bluuebunny/crossref_metadata_2025_split huggingface dataset:
Bacterial polysaccharide capsule synthesis, export and evolution of structural diversity
Authors: Boulnoisl G. J. , Jann K. 
Published: October 2006

SummaryElaboration of a capsule composed of one of a range of acidic polysaccharides is a common feature of many bacteria, particularly those capable of causing serious infections in humans. Biochemical and genet‐cal analyses of capsule biogenesis in Escherichia coli are beginning to reveal new aspects of polysaccharide biosynthesis. Genes have been identified which are thought to encode products responsible for the trans‐location of these high molecular‐weight polysaccharides across the cytoplasmic and outer membranes, and the organization of exported polysaccharide into a capsule. Their further analysis should provide new insights into membrane biology, particularly since the genes in question are absent from the often used laboratory strains of E. coli. Genetic analysis of capsule diversity is beginning to suggest possible mechanisms for the generation of the structural diversity of polysaccharides.
The gene for the precursor of plastocyanin from the cyanobacterium Anabaena sp. PCC 7937: isolation, sequence and regulation
Authors: Van der Plas J. , Bovy A. , Kruyt F. , de Vrieze G. , Dassen E. , Klein B. , Weisbeek P. 
Published: October 2006

SummaryThe gene encoding plastocyanin (petE1) from Anabaena sp. PCC 7937 was isolated using two sets of mixed oligonucleotide hybridization probes derived from conserved regions in the protein. Plastocyanin is encoded as a preprotein of 139 amino acids. The amino‐terminal extension of 34 residues has all the characteristics of a signal peptide and is probably involved in translocation of preplastocyanin over the thylakoid membrane. The level of the petE1 mRNA, a single transcript of about 740 bases, was found to be severely reduced under conditions of Cu2+ deficiency. The petE1 gene was transferred to the genome of Synechococcus sp. PCC 7942, which did not appear to contain a structural gene for plastocyanin itself. The integrated gene becomes expressed at the transcriptional level, regardless of the amount of Cu2+ available.
The major outer membrane protein of Chlamydia trachomatis: critical binding site and conformation determine the specificity of antibody binding to viable chlamydiae
Authors: Conlan J. W. , Kajbaf M. , Clarke I. N. , Chantler S. , Ward M. E. 
Published: October 2006

SummaryThe major outer membrane protein (MOMP) is the prime candidate for the development of a chlamydial vaccine. Antibodies to the subspecies‐specific epitope neutralize chlamydial infection. Monoclonal antibodies (MAbs) to this epitope were prepared either by immunization with whole chlamydiae or with a 16 amino acid synthetic peptide. The critical binding site on the subspecies epitope for these MAbs was determined to single amino acid resolution using several hundred solid‐phase peptides. A frame shift of just one amino acid in critical binding site completely prevented antibody binding to viable chlamydiae. A single MAb to whole organisms was capable of spanning both the surface‐exposed, conformation‐dependent, subspecies epitope and a buried, conformation‐independent species epitope some 10 Å distant. Immunization with peptide generated an MAb with reduced binding constraints which permitted the antibody to bind with broadened species‐specificity at the subspecies binding site. The results show for the first time the importance of both critical binding site and conformation at the subspecies epitope. We suggest that the conformational flexibility of short, epitopic peptide vaccines may in some cases be advantageous, giving rise to extended specificity not attained with the natural protein.
Genetic analysis of phycobilisome mutants in the cyanobacterium Synechococcus species PCC 6301
Authors: Kalia R. , Lind L. K. , Gustafsson P. 
Published: October 2006

SummaryThe chromophoric protein phycocyanin is the major protein in the phycobilisome rod of the cyanobacterium Synechococcus 6301 (formerly designated Ana‐cystis nidulans sp. UTEX 625). The gene clusters coding for the β‐ and α‐subunits of phycocyanin are duplicated on the chromosome of Synechococcus 6301 and separated by an intergenic region 2.5 kb long. The structure of the phycocyanin operons of the phycobilisome mutant strains AN112 and AN135 was compared to that of wild‐type Synechococcus 6301. Both mutants have an altered phycobilisome structure resulting in the appearance of rods of a shorter overall length. Genetic mapping indicated that the mutant strain AN112 completely lacked the intergenic region and carried only one set of phycocyanin subunit genes. No gross structural difference in the genetic organization of the corresponding region of mutant strain AN135 was detected. Northern blot analysis and primer extension experiments were used to monitor the transcriptional pattern of the phycocyanin rod operon. It was found that AN112 had lost the 3.7 kb minor mRNA, which covers the intergenic region, and only produced two major 1.3 and 1.4 kb mRNA species. These transcripts were identified as fusion products of the 5′ end of the transcriptional unit originating from the promoter region of the left‐hand phycocyanin gene cluster and the 3′ end of the transcriptional unit covering the right‐hand phycocyanin gene cluster. The mutant strain AN 135 had a transcriptional pattern very similar to that of the wild type. The level of transcription of the major transcripts covering the phycocyanin gene clusters was similar in the wild‐type and mutant strains. The results indicate that genes coding for two of the linker proteins, namely the 30 and 33 kD poly‐peptides, are located in the intergenic region between the duplicated phycocyanin gene clusters in Synecho‐coccus 6301. The results also show that loss of the right‐hand phycocyanin promoter does not drastically impair phycocyanin synthesis.
Phospholipase C and haemolytic activities of Clostridium perfringens alpha‐toxin cloned in Escherichia coli: sequence and homology with a Baciilus cereus phospholipase C
Authors: Leslie D. , Fairweather N. , Pickard D. , Dougan G. , Kehoe M. 
Published: October 2006

SummaryThe Clostridium perfringens alpha‐toxin (phospholipase C) gene (cpa) has been cloned and expressed in Escherichia coli. The biological activities of the cloned gene product have been analysed and the complete nucleotide sequence of the cpa gene has been determined. The cloned cpa gene product, which is exported to the periplasm in E. coli, possesses both phospholipase C and haemolytic activities. Haemolysis is not apparent when cell extracts are incubated with isotonic suspensions of sheep erythrocytes, but can be detected and quantified readily when dilutions of the same extracts are placed in wells in sheep‐blood agar plates. Like other sequenced clostridial genes, the cpa gene has a high AT content (66.4%), exhibits a strong bias for using codons with A or T in the wobble position, and the 350 base pairs upstream from the gene have a significantly higher AT content (79.5%) than the coding region. The cpa gene encodes a 398 amino acid polypeptide with a deduced molecular weight of 45481D. This is very similar to the estimated molecular weight (Mr) of the cpa primary gene product expressed in an in vitro transcription‐translation system (Mr 46000), but larger than the cpa gene product detected in E. coli minicells, E. coli whole cells or in C. perfringens cells (Mr 43000), suggesting post‐translational processing. The 28 N‐terminal residues of the deduced alpha‐toxin sequence possess the consensus features of a signal peptide and may be removed during secretion. The deduced alpha‐toxin sequence shares significant structural homology with the phosphatidylcholine‐preferring phospholipase C of Bacillus cereus.
Molecular and mutational analysis of three genes preceding pyrE on the Escherichia coli chromosome
Authors: Poulsen P. , Andersen J. T. , Jensen K. F. 
Published: October 2006

SummaryThe nucleotide sequence of two kilobase pairs (kb) 5′ to the orfE‐pyrE operon has been determined. The sequence revealed two open reading frames, orfX and orfY, consisting of 286 and 274 codons, respectively, and having a transcriptional orientation opposite that of the orfE‐pyrE operon. Analysis of transcription initiations showed that the promoters of orfE and orfX constitute a pair of divergent promoters with overlapping ‐35 regions and that orfY is transcribed from an independent promoter. Translational analysis indicated that the orfs are expressed in Escherichia coli.The orfE, orfX, and orfY genes were inactivated on the bacterial chromosome by deletion‐insertion mutagenesis using a kanamycin resistance cassette. The mutants were all viable. However, the orfE deletion caused a dramatic reduction in the level of pyrE expression and a partial pyrimidine requirement, because this mutation prevented transcription of pyrE. The orfE protein seemed without significance for pyr‐gene expression in E. coli, and the mutations in orfX and orfY were without detectable phenotypes.
Gene structure and precursor processing of a novel Bacillus subtilis spore coat protein
Authors: Aronson A. I. , Song H.‐Y. , Bourne N. 
Published: October 2006

SummaryThe gene for an unusual 8kD Bacillus subtilis spore coat polypeptide has been cloned and sequenced. It contains high percentages of proline, glycine and tyrosine, lacks thirteen amino acids, and is present as the carboxyl two‐thirds of an open reading frame encoding a 12 kD polypeptide. Two presumptive precursors which could be converted to the 8kD antigen by incubation with trypsin were found in extracts of cells or spores of a strain containing multiple copies of this gene. Large amounts of these coat antigens were also present in extracts of a germination‐defective mutant which is altered in spore coat structure. There was little 8kD coat protein in the mutant, however, implying that processing is dependent on proper coat assembly. This gene was mapped to the metA region of the B. subtilis chromosome, a unique location as is true for other spore coat genes. Transcription and translation occurred late in sporulation (stage V) and the upstream region contained sequences similar to those found in other spore coat genes.
Cloning and expression of plasmid DNA sequences involved in Salmonella serotype Typhimurium virulence
Authors: Norel F. , Coynault C. , Miras I. , Hermant D. , Popoff M. Y. 
Published: October 2006

SummaryA 22 kb region of the 90kb virulence‐associated plasmid, plP1350, of Typhimurium strain C52 was cloned into the mobilizable vector pSUP202, yielding plasmid plP1352. This recombinant plasmid restored full virulence to plasmidless strain C53 in a mouse model. Transposon Tn5 insertion mutagenesis demonstrated the existence of two DNA sequences in plP1352 designated VirA and VirB, both of which are essential for the expression of virulence. A recombinant plasmid containing only the VirA and VirB regions markedly increased the virulence of the plasmidless strain C53, but did not confer full virulence. These results suggested that a third virulence‐associated region might be present on plP1352. Eleven proteins encoded by the 22 kb insert sequence of pl P1352 were identified in Escherichia coli SE5000 maxicells. The VirA region encoded at least two proteins with apparent molecular weights of 71000 and 28000 and the VirB region encoded two proteins of 43000 and 38000.
The Escherichia coli enterobactin biosynthesis gene, entD: nucleotide sequence and membrane localization of its protein product
Authors: Armstrong S. K. , Pettis G. S. , Forrester L. J. , Mclntosh M. A. 
Published: October 2006

SummaryThe nucleotide sequence of the Escherichia coli enterobactin biosynthesis gene entD has been determined. entD specifies a predicted 23579 Dalton protein containing several helical regions, a trans‐membrane segment and one positively charged domain. The EntD polypeptide was overexpressed and identified in electrophoretic gels as a membrane protein. Although results of conventional membrane fractionation techniques were inconclusive, protease accessibility studies provided evidence that EntD domains are exposed on the inner leaflet of the cytoplasmic membrane. The presence of repetitive extragenic palindromic (REP) sequences within the fepA‐entD intercistronic region was confirmed. Lack of a canonical promoter and an iron control region 5’to entD, along with RNA hybridization data, suggest that an iron‐regulated transcript contains both fepA and entD.
RFLP‐based genetic map of Phanerochaete chrysosporium ME446: lignin peroxidase genes occur in clusters
Authors: Raeder U. , Thompson W. , Broda P. 
Published: October 2006

SummaryWe describe the construction of an RFLP‐based genetic map of the white rot fungus Phanerochaete chrysosporium ME446. The map is deduced from the allele distributions of 38 RFLP markers in a test set of 53 meiotically derived haploid recombinants of this strain. The map includes a cellulase gene, a mating‐type locus and a family of lignin peroxidase (and related) genes that are arranged in two unlinked clusters.
The nifA gene product from Rhizobium leguminosarum biovar trifolii lacks the N‐terminal domain found in other NifA proteins
Authors: Iismaa S. E. , Watson J. M. 
Published: October 2006

SummaryThe nifA gene has been identified between the fixX and nifB genes in the clover microsymbiont Rhizobium leguminosarum biovar trifolii (R.I. bv. trifolii) strain ANU843. Expression of the nifA gene is induced in the symbiotic state and site‐directed mutagenesis experiments indicate that nifA expression is essential for symbiotic nitrogen fixation. Interestingly, the predicted R.I. bv. trifolii NifA protein lacks an N‐terminal domain that is present in the homologous proteins from R.I. bv. viciae, Rhizobium meliloti, Brady rhizobium japonicum, Klebsiella pneumoniae and all other documented NifA proteins. This indicates that this N‐terminal domain is not essential for NifA function in R.I. bv. trifolii.
Specificity of insertion of IS91, an insertion sequence present in α‐haemolysin plasmids of Escherichia coli
Authors: Mendiola M. V. , de la Cruz F. 
Published: October 2006

SummaryWe have determined the DNA sequences of eight different insertions of IS91 in a specifically engineered recipient plasmid of known DNA sequence (pSU300). The sequences at the termini of IS91 are 5′ ‐CGAG‐TAGG… CCTATCGAT. IS91 inserts specifically 5′ to either one of the tetranucleotides 5′‐GAAC or 5′‐CAAG, and always in the same relative orientation with respect to the sequence of the target. Except in one special case, no duplications of the recipient DNA were produced at the site of insertion.
Genetic evidence that DicF, a second division inhibitor encoded by the Escherichia coli dicB operon, is probably RNA
Authors: Bouché F. , Bouché J.‐P. 
Published: October 2006

SummaryThe dicB operon of Escherichia coli, which was previously shown to code for a small protein inhibiting cell division, expresses a second inhibitor, DicF. Inhibition by DicF requires the transcription of a short (at the most 65 nucleotides long) stretch of DNA, acts in trans, and does not require the expression of other components of the dicABCF locus. The characteristics of the DNA sequence strongly suggest that division inhibition does not involve the translation of dicF mRNA into protein.
The terminus of the Escherichia coli chromosome is flanked by several polar polar replication pause sites
Authors: François V. , Louam J. , Louarn J.‐M. 
Published: October 2006

SummaryReplication of two small ‘constrained’ regions of the Escherichia coli chromosome, one bordered by replication terminator T1 and the other by T2, displays normal velocity in the normal direction whereas it is much slower in the opposite direction (de Massy et al., 1987). The presence of multiple polar terminators has been investigated, using a bacteriophage λ derivative which provides a replication origin movable to predetermined loci and inducible on demand. The amount of DNA made from this induced origin was determined by in vivo labelling and hybridization to probes of the surrounding region. A redundancy of terminator‐like sequences, or pause sites, has been disclosed. So far, two polar pause sites, in the same orientation and separated by 50 or 80 kb, have been localized on each side of the terminus region. The results are discussed in relation to previously observations indicating that these regions are refractory to genomic inversions.
Yeast 2μm vectors replicate and undergo recombination in Torulaspora delbrueckii
Authors: Compagno C. , Ranzi B. M. , Martegani E. 
Published: October 2006

SummaryIn order to develop a procedure for transformation of the industrial yeast Torulaspora delbrueckii, we have constructed a set of recombinant plasmids carrying Saccharomyces cerevisiae ARS and 2μm origin of replication and kanamycin‐G418 resistance gene of Tn903(601) as a selective marker. In this paper we show that S. cerevisiae ARS vectors can replicate autonomously and that vectors bearing the whole S. cerevisiae 2 μ sequence yield stable transform ants. We also present evidence to show that 2μm vectors undergo an FLP‐mediated inter‐ and intramolecular recombination, which suggests that T. delbrueckii can support the amplification and partition mechanisms of these plasmids.
Identification of Bacillus subtilis genes expressed early during sporulation
Authors: Mathiopoulos C. , Sonenshein A. L. 
Published: October 2006

SummaryLabelled cDNA transcribed in vitro from early‐sporulation RNA was enriched for sporulation‐specific sequences by subtractive hybridization to an excess of vegetative RNA and used to probe libraries of Bacillus subtilis chromosomal DNA. From the initial collection of clones that coded for RNAs transcribed preferentially during sporutation, several were subcloned and studied in more detail. It was found that two clones contained sequences (dciA and dciB) that had an undetectable level of transcription during vegetative growth but had transcripts that started to appear no later than eight minutes after induction of sporulation. A third DNA segment (dciC) was expressed at a low level in vegetative cells and increased within four minutes after induction of sporulation.The effects of spo0 mutations, i.e. mutations that prevent cells from reaching stage I of the sporulation process, were tested. Induction of the dciA and dciB transcripts was significantly reduced in strains carrying mutations in the spo0A and spo0H genes but not in a spo0B mutant strain. In addition, a product of the abrB locus, a locus in which mutations are known to partially overcome the pleiotropic effect of spo0A and spo0B mutations, seemed to be required for dciA and dciB expression.
The nucleotide sequence of the rodC operon of Bacillus subtilis
Authors: Honeyman A. L. , Stewart G. C. 
Published: October 2006

SummaryThe rodC1 mutation of Bacillus subtilis is a temperature‐sensitive marker which affects the levels of teichoic acid synthesis and the cellular morphology. We have determined the nucleotide sequence of the bicistronic operon which contains the rodC gene and the nucleotide sequence of the rodC1 mutant allele. The temperature‐sensitive phenotype of the rodC mutant is the result of a single base‐pair change. A cytosine to thymine transition in the non‐coding strand results in the replacement of a serine residue in the wild‐type protein with a phenylalanine residue in the mutant protein. The other gene in the operon, the rodD gene, appears to be equivalent to the gtaA gene which encodes uridine diphosphate‐glucose poly‐(glycerol phosphate) α‐glucosyl transferase, an enzyme involved in techoic acid synthesis. This is the first nucleotide sequence analysis of both the wild‐type and mutant alleles of a morphogene in B. subtilis.
Iron and virulence in Shigella
Authors: Payne S. M. 
Published: October 2006

SummaryIron limitation, a condition encountered within mammalian hosts, induces the synthesis of a number of proteins in pathogenic Shigella species. These include several outer membrane proteins, Shiga toxin, and proteins involved in the biosynthesis and transport of high‐affinity iron‐binding compounds or siderophores. Although siderophores have been shown to play a major role in the virulence of some bacterial pathogens, these compounds do not appear to be essential for the virulence of Shigella species. Unlike those pathogens which are restricted to the extracellular compartments of the host, the Shigella species invade and multiply within host cells. Alternative iron‐acquisition systems, such as the ability to utilize haem‐iron, permit growth of the intracellular bacteria. Virulent shigellae also possess a cell‐surface haem‐binding protein, and synthesis of this protein correlates with infectivity and virulence. This protein does not appear to be involved in iron acquisition. Rather, it may allow the bacteria to coat themselves with haem compounds, thus enhancing their ability to interact with target host cells.
Isolation, molecular characterization and expression of the ushB gene of Salmonella typhimurium which encodes a membrane‐bound UDP‐sugar hydrolase
Authors: Garrett A. R. , Johnson L. A. , Beacham I. R. 
Published: October 2006

SummaryThe UDP‐sugar hydrolase of Salmonella typhimurium has previously been reported to be located in both the inner and the outer membrane. We have cloned the gene, designated ushB, encoding this enzyme and determined its nucleotide sequence. No significant sequence homology with the periplasmic UDP‐sugar hydrolase of Escherichia coli was found at either the DNA or protein level. However, a sequence is detectable, in the E. coli genome, which weakly hybridizes with a specific ushB probe. Polypeptide analysis has allowed the identification of the Salmonella hydrolase which has an Mr of 28349 as compared to an Mr of 60767 for the E. coli hydrolase. Most of the protein (∼90%) is located in the inner membrane. Two independent membrane fractionation procedures indicate that the remainder may be associated with the outer membrane. The deduced primary structure indicates the presence of an N‐terminal signal peptide, although certain features of the region surrounding the putative processing site indicate that processing may be inefficient, or may not occur. Experiments with several inhibitors of signal peptidase function fail to demonstrate the appearance of a precursor form.
Receptor for IgA in group A streptococci: cloning of the gene and characterization of the protein expressed in Escherichia coli
Authors: Lindah G. , Åkerström B. 
Published: October 2006

SummaryThe gene for an IgA‐binding protein from a group A streptococcal strain was cloned and expressed in Escherichia coli. The IgA‐binding protein, called protein Arp, was purified on IgA‐Sepharose, allowing complete purification in a single step. Analysis of protein Arp by Western immunoblotting demonstrated a major IgA‐binding band, with an apparent molecular weight of 42kD. The purified protein was shown to bind serum IgA and secretory IgA, as well as monoclonal IgA of both subclasses. There was no binding to IgM, IgD or IgE, but a weak binding to IgG. Inhibition experiments with whole bacteria indicated that IgA and IgG bind at separate sites. Experiments with immunoglobulin fragments showed that protein Arp binds to the Fc region of both IgA and IgG. The equilibrium constant of the reaction between protein Arp and polyclonal human IgA was determined to be 5.6 × 108 M‐1. Amino acid sequencing of protein Arp demonstrated a direct repeat of 7 amino acids in the NH2‐terminal region, a feature previously found in several streptococcal M proteins. This suggests that protein Arp, like M proteins, may be a streptococcal virulence factor.