bluuebunny/crossref_metadata_2025_split huggingface dataset:
Sequence of the fhuE outer‐membrane receptor gene of Escherichia coli K12 and properties of mutants
Authors: Sauer M. , Hantke K. , Braun V. 
Published: October 2006

SummaryThe fhuE gene of Escherichia coli codes for an outer‐membrane receptor protein required for the uptake of iron(III) via coprogen, ferrioxamine B and rhodotorulic acid. The amino acid sequence, deduced from the nucleotide sequence, consisted of 729 residues. The mature form, composed of 693 residues, has a calculated molecular weight of 77453, which agrees with the molecular weight of 76000 determined by poly‐acrylamide gel electrophoresis. The FhuE protein contains four regions of homology with other TonB‐dependent receptors. A valine to proline exchange in the ‘TonB box’ absolished transport activity. Phenotypic revertants with substitutions of arginine, glutamine, or leucine at the valine position exhibited Increasing iron‐coprogen transport rates. Point mutations resulting in the replacement of glycine (127) in the second homology region with either alanine, aspartate, valine, asparagine or histidine exhibited decreased transport rates (listed in descending order). A truncated FhuE protein lacking 24 amino acids at the C‐terminal end was exported to the periplasm but failed to be inserted into the outer membrane.
The regulatory role of the IS 1‐encoded InsA protein in transposition
Authors: Zerbib D. , Polard P. , Escoubas J. M. , Galas D. , Chandler M. 
Published: October 2006

SummaryWe show here that the protein InsA, which is encoded by IS 1 and binds specifically to the terminal inverted repeats of this insertion sequence, negatively regulates IS 1 transposition activity. We demonstrate that it inhibits both IS 1‐mediated cointegrate formation and transposition of a synthetic IS 1‐based transposon (‘omegon’Ω‐on). These results also indicate that the Ω‐on which does not itself encode IS 1 transposition functions can be complemented in trans, presumably by the copies of IS 1 resident in the Escherichia coli chromosome. Using insA‐lacZ gene fusions, we show that at least part of this effect can be explained by the ability of InsA to repress expression of IS 1‐encoded genes both in cis or in trans. The experiments involving Ω‐on transposition raise the possibility that InsA inhibits transposition directly by competition with the transposase for their cognate site within the ends of IS 1.
The purine‐cytosine permease gene of Saccharomyces cerevisiae: primary structure and deduced protein sequence of the FCY2 gene product
Authors: Weber E. , Rodriguez C. , Chevallier M. R. , Jund R. 
Published: October 2006

SummaryA 2.1 kb DNA segment carrying the purine‐cytosine permease gene (FCY2) of Saccharomyces cerevisiae was sequenced, the primary structure of the protein (533 amino acids) deduced and a folding pattern in the membrane is proposed for the permease protein. Expression of the FCY2 gene product requires a functional secretory pathway and is reduced in mnn9, a mutant strain deficient in outer chain glycosylation. The FCY2 gene was mapped on the right arm of chromosome V close to the HIS1 gene.
Mechanism of polysialic acid chain elongation in Escherichia coli K1
Authors: Steenbergen S. M. , Vimr E. R. 
Published: October 2006

SummaryUnderstanding the mechanisms of polysialic acid synthesis in Escherichia coli K1 requires a molecular description of the polymerase complex. Since the number of potential models explaining polysialic acid assembly would be constrained if only one sialyltransferase were required for this process, the phenotypes of a sialyltransferase null mutation generated by transposon mutagenesis were investigated. The chromosomal insertion mutation was mapped by Southern hybridization analysis and by complementation with plasmid subclones, demonstrating that sialyltransferase is encoded by neuS, a gene implicated previously as coding for the polymerase (Vimr et al., 1989). As expected, if only one gene encoded sialyltransferase, the null mutant had undetectable polymerase activity when assayed with endogenous or exogenous acceptors, and accumulated sugar nucleotide precursors intracellularly. Nested deletion analysis of neuS ruled out polarity effects of transposon insertion mutation and provided more precise mapping of the sialyltransferase structural gene. Maxicell analysis of the nested deletion set implicated a 34000 molecular weight polypeptide as the neuS gene product. These results, together with biochemical characterization of sialyltransferase reaction products in the wild type, indicated that CMP‐sialic acid is the probable sialosyl donor for polysialic acid elongation and that chain growth is by sequential addition of monomeric units.
LamB (maltoporin) of Salmonella typhimurium: isolation, purification and comparison of sugar binding with LamB of Escherichia coli
Authors: Schülein K. , Benz R. 
Published: October 2006

Summary LamB (maltporin) of Salmonella typhimurium was found to be more strongly associated with the murein than OmpF. It was purified in one step using a hydroxyapatite (HTP) column. Reconstitution of the pure protein with lipid bilayer membrane showed that LamB of S. typhimurium formed small ion‐permeable channels with a single channel conductance of about 90 pS in 1 M KCI and some preference for cations over anions. The conductance concentration curve was linear, which suggested that LamB of S. typhimurium does not contain any binding site for ions. Pore conductance was completely inhibited by the addition of 20mM maltotriose. Titration of the LamB‐induced membrane conductance with different sugars, including all members of the maltooligosaccharide series up to seven glucose residues, suggested that the channel contains, like LamB (maltoporin) of Escherichia coli, a binding site for sugars. The binding constant of sugars of the maltooligosaccharide series increased with increasing number of glucose residues up to five (saturated). Small sugars had a higher stability constant for sugar binding relative to LamB of E. coli. The advantage of a binding site inside a specific porin for the permeation of solutes is discussed with respect to the properties of a general diffusion porin.
The ami locus of the Gram‐positive bacterium Streptococcus pneumoniae is similar to binding protein‐dependent transport operons of Gram‐negative bacteria
Authors: Alloing G. , Trombe M.C , Claverys J.P. 
Published: October 2006

SummaryThe complete nucleotide sequence of the ami locus of Streptococcus pneumoniae revealed the presence of six open reading frames, amiABCDEF. The predicted Ami proteins are probably involved in a transport system. The Ami A, C, D, E, and F proteins exhibit homology with components of the oligopeptide permeases (opp) of Salmonella typhimurium and Escherichia coli. Intriguingly, the AmiB protein is homologous to ArsC, a cytosolic modifier subunit of the anion pump encoded by the arsenical resistance operon of the R‐factor R773 from E. coli. Data are presented which indicate that Ami is indeed a transport system.
The cytotoxic protein YopE of Yersinia obstructs the primary host defence
Authors: Rosqvist R. , Forsberg Å. , Rimpiläinen M. , Bergman T. , Wolf‐Watz H. 
Published: October 2006

SummaryIt has previously been shown that the plasmid‐encoded YopE protein of Yersinia pseudotubercuiosis is a virulence determinant. In this study, HeLa cells, macrophages and mice were used as different model systems to determine the actual role of YopE in the virulence process. The YopE protein mediates a cytotoxic response on a confluent layer of HeLa cells. A prerequisite of this activity is that the pathogen binds to the cell surface. YopE also induces a cytotoxic response on mouse macrophages where it influences the ability of the pathogen to resist phagocytosis. Bacterial mutants defective in their ability to express YopE are avirulent after oral or Intra peritonea I infection but virulent following intravenous injection. On the basis of these results, we propose a role for YopE in the virulence process of Yersinia.
Ureaplasma urealyticum urease genes; use of a UGA tryptophan codon
Authors: Blanchard A. 
Published: October 2006

SummaryNucleotide sequence analysis of a Ureaplasma urealyticum DNA fragment, homologous to cloned urease genes of other prokaryotes, revealed three consecutive open reading frames. The molecular weights of the three deduced polypeptides are 11.2kD, 13.6kD and 66.6 kD. These values are consistent with the size of the three subunits previously reported for purified native urease. A significant sequence homology was found between the three polypeptides of the ureaplasmal urease and the single polypeptide of jack bean (Canavalia ensiformis) urease. Codon usage indicates that UGA is a tryptophan codon in this mollicute. Use of polymerase chain reactions has disclosed the existence of genetic polymorphism among the urease genes of different serotypes of U. urealyticum.
Genetic functions and cell–cell interactions in the pheromone‐inducible plasmid transfer system of Enterococcus faecalis
Authors: Dunny G. M. 
Published: October 2006

SummaryPheromone‐inducible plasmid transfer is a novel form of bacterial conjugation which has, to date, been observed only in Enterococcus (Streptococcus) faecalis. This process includes several important stages of interaction between the donor and recipient cell. The initial interaction is the transmission of a chemical signal from the recipient to the donor cell. Recent evidence has shown that the signal is in the form of a small hydrophobic peptide, which is capable of inducing a complex mating response in the donor cell at concentrations as low as 1–5 molecules per responder cell. Most E. faecalis strains produce multiple pheromones, each of which induces a response only in cells carrying a particular plasmid (or member of a family of related plasmids). Genetic functions ascribed to the pheromone response include: (i) cell‐cell aggregation, which promotes initial close contact between mating cells; (ii) surface exclusion, which prevents plasmid transfer between aggregated donor cells; and (iii) highly efficient DNA transfer, which requires other unidentified functions in addition to aggregation. The first two processes appear to be mediated by proteinaceous surface antigens.
The ‘Bayer bridges’ confronted with results from improved electron microscopy methods
Authors: Kellenberger E. 
Published: October 2006

SummaryIn electron micrographs of conventionally prepared thin sections of Escherichia coli one observes (i) a wavy appearance of the two membranes showing frequent appositions (named adhesion sites) and (ii) intermembrane bridges after plasmolysis which, it is claimed, occur at the adhesion sites and are related to intermembrane protein transport (transmigration). When chemical fixation is replaced by cryofixation, the observations are very different, (a) The two membranes are equally spaced and no contacts, adhesions or other sorts of connections are visible, (b) After plasmolysis the protoplast is shrunken, but the typical bridges are no longer produced. (c) In addition, when peptidoglycan is stained on conventionally prepared sections, it is revealed as a 7‐nm‐thick sacculus which is not interrupted at the sites of apposition. In view of the new observations, the structural concepts derived from conventionally prepared material must be revised. It is proposed that the intermembrane space is entirely filled by a gel, the outer part of which is the 7 nm thick, very stable, chemically resistant peptidoglycan (or murein). The inner part is much less stable and is proposed to undergo rapid autolytic changes upon cell death. The large ‘Bayer bridges’ might then tentatively be explained as an artificial post‐mortem enhancement of either a stream of proteins transmigrating across the periplasm or of a pre‐existing, but not yet resolved, structure. This enhancement probably occurs during the 7‐10 min between plasmolysis and fixation that are prescribed for the procedure necessary for revealing‘Bayer bridges'.
Integrity of Escherichia coli P pili during biogenesis: properties and role of PapJ
Authors: Tennent J. M. , Lindberg F. , Normark S. 
Published: October 2006

SummaryThe papJ gene of uropathogenic Escherichia coli is required to maintain the integrity of Galα(1‐4)Gal‐binding P pili. Electron microscopy and ELISA have established that strains carrying the papJ1 mutant allele have a large amount of pilus antigen free of the cells. In contrast to the whole pili released by strains unable to produce the PapH pilus anchor, the free papJ1 pili consist of variably sized segments that appear to result from Internal breakages to the pilus. The DNA sequence of papJ is presented and its gene product identified as an 18kD periplasmic protein that possesses homology with nucleotide‐binding proteins. PapJ may function as a ‘molecular chaperone’ directly or indirectly establishing the correct assembly of PapA subunits in the P pilus.
Cloning of a large gene cluster involved in Erwinia amylovora CFBP1430 virulence
Authors: Barny M. A. , Guinebretière M. H. , Marçais B. , Coissac E. , Paulin J. P. , Laurent J. 
Published: October 2006

SummaryPhage MudIIPR13 insertional mutagenesis of Erwinia amylovora CFBP1430 allowed us to isolate 6900 independent CmR mutants. The frequencies of different auxotrophs in this population indicated that MudIIPR13 had inserted randomly in E. amylovora. Screening of 3500 CmR mutants on (i) apple calli and (ii) pear and apple seedlings led to the isolation of 19 non‐pathogenic prototrophic single mutants, four of which expressed a LacZ+ hybrid protein. Expression of the fusion proteins was temperature sensitive. The 19 mutants could be separated into two classes according to their behaviour on tobacco: 13 were unable to elicit the hypersensitive response on tobacco (Hrp−) while six still could (Dsp−). The 19 MudIIPR13 insertions all mapped in the same virulence region. The MudIIPR13 insertions of Hrp− mutants were all clustered on the left part of this region, white the MudIIPR13 insertions of Dsp− mutants were located on the right part. All of the mutants except one, which proved to have a large deletion of the entire virulence region, could be complemented functionally by cosmids from an E amylovora CFBP1430 genomic library. No hybridization was observed between the cosmid pPV130, which complemented 12 hrp::MudIIPR13 mutations, and the hrp genes from Pseudomonas syringae pv. phaseolicola (Lindgren et al., 1986), P. syringae pv. tomato (N. J. Panopoulos, unpublished data) or P. solanacearum (Boucher et al., 1987). Further analysis of the large virulence region will allow mapping of the border of the virulence region and facilitate the study of the function and regulation of the hrp and dsp genes.
Genetic characterization of Bordetella pertussis filamentous haemagglutinin: a protein processed from an unusually large precursor
Authors: Domenighini M. , Relman D. , Capiau C. , Falkow S. , Prugnola A. , Scarlato V. , Rappuoli R. 
Published: October 2006

SummaryThe nucleotide sequence of the structural gene for filamentous haemagglutinin (FHA), fhaB, a crucial adherence factor for Bordetella perfussis, has been determined. Its 10774 nucleotides are far more than necessary to encode the 220kD biologically active, mature polypeptide product, suggesting a role for co‐or post‐translational processing. Fusion proteins derived from various portions of the fhaB open reading frame (ORF) were used to generate polyclonal antisera. Western immunoblot analysis of purified FHA and Bordetella sp. whole cell extracts with these antisera indicated that the 220kD product is encoded by the 5 portion of the ORF and that the smaller polypeptide species are breakdown products of this polypeptide. These data, as well as N‐terminal amino acid sequencing of the major polypeptide species, suggest a scheme for the proteolytic processing of an FHA precursor polypeptide.
Recent advances in peptide chain termination
Authors: Craigen W. J. , Lee C. C. , Caskey C. T. 
Published: October 2006

SummaryPeptide chain termination occurs when a stop codon is decoded by a release factor. In Escherichia coli two codon‐specific release factors (RF1 and RF2) direct the termination of protein synthesis, while in eukaryotes a single factor is required. The E. coli factors have been purified and their genes isolated. A combination of protein and DNA sequence data reveal that the RFs are structurally similar and that RF2 is encoded in two reading frames. Frame‐shifting from one reading frame to the next occurs at a rate of 50%, is regulated by the RF2‐specific stop codon UGA, and involves the direct interaction of the RF2 mRNA with the 3’end of the 16S rRNA. The RF genes are located in two separate operons, with the RF1 gene located at 26.7 min and the RF2 gene at 62.3 min on the chromosome map. Ribosomal binding studies place the RF‐binding region at the interface between the ribosomal subunits. A possible mechanism of stop‐codon recognition is reviewed.
DNA from diverse sources manifests cryptic low‐level transcription in Escherichia coli
Authors: Miller W. G. , Simons R. W. 
Published: October 2006

SummaryWe present evidence that DNA from diverse prokaryotic and eukaryotic sources gives rise to low‐level fusion expression in Escherichia coli promoter‐probe vectors. This expression may be as high as 10% of the E coli lac UV5 promoter. Although expression does not correlate with the presence of obvious E. coli promoter‐like sequences, it is blocked by transcriptional terminators. Furthermore, transcription across the fusion junction is detected at levels that correlate with fusion expression. We suggest that this Mow‐level transcription'(LLT) results from infrequent initiation by RNA polymerase at random sites and/or weak promoters. We propose that LLT has biological significance. In some instances, it may provide an advantageous basal level of gene expression, and we suggest that this may be true for the E. coli lac Y gene. In other instances, LLT may be detrimental, in which case it may be blocked by mechanisms such as RNA secondary structure or transcriptional polarity. We present evidence to show that activation of the IS 10 transposase gene by LLT is blocked at the translational level.
Sequence analysis of Enterococcus faecalis aggregation substance encoded by the sex pheromone plasmid pAD1
Authors: Galli D. , Lottspeich F. , Wirth R. 
Published: October 2006

SummaryThe location of the structural gene for aggregation substance on the sex pheromone plasmid pAD1 of Enterococcus faecalis was determined using an oligonucleotide deduced from the N‐terminal amino acid sequence of the purified protein. The nucleotide sequence was determined for the corresponding region and two open reading frames (ORFs) could be identified. ORF1 codes for a small (Mr 13160) acidic protein of unknown function. The gene for aggregation substance (named asa1 was found to code for a protein of 1296 amino acids (Mr 142248). The protein has a signal peptide of 43 amino acids (the resulting (Mr for mature aggregation substance is 137429) and contains in its C‐terminal region a proline‐rich sequence, previously characterized as being involved in cell wall association, which is followed by a membrane anchor. The membrane anchor showed significant similarity to that of other Gram‐positive organisms, but no other similarities to surface proteins from Gram‐positive bacteria were found. In particular, no repeats on the DNA or protein level could be detected for pAD1‐specific aggregation substance. The protein contains the amino acid motifs Arg‐Gly‐Asp‐Ser and Arg‐Gly‐Asp‐Val (once each), which, it is proposed, play a crucial role in adherence to eukaryotic cells.
Insertion of an extra amino acid is the main cause of the low affinity of penicillin‐binding protein 2 in penicillin‐resistant strains of Neisseria gonorrhoeae
Authors: Brannigan J. A. , Tirodimos I. A. , Zhang Q.‐Y. , Dowson C. G. , Spratt B. G. 
Published: October 2006

SummaryNon‐β‐lactamase‐ producing, penicillin‐resistant strains of Neisseria gonorrhoeae (CMRNG strains) produce altered forms of penicillin‐binding protein 2 (PBP2) that have decreased affinity for penicillin. A feature of PBP2 from all CMRNG strains is the presence of an additional residue (Asp‐345A) that is absent from PBP2 of penicillin‐sensitive strains. The role of the additional aspartic acid residue in the decreased affinity of PBP2 is unclear as PBP2 of all previously examined CMRNG strains possess several other amino acid sequence alterations, in addition to the insertion of Asp‐345A, compared to PBP2 of penicillin‐sensitive strains. Site‐directed mutagenesis has been used to insert the Asp‐345A codon into the penA gene from a penicillin‐sensitive gonococcus. The resulting penA gene expressed an altered form of PBP2 that had a decreased affinity for benzylpenicillin and was able to transform a pencillin‐sensitive strain of N. gonorrhoeae to an increased level of resistance to benzylpenicillin. Insertion of amino acids other than aspartic acid did not produce forms of PBP2 that provided increased resistance to penicillin. Removal of the Asp‐345A codon from the penA gene of a CMRNG strain reduced its ability to transform a penicillin‐sensitive strain to an increased level of penicillin resistance. The reduction in the affinity of PBP2 in CMRNG strains is therefore largely, although not exclusively, due to the insertion of Asp‐345A. Clinical isolates that produce altered forms of PBP2 that differ from that of penicillin‐sensitive strains only in the insertion of Asp‐345A have been identified.
Octanoylation of the lipoyl domains of the pyruvate dehydrogenase complex in a lipoyl‐deficient strain of Escherichia coli
Authors: Ali S. T. , Moir A. J. G. , Ashton P. R. , Engel P. C. , Guest J. R. 
Published: October 2006

SummaryThe overexpression of a subgene encoding a hybrid lipoyl domain of the dihydrolipoamide acetyltransferase component of the pyruvate dehydrogenase complex of Escherichia coli has previously bee shown to result in the formation of lipoylated an unlipoylated products. Overexpression of the same subgene in a lipoic acid biosynthesis mutant growing under lipoate‐deficient conditions has now bee shown to produce domains modified by octanoylation as well as unmodified domains. It was concluded from the mass of a lipoyl‐binding‐site peptide that the modification involves N6‐octanoylation of the lysin residue (Lys244) that is normally lipoylated, and this was confirmed by the trypsin‐insensitivity of the corresponding Lys244‐Ala245 bond, and the absence c modification in a mutant domain in which Lys244 is replaced by Gin. This novel protein modification raise interesting questions concerning the pathway of lipoic acid biosynthesis and the mechanism of enzyme lipoylation.