bluuebunny/crossref_metadata_2025_split huggingface dataset:
Export and secretion of the lipoprotein Pullulanase by Klebsiella pneumoniae
Authors: D'Enfert C. , Chapon C. , Pugsley A. P. 
Published: October 2006

SummaryPullulanase, a secreted lipoprotein of Klebsiella pneumoniae, is initially localized to the outer face of the outer membrane, as shown by protease and substrate accessibility and by immunofluorescence tests. Freeze‐thaw disruption of these cells released both membrane‐associated and apparently soluble forms of Pullulanase. Membrane‐associated Pullulanase co‐fractionated with authentic outer membrane vesicles upon isopycnic sucrose‐gradient centrifugation, whereas the quasi‐soluble form had the same equilibrium density as inner membrane vesicles and extracellular Pullulanase aggregates. The latter also contained outer membrane maltoporin, but were largely devoid of other membrane components including LPS and lipids. K. pneumoniae carrying multiple copies of the Pullulanase structural gene (pulA) produced increased amounts of cell‐associated and secreted Pullulanase, but a large proportion of the enzyme was neither exposed on the cell surface nor released into the medium, even after prolonged incubation. This suggests that factors necessary for Pullulanase secretion were saturated by the over‐produced Pullulanase. When pulA was expressed under lacZ promoter control, the Pullulanase which was produced was not exposed on the cell surface at any time, suggesting that Pullulanase secretion genes are not expressed constitutively, and raising the possibility that they, like puM, may be part of the maltose regulon.
Nucleotide sequence of the virG locus of the Agrobacterium tumefaciens plasmid pTiC58
Authors: Powell B. S. , Powell G. K. , Morris R. O. , Rogowsky P. M. , Kado C. I. 
Published: October 2006

SummaryThe nucleotide sequence of the virG locus of the nopaline type plasmid pTiC58 of Agrobacterium tumefaciens has been determined. It contains an open reading frame (ORF) of 759 nucleotides and has 77% homology to the virG sequences of octopine type plasmids. Differences between the sequences of the two types of Ti plasmids in the region of virG are located predominantly outside the ORF. The amino acid sequences inferred from the two virG genes show 80% homology to each other and each shows the same moderate homologies to amino acid sequences derived from genes in a family of two‐component regulatory systems. Specific differences in nucleotide and amino acid sequences as well as a structure‐function model for the gene product are discussed.
A RAS‐encoded protein in Dictyostelium discoideum is acylated and membrane‐associated
Authors: Weeks G. , Lima A. F. , Pawson T. 
Published: October 2006

Summary Dictyostelium dfscoideum synthesizes a 23000 Mr protein, p23dd‐ras, closely related to the mammalian oncogene‐encoded protein p21ras. To investigate the subcellular localization of P23dd‐ras, conditions were optimized to reduce protein degradation following cell breakage. Subcellular fractionation of D. discoideum showed that p23dd‐ras was associated predominantly with the membrane fraction during both vegetative growth and differentiation, in the absence of suitable protease inhibitors considerable amounts of a truncated form of p23dd‐ras were recovered in the cytosol fraction, suggesting that intact p23dd‐ras is attached to the membrane by a short terminal peptide sequence. Radio‐isotope labelilng of D. discoideum with myristic acid or palmitic acid in the presence of excess un‐labelled acetate resulted in radio‐isotope incorporation into a select group of proteins including p23dd‐ras. No acyl label appeared in the truncated cytoplasmic form of p23dd‐ras when ceil breakage was performed In the absence of suitable protease inhibitors, indicating that the acyl group is associated with the short terminal peptide that is cleaved. These data suggest that p23dd‐ras like its mammailan counterpart, is acylated and associated with the plasma membrane. There was no evidence during a 30‐minute pulse of methionine for a cytoplasmic precursor to the membrane‐bound p23dd‐ras suggesting that the turnover of the presumptive precursor must be much more rapid in D. discoideum than for pro‐p21ras in mammalian cells.
Penicillin‐binding proteins in β‐lactam‐resistant laboratory mutants of Streptococcus pneumoniae
Authors: Lalble G. , Hakenbeck R. 
Published: October 2006

SummaryThe increasing number of penicillin‐resistant clinical strains of Strepfococcus pneumoniae has raised questions about the mechanism involved. We have isolated a large number of independent, spontaneous laboratory mutants with increasing resistance against either piperacillin or cefotaxime. Both classes of mutants showed a different pathway of penicillin‐binding protein (PBP) alterations, and within each group of mutants the individual PBPs appeared to have changed at different resistance levels and in different sequences. The mutations led to decreased β‐lactam affinity and possibly to a reduction in the amount of protein present in the cell, but differences in apparent molecular weight, like those reported in low‐ and high‐level resistant pathogenic strains, were not found. Some mutants showed a high degree of cross‐resistance to a variety of pencillins and cephaiosporins independently of the acquired PBP alterations, indicating that different genotypes can be responsible for the same phenotypic expression of resistance.
DNA sequence of the gene scrA encoding the sucrose transport protein EnzymellScr of the phosphotransferase system from enteric bacteria: homology of the EnzymellScr and EnzymellBgl proteins
Authors: Ebner R. , Lengeler J. W. 
Published: October 2006

SummaryThe nucleotide sequence of the structural gene, scrA, which codes for sucrose‐specific EnzymellScr (EIIScr) of the phosphoenolpyruvate‐dependent carbohydrate: phosphotransferase system (PTS), was determined. EllScr requires an Enzymelll, the product of the gene crr, for full activity. The gene scrA is preceded immediately by a classical Shine‐Dalgarno sequence (AAGAGGGTA). It contains 1368 nucleotides with an increased GC‐content (58%) corresponding to a polypeptide of 455 amino acid residues (Mr 47 500). The protein has the hydropathic profile (average hydropathy +0.82) of an integral membrane protein lacking extended α‐helical structures and a signal peptide. Comparison with the sequence of the β‐glucoside‐specific Enzymell (EllBgl, 625 amino acids, Mr 66480; Bramley and Kornberg, 1987a; Schnetz et al., 1987) revealed strong homologies between EllScr and the first 458 residues of EllBgl. The 162 carboxyterminal residues of EllBgl, however, showed a high homology with the sequence of Enzymelll (Nelson et al., 1984), a homology also described recently by Bramley and Kornberg (1987b). The evolutionary and functional significance of the similarities with four other Enzymesll is discussed.
Nucleotide sequence and fine structural analysis of the Corynebacterium glutamicum hom‐thrB operon
Authors: Peoples O. P. , Liebl W. , Bodis M. , Maeng P. J. , Follettie M. T. , Archer J. A. , Sinskey A. J. 
Published: October 2006

SummaryThe complete nucleotide sequence of the Corynebacterium glutamicum hom‐thrB operon has been determined and the structural genes and promoter region mapped. A polypeptide of Mr 46136 is encoded by hom and a polypeptide of Mr, 32618 is encoded by thrB. Both predicted protein sequences show amino acid sequence homology to their counterparts in Escherichia coli and Bacillus subtilis. The promoter region has been mapped by S1‐nuclease and deletion analysis. Located between −88, RNA start site and −219 (smallest deletion clone with complete activity) are sequence elements similar to those found in E. coli and B. subtills promoters. Although there are no obvious attenuator‐like structures in the 5′‐untranslated region, there is a dyad‐symmetry element, which may act as an operator.
Biogenesis of F71 and F72 fimbriae of uropathogenic Escherichia coli: influence of the FsoF and FstFG proteins and localization of the Fso/FstE protein
Authors: Riegman N. , van Die I. , Leunissen J. , Hoekstra W. , Bergmans H. 
Published: October 2006

SummaryThe F71 and F71 P‐fimbriae of Escherichia coli are encoded by the fso (F seven one) and fst (F seven two) gene clusters, respectively (Van Die et al., 1984; 1985).With the immunocytochemical gold‐labelling technique it was demonstrated that both the FsoE and FstE proteins are non‐adhesive minor fimbrial sub‐units located at the tip of the fimbrial structure. The FsoF and FstFG proteins play an important role in the initiation of polymerization of the minor and major subunits into the fimbrial structure.
Role of the intercistronic region in post‐transcriptional control of gene expression in the histidine transport operon of Salmonella typhimurium: involvement of REP sequences
Authors: Stern M. J. , Prossnitz E. , Ames G. Ferro‐Luzzi 
Published: October 2006

SummaryThe high‐affinity histidine permease of Salmonella typhimurium is encoded by a four‐gene operon containing a large intercistronic region located between the first gene (hisJ) and the three distal genes (hisQ, hisM, hisP). The level of expression of hisJ is 30‐fold greater than that of hisP. In order to investigate the role of the intercistronic region in intra‐operonic control of gene expression, we have isoiated MudII‐mediated IacZ gene fusions to hisQ, hisM and hisP. We have used these fusions to isolate and analyse mutants that have altered levels of expression of the hisQ gene, the first gene downstream from the intercistronic region. The results indicate that intra‐operonic regulation is due to a combination of factors including efficiency of translational initiation, mRNA degradation, and retroregulation of hisJ expression. They also suggest that the REP (Repetitive Extragenic Palin‐dromic) sequences, which are located in the hisJ‐hisQ intercistronic region, may interfere with translation of the hisQ gene and affect upstream messenger RNA stability by protecting it from 3’to 5’nuclease degradation (in agreement with data presented by Newbury et al., 1987).
Functional analysis of different sequence elements in the Escherichia coli galactose operon P 2 promoter
Authors: Ponnambalam S. , Chan B. , Busby S. 
Published: October 2006

SummaryStarting with a DNA fragment containing the galactose operon P 2 promoter, we made a series of deletions that progressively replaced DNA sequences upstream of the transcription startpoint and determined their effects on P 2 activity. The results show that specific sequences upstream of −32 are not important. Removal of the sequence 5′‐CACA‐3′ from −32 to −28 reduces P 2 activity by 50%: longer deletions to −16 further reduce activity but do not remove the information specifying the transcription startpoint. DNA sequences between −32 and −16 at galP 2 assist the isomerization of RNA polymerase from closed to open complexes rather than contributing to the initial binding of RNA polymerase. The activity of gal P 2 in the absence of −35 region sequences is dependent on the sequence TG just upstream of the − 10 hexamer, TATACT: a mutation at −14 changing the TG sequence to TT totally inactivates P 2. However, P 2 activity can be restored if the consensus −35 region sequence TTGACA is cloned 17 bp upstream of the −10 hexamer. Thus, for transcription initiation, the −10 hexamer, TATACT, must ‘cooperate’ with upstream sequences that may be located either around −35 or −14.
Characterization of the Rhizobium leguminosarum genes nodLMN involved in efficient host‐specific nodulation
Authors: Surin B. P. , Downie J. A. 
Published: October 2006

SummaryThree nodulation genes, nodL, nodM and nodN, were isolated from Rhizobium leguminosarum and their DNA sequences were determined. The three genes are in the same orientation as the previously described nodFE genes and the predicted molecular weights of their products are 20105 (nodL), 65795 (nodM) and 18 031 (nodN.) Analysis of gene regulation using operon fusions showed that nodL, nodM and nodN are induced in response to flavanone molecules and that this induction is nodD‐dependent. In addition, it was shown that the nodM and nodW genes are in one operon which is preceded by a conserved ‘nod‐box’ sequence, whereas the nodL gene is in the same operon as the nodFE genes. DNA hybridizations using specific gene probes showed that strongly homologous genes are present in Rhizobium trifolii but not Rhizobium meliloti or Bradyrhizobium japonicum. A mutation within nodL strongly reduced nodulation of peas, Lens and Lathyrus but had little effect on nodutation of Vicia species. A slight reduction in nodulation of Vicia hirsuta was observed with strains carrying mutations in nodM or nodN.
Uropathogenic, Escherichia coli can express serologically identical pili of different receptor binding specificities
Authors: Lund B. , Marklund B.‐I. , Strömberg N. , Lindberg F. , Karlsson K.‐A. , Normark S. 
Published: October 2006

SummaryUropathogenic Escherichia coli frequently express P‐pilus adhesins that recognize Galα(1–4)Gal‐containing glycoconjugates. The P‐pilus adhesin of the, E. coli isolate J96 is encoded by the pap gene cluster and has been shown to agglutinate P1‐erythrocytes. We now describe a novel gene cluster from J96, prs, which is responsible for the agglutination of sheep erythrocytes. The structurally related gene clusters both expressed pili exhibiting the F13 antigen. Analysis of mutants of cloned prs sequences, together with trans‐complementation of pap and prs genes, identified the sheep‐specific adhesin as the 37‐kD PrsG protein. The prsG gene occupies the equivalent position in prs as occupied by papG, which specifies the Galeα(1–4)Gal‐specific adhesion of pap. PrsG was shown to be structurally distinct from PapG since PapG‐specific antiserum did not cross‐react with PrsG. Using a solid phase glycolipid receptor binding assay, PrsG was found to specify preferential binding to the Forssman antigen, a major constituent of sheep erythrocyte membranes. The binding epitope was identified as the GaINAcα(1–3)GaINAc moiety. This is the first direct evidence that serologically identical pili may present antigenically distinct adhesins, each capable of binding to a specific receptor.
Cloning and CO2‐dependent expression of the genetic region for encapsulation from Bacillus anthracis
Authors: Makino S. , Sasakawa C. , Uchida I. , Terakado N. , Yoshikawa M. 
Published: October 2006

SummaryThe capsule of Bacillus anthracis is an important virulence factor consisting of poly‐o‐glutamic acid. The genetic region required for the encapsulation was cloned in Escherichia coli from the capsule plasmid pTE702, using a selection procedure based on an immunodlffusion assay. The cloned region directed synthesis of the capsule both In E. coli and B. anthracis. Capsule synthesis from these clones, as in the wild type, was dependent upon the presence of CO2. However, encapsulation directed by the cloned fragment was less marked than from pTE702. Another region enhancing capsulation was shown to exist on pTE702. The minimum size of the encapsulation region was defined to within 2.7 kb DNA and shown to be essential for the encapsulation in B. anthracis.
Aerobic expression of the nar operon of Escherichia coli in a fnr mutant
Authors: Bonnefoy V. , Fons M. , Ratouchnlak J. , Pascal M.‐C. , Chippaux M. 
Published: October 2006

SummaryMutations allowing aerobic expression of the anaerobically controlled nar operon have been located in the autoregulated fnr gene. Cloning and sequencing of the mutant fnrd20 allele, and fnr mRNA quantitation by dot blot assay, revealed that the mutation was the result of an IS5 insertion into the control region of fnr that enhanced transcription of the fnr gene at least ten‐fold. Examination of the regulatory region of the negatively autoregulated fnr gene indicated that it shared homologous sequences with the positively Fnr‐controlled frd and nar operons. The increase in fnr transcription in the fnrd20 mutated allele could be partly the result of loss of autoregulation, since the IS5 separated the Fnr target site from the ‘−35’ region of the promoter.
Cloning and characterization of an albicidin resistance gene from Klebsiella oxytoca
Authors: Walker M. J. , Birch R. G. , Pemberton J. M. 
Published: October 2006

SummaryA DNA fragment containing a gene for resistance to the antibiotic albicidin was isolated from Klebsiella oxytoca and shown to be expressed in Escherichia coli, where it also protected bacteriophage T7 replication from inhibition by albicidin. In vivo translation analysis demonstrated that the cloned 2.2kb DNA fragment coded for a 36 kiloDalton (kD) protein and a 25 kD protein.The DNA sequence was determined for a 654‐base‐pair open reading frame contained within a 1.2 kb subcloned DNA fragment encoding albicidin resistance. The predicted molecular weight of the polypeptide translated from the open reading frame was 25.8 kD. A putative Shine‐Dalgarno sequence precedes the open reading frame but a potential promoter sequence was not detected. A possible rho‐independent transcription termination signal was found directly following the stop codon. The functional protein for albicidin resistance was isolated and purified. Both the molecular weight and NHg‐terminal amino acid sequence of this protein correspond with that predicted from the DNA sequence of the open reading frame. The cloned albicidin resistance gene had no effect on the tsx (nupA) nucleoside uptake gene associated with spontaneous albicidin resistance in E. coli; also, it did not complement any of a range of E. coli DNAts mutants at restrictive temperatures. The cloned resistance gene product remained intracellular in exponential cultures of K. oxytoca and E. coli. Cell‐free extracts from E. coli containing the resistance gene protected a sensitive strain of E. coli from inhibition by albicidin, as did the purified albicidin resistance protein. The mechanism of this albicidin resistance protein involved binding to albicidin to form a complex without antibiotic activity, but without catalysing further chemical modification of the antibiotic.
Determinants of translational initiation efficiency in the atp operon of Escherichia coli
Authors: McCarthy J. E. G. , Bokelmann C. 
Published: October 2006

SummaryTranscription and translation of the atp genes encoding the subunits δ, α, γ, and ɛ of the Escherichia coli H+‐ATPase were studied. The nature and quantities of the respective transcripts initiated from different promoters were compared with overall expression rates thus yielding accurate information about relative translational efficiency and its coupling to mRNA levels. Part of the highly efficient subunit c gene translational initiation region (TIR) was used as a tool in manipulating the TIRs of the other genes. Rate control of atp cistron translation occurs at the initiation level and is determined locally by each gene's TIR. In this way, individual subunit synthesis rates are set to match the requirements for H+‐ATPase assembly. There is no (or very restricted) translational coupling between the cistrons. Translational initiation rates of the normally weakly expressed atp genes could be increased by up to a factor of 27 by manipulating the sequences upstream of the start codons, despite biased codon usages. In the presence of an improved upstream sequence, the N‐terminal sequence of the subunit γ gene exerted a limiting effect. This could be relieved by altering the sequence of the first seven codons. The levels of subunit γ mRNA were more sensitive to changes in translational efficiency than the concentrations of the other atp mRNAs. The relationships between initiation efficiency and primary and secondary structure in the natural and manipulated atp TIRs are discussed in detail.
Molecular characterization of malQ, the structural gene for the Escherichia coli enzyme amylomaltase
Authors: Pugsley A. P. , Dubreuil C. 
Published: October 2006

SummaryThe structural gene for the Escherichia coli enzyme amylomaltase, malQ, is the second gene in the malPQ operon. The nucleotide sequence of malQ shows that the gene encodes an Mr 78360 protein close to the experimentally determined Mr of purified amylomaltase (72000–74000). The malQ initiation codon was identified by sequence analysis of clustered deletions around the 5′ end of the gene. One of these deletions removed the first 5 bases from the malQ coding sequence. Strains carrying a plasmid with this truncated malQ gene under lacZ promoter control and out‐of‐frame with the first four codons of lacZ were Mal−. The Mal+ phenotype could be restored by inserting small, random fragments of E. coli chromosomal DNA into the unique EcoRI site. Nucleotide sequencing showed that the inserts either joined the lacZ and malQ sequences in frame, or contained a new translation start signal and coding sequence in frame with malQ. These results indicate that amylomaltase could be useful as a reporter protein in gene fusion studies.
Extracellular proteins of Vibrio cholerae: nucleotide sequence of the structural gene (hlyA) for the haemolysin of the haemolytic El Tor strain 017 and characterization of the hlyA mutation in the non‐haemolytic classical strain 569B
Authors: Aim R. A. , Stroeher U. H. , Manning P. A. 
Published: October 2006

SummaryThe El Tor haemolysin, product of hlyA, is exported from Vibrio cholerae as a Mr 80000 protein which can be subsequently cleaved to give two proteins of Mr 65000 and 15000. Nucleotide sequence analysis has demonstrated that hlyA encodes a protein of Mr 82250 with a potential 18‐amino‐acid signal sequence.The non‐haemolytic classical strain 569B has been shown to have a structural gene defect rather than a defect in secretion. By non‐reciprocal recombination it was possible to transfer this defect onto a plasmid and show that a truncated hlyA product of Mr 27000 is made in Escherichia coli K‐12 minicells. Nucleotide sequence analysis demonstrates an 11‐base‐pair deletion which would result in a Mr 26940 protein probably loosely associated with the membrane.
Expression of four virulence antigens of Shigella flexneri is positively regulated at the transcriptional level by the 30 kilo Dalton virF protein
Authors: Sakai T. , Sasakawa C. , Yoshikawa M. 
Published: October 2006

SummaryOn the virulence plasmid of Shigella flexneri the virG region required for cell‐to‐cell spread of the bacteriumencodes a 130 kiloDalton (kD) antigen and Region‐2essential for the bacterial invasion of epithelial cells encodes 57, 43 and 39 kD antigens. The expression of these four antigens is positively regulated by the 30 kD protein encoded by virF, whose nucleotide sequence had been determined and which was previously found to be essential for virulence. An approximately 3.8kilobase (kb) RNA transcript is found to be transcribed by the virG region and is positively regulated by the virF protein resulting in increased production of the 130 kD antigen. The virF sequence is conserved among all shigellae and enteroinvasive Escherichia coli.
Application of electroporation for transfer of plasmid DNA to Lactobacillus, Lactococcus, Leuconostoc, Listeria, Pediococcus, Bacillus, Staphylococcus, Enterococcus and Propionibacterium
Authors: Luchansky J. B. , Muriana P. M. , Klaenhammer T. R. 
Published: October 2006

SummaryPlasmid DNA was introduced by electroporation into Bacillus, Enterococcus, LactobacHlus, Lactococcus, Leuconostoc, Listeria, Pediococcus, Propionibacterium and Staphylococcus as an alternative to competent‐cell or protoplast transformation. Plasmid‐containing transformants were recovered in these recipients at frequencies ranging from 101 105 transformants μg−1 of pGK12. Several parameters of the protocol, including DNA concentration, voltage, plating regimen and electroporation buffers were evaluated to determine conditions that improved transformation frequencies for Lactobacillus acidophilus. Using optimized conditions, the following plasmids were introduced into L. acidophilus: pAMB1, pC194, pGB354, pGKV1, pSA3, pTRK13, pTV1 andpVA797. The ability to transfer plasmid DNA via electroporation will greatly facilitate the application of recombinant DNA methodology and transposon technology to Gram‐positive bacteria for cloning and analysis of significant genes.