled transport of iron siderophores, of vitamin B12, and of colicins of the B‐group across the outer membrane of Escherichia coli. In order to obtain more data about which of its particular amino acid sequences are necessary for TonB function, we have cloned and sequenced the tonB gene of Serratia marcescens. The nucleotide sequence predicts an amino acid sequence of 247 residues (Mr 27389), which is unusually proline‐rich and contains the tandem sequences (Glu‐Pro)5 and (Lys‐Pro)5. In contrast to the TonB proteins of E. coli and Salmonella typhimurium, translation of the S. marcescens TonB protein starts at the first methionine residue of the open reading frame, which is the only amino acid removed during TonB maturation and export. Only the N‐terminal sequence is hydrophobic, suggesting its involvement in anchoring the TonB protein to the cytoplasmic membrane. The S. marcescens tonB gene complemented an E. coli tonB mutant with regard to uptake of iron siderophores, and sensitivity to phages T1 and φ 80, and to colicins B and M. However, an E. coli tonB mutant transformed with the S. marcescens tonB gene remained resistant to colicins la and Ib, to colicin B derivatives carrying the amino acid replacements Val/Ala and Val/Gly at position 20 in the TonB box, and they exhibited a tenfold lower activity with colicin D. In addition, the S. marcescens TonB protein did not restore T1 sensitivity of an E. coli exbB tolQ double mutant, as has been found for the overexpressed E. coli TonB protein, indicating a lower activity of the S. marcescens TonB protein. Although the S. marcescens TonB protein was less prone to proteolytic degradation, it was stabilized in E. coli by the ExbBD proteins. In E. coli, TonB activity of S. marcescens depended either on the ExbBD or the TolQR activities.
The C‐terminal domain of the toxic fragment of a Bacillus thuringiensis crystal protein determines receptor binding
Authors: Honée G. , Convents D. , Van Rie J. , Jansens S. , Peferoen M. , Visser B. 
Published: October 2006

SummaryThe insecticidal crystal proteins of Bacillus thuringiensis show a high degree of specificity. In vitro binding studies with several crystal proteins demonstrated a correlation between toxicity and binding to receptors of larval midgut epithelial cells. In order to study the domain‐function relationships of the toxic fragment, hybrid crystal proteins based on CrylA(b) and CrylC were constructed. Two out of 11 hybrid proteins constructed exhibited insecticidal activity. Both displayed an insectidial spectrum similar to that of the parental crystal protein from which the C‐terminal part of the toxic fragment originated. In addition, in vitro binding studies directly demonstrated the involvement of the C‐terminal part of the toxic fragment in receptor binding. These results demonstrate that the C‐terminal part of the toxic fragment determines specific receptor binding, which in turn determines, to a large extent, the insect specificity.
The 120 kilodalton outer membrane protein (rOmp B) of Rickettsia rickettsii is encoded by an unusually long open reading frame: evidence for protein processing from a large precursor
Authors: Gilmore R. D. , Cieplak W. , Policastro P. F. , Hackstadt T. 
Published: October 2006

SummaryA Rickettsia rickettsii outer surface membrane protein (rOmp B), of an apparent molecular mass of 120 kilodaltons, is a major surface antigen of the Rickettsiae that displays genus, species, and sub‐species specific antigenic determinants. The 5′ portion of this gene was found to be unstable in plasmids, but was stably cloned in a lambda vector. The nucleotide sequence of the 5′ terminus has been determined, thus completing the DNA sequence of the entire gene. Genetic analysis revealed an unusually large open reading frame with the capacity to encode a product much larger than the mature protein. A 32 kilodalton peptide from purified rickettsiae was isolated and the amino terminus was sequenced, which revealed that the peptide is encoded by the 3′ portion of this large open reading frame. This suggests a role for post‐translational processing of rOmp B from a large precursor molecule.
Differential gene expression from the Escherichia coli atp operon mediated by segmental defferences in mRNA stability
Authors: McCarthy J. E. G. , Gerstel B. , Surin B. , Wiedemann U. , Ziemke P. 
Published: October 2006

SummaryThe atp operon of Escherichia coli directs synthesis rates of protein subunits that are well matched to the requirements of assembly of the membrane‐bound H+‐ATPase (α3β3γ1δ1σ1a1b2c10–15). Segmental differences in mRNA stability are shown to contribute to the differential control of atp gene expression. The first two genes of the operon, atpl and atpB, are rapidly inactivated at the mRNA level. The remaining seven genes are more stable. It has previously been established that the translational efficiencies of the atp genes vary greatly. Thus differential expression from this operon is achieved via post‐transcriptional control exerted at two levels. Neither enhancement of translational efficiency nor insertion of repetitive extragenic palindromic (REP) sequences into the atplB intercistronic region stabilized atpl. We discuss the implications of these results in terms of the pathway of mRNA degradation and of the role of mRNA stability in the control of gene expression.
Analysis of Haemophilus influenzae type b lipooligosaccharide‐synthesis genes that assemble or expose a 2‐keto‐3‐deoxyoctulosonic acid epitope
Authors: Abu Kwaik Y. , McLaughlin B. E. , Apicella M. A. , Spinola S. M. 
Published: October 2006

SummaryWe recently isolated a recombinant phage from a Haemophilus influenzae type b (Hib) library that assembles an oligosaccharide with an apparent molecular weight of 1400 (1.4 K) on a 4.1 K Escherichia coli lipopolysaccharide (LPS) structure, producing a 5.5 K LPS species that contains a KDO (2‐keto‐deoxyoctulosonic acid) epitope. Subcloning and deletional analysis of the 14 kb Haemophilus insert showed that three overlapping restriction fragments contained within a 7.2 kb pstl–Bam HI fragment sequentially modified an E. coli 4.1 K LPS structure, generating novel species of 4.5K, 5.1K and 5.5K. Only the 5.5K species contained the KDO epitope. We confirmed the relationship between the cloned genes and Hib lipooligosaccharide (LOS) biosynthesis by constructing a mutant that expressed an altered LOS. Thus, the Hib 7.2 kb Pstl‐Bam Hl restriction fragment contained a cluster of at least three genetic loci whose products acted sequentially in LOS biosynthesis.
The bvg‐dependent promoters show similar behaviour in different Bordetella species and share sequence homologies
Authors: Scarlato V. , Prugnola A. , Aricò B. , Rappuoli R. 
Published: October 2006

SummaryThe expression of the virulence‐associated genes in Bordetella species is co‐ordinately regulated by the gene products encoded by the bvg locus. In Bordetella pertussis the expression of this locus is regulated by the P1, P2, P3 and P4 promoters which are located in a 350bp DNA fragment also containing the PFHA promoter. Here we report the transcriptional regulation of the bvg locus and the fha gene in Bordetella paraper‐tussis and a sequence analysis of the byg‐regulated promoters. The Pp1, Pp2, Pp4 and PpFHA promoters are indistinguishable, both in transcription initiation sites and environmental regulation, from the corresponding promoters of B. pertussis, while the Pp3 promoter is not active. Sequence homologies from nine bvg‐regulated promoters show a conserved dinucleotide, 5′‐TG‐3′, at approximately one turn of helix upstream of the ‐10 5′‐A.AaTat‐3’region, and a 5′‐TTTCC‐3’sequence in the ‐90 region. Since the nucleotide sequence of the inactive Pp3 promoter shows several base substitutions with respect to the found sequence homologies, it is likely that some of these bases play an essential role in promoter activity.
Role of plasmid multimers in mutation to tetracyline resistance
Authors: Boe L. , Marinus M. G. 
Published: October 2006

SummaryAs an additional system for analysing mutations that appear to be specifically induced or directed, we have used a plasmid that contains the mnt repressor gene inserted as an operon fusion with the tet gene of the plasmid pBR322. Thus, the mnt gene product acts as a negative transcriptional regulator of tet gene expression. Mutations inactivating the Mnt repressor are recessive while those destroying operator recognition (Oc) are dominant in conferring tetracycline resistance on the host. When resistance mutations were isolated on plates with high levels of tetracycline they were preferentially mnt‐ and the plasmids were monomers. Pre‐exposure to low concentrations increased the frequency of resistant mutants by 100‐ to 1000‐fold, and the mutations were now mostly Oc, located on one unit of a plasmid multimer. Recessive repressor mutations on one unit would not have been selected. We suggest that the high frequency of mutation in tandem multimeric plasmids may be caused by the formation of single‐stranded and hence highly mutable regions by homologous pairing out of register. The role of tetracycline in promoting mutations is discussed.
Distinguishing between the extracellular DNases of Vibrio cholerae and development of a transformation system
Authors: Focareta T. , Manning P. A. 
Published: October 2006

Summary Vibrio cholerae is known to secrete DNase(s) into the extracellular environment. These proteins have been thought to be responsible for the difficulties in transforming this organism. In this work we demonstrate that the dns and xds genes differ and that their products are solely responsible for the extracellular DNase activity. By site‐directed mutagenesis, strains have been constructed which are mutant in one or both genes. These strains have been assessed for their ability to be transformed with plasmid DNA and for their virulence in the infant mouse cholera model. DNase‐deficient mutants can be readily transformed and the product of dns appears to be the more significant barrier. No effect on virulence was observed with the mutants.
The cell envelope of the hyperthermophilic archaebacterium Pyrobaculum organotrophum consists of two regularly arrayed protein layers: three‐dimensional structure of the outer layer
Authors: Phipps B. M. , Huber R. , Baumeister W. 
Published: October 2006

SummaryThe cell envelope of the hyperthermophilic sulphur‐reducing archaebacterium Pyrobaculum organotrophum H10 was found to be composed of two distinct hexagonally arranged crystalline protein arrays. Electron microscopic analysis of freeze‐etched cells and isolated envelopes in conjunction with image processing showed that the inner layer (lattice centre‐to‐centre spacing 27.9 nm) is essentially identical to the protein array of Pyrobaculum islandicum GE03, a complex, rigid structure implicated in the maintenance of cell shape. The outer layer has clear p6 symmetry and a lattice spacing of 20.6 nm. Its three‐dimensional structure was reconstructed from a negative stain tilt series of an intact double‐layered envelope using Fourier filtration to separate the desired information from the other lattices present. The outer layer is a unique, porous network of block‐like dimers disposed around six‐fold axes, and exhibits minimal asymmetry between its inner and outer faces. It appears Xo be rather loosely associated with the outer surface of the inner layer. In most H10 envelopes, the inner layer is orientated with one base vector exactly perpendicular to the long axis of the cell, so that the cylindrical portion is composed of a series of parallel cell‐girdling hoops of hexameric morphological units. All the other known Pyrobaculum strains were found to have a GEO3‐type envelope structure, consisting of a single rigid protein array and a fibrous capsule. Although H10 does not possess a capsule, fibrils appear to be sandwiched between the two protein layers.
Molecular cloning, partial purification, and characterization of a haemin‐binding lipoprotein from Haemophilus influenzae type b
Authors: Hanson M. S. , Hansen E. J. 
Published: October 2006

SummaryA library of genomic DNA fragments from Haemophilus influenzae type b (Hib) DL42 was constructed in plasmid pBR322, transformed into Escherichia coli strain RR1, and screened for recombinant clones with haemin‐binding activity by plating onto haemin‐containing agar. Expression of haemin‐binding activity by clones correlated with the expression of a protein with an apparent molecular weight of 51000 (51K) that was also recognized by anti‐Hib strain DL42 serum in immunoblots. One recombinant clone, designated pHM2, with the smallest DNA insert (3.62kb) was characterized further. Ethanol inhibition of expression of pHM2 in minicells revealed that the 51K protein was the result of a processing event involving a larger precursor. E. coli RR1(pHM2) adsorbed haemin in liquid suspensions as well as from solid media. Subcloning of a 2.6 kb fragment of pHM2 into a shuttle vector permitted the construction of a recombinant Hib clone, DL42(pHM1002), which overexpressed the 51K haemin‐binding protein. This 51K protein appears to be peripherally associated with the inner, and possibly outer, membranes of Hib. Affinity chromatography on haemin‐agarose was utilized to purify the haemin‐binding protein from both E. coli RR1(pHM2) and Hib DL42(pHM1002) to near homogeneity. The use of the antibiotic globomycin in a minicell expression system and radioimmunoprecipitation analysis of Hib proteins intrinsically radiolabelled with [3H]‐palmitate indicated that the 51K haemin‐binding protein is a lipoprotein.
Pilin expression and processing in pilus mutants of Neisseria gonorrhoeae: critical role of Gly‐1 in assembly
Authors: Koomey M. , Bergstrom S. , Blake M. , Swanson J. 
Published: October 2006

SummarySpontaneous mutants of Neisseria gonorrheae failing to express pili or having diminished levels of piliation were studied with regard to pilin expression. All mutants displayed altered pilin processing detectable as the release of soluble, truncated pilin molecules (S‐pilin). Of particular interest was the finding, in one mutant, that substitution of serine for glycine at position ‐1 of propilin, a highly conserved residue among N‐metPhe and related pilins, abolished pilus expression but not S‐pilin release. The degree of S‐pilin processing and the levels of membrane‐associated pilin varied among the different classes of mutants, suggesting that each was blocked at a distinct step of pilus biogenesis. The data support a model in which increased S‐pilin processing is a result of a decreased rate of pilus polymerization.
Genetic analysis of superoxide dismutase, the 23 kilodalton antigen of Mycobacterium tuberculosis
Authors: Zhang Y. , Lathigra R. , Garbe T. , Catty D. , Young D. 
Published: October 2006

SummaryThe gene encoding a 23 kilodalton protein antigen has been cloned from Mycobacterium tuberculosis by screening of a recombinant DNA library with monoclonal antibodies. The product of the gene has been identified as the superoxide dismutase (SOD) of M. tuberculosis on the basis of sequence comparison and by expression of the recombinant protein in a functionally active form. The derived amino acid sequence of M. tuberculosis SOD reveals a close similarity to manganese‐containing SODs from other organisms, in spite of the fact that previous studies using the purified enzyme have identified iron as the preferred metal ion ligand. SOD is present in the extracellular fluid of logarithmic‐phase cultures of M. tuberculosis, but the structural gene is not preceded by a signal peptide sequence. Insertion of the M. tuberculosis SOD gene into a novel shuttle vector demonstrated the presence of a promoter which functions efficiently in mycobacteria but is ineffective in Escherichia coli
Sequence and expression analysis of a cAMP‐responsive gene regulated during late development of Dictyostelium discoideum
Authors: Ramji D. P. , Donovan J. T. J. , Hames B.D. 
Published: October 2006

SummaryThe 7E gene is expressed late in normal development of Dictyostelium discoideum after pseudoplasmodium formation. After disaggregation of the developing cells, transcription of this gene depends entirely on exogenous 3′5′ cyclic AMP (cAMP). The 5′ flanking region of the 7E gene contains two TATA box‐oligo (dT) promoter motifs but analysis of 7E gene expression by primer extension shows only a single primary transcript with transcription initiating immediately after the most proximal promoter motif during development or in disaggregated cells in the presence of exogenous cAMP. Four C‐rich sequence lie within 350 bp upstream of the cap site, analogus to the upstream elements implicated in the cAMP regulation of several other Dictyostelium genes expressed in development.
Two global regulators repress the anaerobic expression of MnSOD in Escherichia coli: Fur (ferric uptake regulation) and Arc (aerobic respiration control)
Authors: Tardat B. , Touati D. 
Published: October 2006

SummaryThe expression of sodA, the Escherichia coli gene encoding manganese superoxide dismutase (MnSOD) is induced by aerobiosis and superoxide generators such as paraquat. Analysis of variants expressing sodA in the absence of oxygen has revealed that mutations in genes for two global regulatory systems, Fur (ferric uptake regulation) and Arc (aerobic respiration control), are simultaneously required for the expression of sodA in anaerobiosis. The Fur protein still represses sodA in an iron‐dependent fashion in aerobiosis. Moreover, all mutants remain inducible by paraquat, indicating that the positive control of SoxR, which mediates the response to superoxide in E. coli, is still operative. Thus, in addition to the response to the superoxide‐mediated oxidative stress which depends on SoxR, two global controls regulate MnSOD expression: ArcA couples MnSOD expression to respiration, and Fur couples it to the intracellular concentration of iron.
Roles of DNA adenine methylation in controlling replication of the REPI replicon of plasmid pColV‐K30
Authors: Gammie A. E. , Crosa J. H. 
Published: October 2006

SummaryDNA adenine methylation controls DNA replication of plasmids containing the prototypic REPI replicon by affecting protein recognition and by altering the helical stability of the origin. Denaturing gradient gel electrophoresis shows that adenine methylated origin DNA is more easily melted than unmethylated. However, because an added DNA adenine methylation (dam) site at the origin, whether in or out of phase with other helically aligned dam sites, actually prevents replication, we conclude that destabilization of the helix is not the exclusive function of adenine methyfation in REPI replication. We find that the conformation and degree of methylation at the origin, features which are important for protein recognition, are essential for replication. In fact, RepI, a protein required for replication initiation at REPI replicons, contains a region homologous with a domain in proteins which specifically recognize and bind 5′‐GATC‐3′. We propose that the dam sites in the origin play a dual role: one is destabilization of the helix, and the other is protein recognition.