bluuebunny/crossref_metadata_2025_split huggingface dataset:
Analysis of the membrane‐binding domain of penicillin‐binding protein 5 of Escherichia coli
Authors: Jackson M. E. , Pratt J. M. 
Published: October 2006

SummaryInternal deletions close to the C‐terminus of the Escherichia coli penicillin binding protein 5 (PBP5, DacA) have defined the C‐terminal 18 residues of the protein as essential for membrane binding. This C‐terminal sequence is capable of forming a strongly amphiphilic α‐helix. In this paper we show that the PBP5 amphiphilic helix is able to anchor the periplasmic TEM‐β‐lactamase to the inner membrane. In addition, we have demonstrated that mature PBP5 (lacking the N‐terminal signal sequence) possesses the ability to bind to the membrane from a soluble form of the protein, showing that translocation across the membrane is unnecessary for anchoring to be established.
Transcriptional analysis of the 16S rRNA gene of the rrnD gene set of Streptomyces coelicolor A3(2)
Authors: Baylis H. A. , Bibb M. J. 
Published: October 2006

SummaryThe nucleotide sequence of 2.5 kb of the Streptomyces coelicolor A3(2) rRNA gene set rrnD, extending from upstream of the 16S rRNA gene to the putative 5′ end of the 23S rRNA gene, has been determined (Baylis and Bibb, 1987; this paper). In addition to locating the 5′ end of the 16S rRNA gene, nuclease S1 mapping identified seven RNA 5′ end‐points upstream of the 16S rRNA gene; four of these were coincident with transcriptional initiation points for S. coelicolor A3(2) RNA poiymerase in vitro and were consequently regarded as in vivo transcription start points for promoters p1 to p4. One end‐point identified by nuclease S1 mapping localized a putative processing site analogous to those found upstream of 16S rRNA genes in other eubacteria. Sequence motifs similar to those discovered in low G+C Gram‐positive bacteria were found associated with two of the promoters and the processing site. A probable protein coding region was observed upstream of the promoter region.
Mapping and characterization of mutants of the Escherichia coli cell division gene, ftsA
Authors: Robinson A. C. , Begg K. J. , Donachie W. D. 
Published: October 2006

SummaryEight independent temperature‐sensitive mutants of the cell division protein FtsA have been studied. They fall into two classes in terms of their behaviour at 42°C and recovery at 30°C. The first class shows salt dependent temperature‐sensitivity and reversible in activation of FtsA protein at 42°C. The second shows irreversible inactivation which is not prevented by salt. Recovery of the ability to divide at 30°C is rapid in mutants of the first group, but is delayed for approximately a generation time in the second group. This suggests that irreversible inactivation of FtsA causes extensive damage to the division machinery. The amino acid substitutions show clustering to a limited domain of the protein, and one particular substitution is found in three of the mutants.
Nucleotide sequence of Acinetobacter baumannii aphA‐6 gene: evolutionary and functional implications of sequence homologies with nucleotide‐binding proteins, kinases and other aminoglycoside‐modifying enzymes
Authors: Martin P. , Jullien E. , Courvalin P. 
Published: October 2006

SummaryA new kanamycin‐resistance gene, detected in Acinetobacter baumannii and designated aphA‐6, was sequenced. It specifiesa 30319 Dalton 3′‐aminoglycosidephospliotransferase (APH(3′)) that mediates resistance to kanamycin and structurally related aminogiycosides, including amikacin. Pairwise comparisons of the six types of APH(3′) so far detected inhuman pathogens (types I, II, IM and VI) and in aminoglycoside‐producing microorganisms (types IV and V), confirm that APH(3) enzymes have diverged from a common ancestor. Three highly retained motifs (1:V–HGD—N; 2: G–D‐GR/K‐G and 3: D–K/R‐Y/F—LDE) located in the C‐terminal part of the enzymes were defined. Screening of protein sequence databases for each of these motifs revealed that motifs 1and 2 are both found in nucleotide‐binding phosphotransferases associated with a variety of biological processes, namely adenylate kinase, viral oncogenic protein kinases, elongation factors, Na+/K+‐transporting ATPase, myosin and antibiotic‐modifying enzymes. Motif 2 probably corresponds to the MgATP binding site, while motifs 3 and 1 could be involved in the splitting of the phosphodiester bond and in the phosphate transfer, respectively. Moreover, an additional motif, almost invariably centrally located, was found in all aminoglycoside‐modifying enzymes. The occurrence of this motif, possibly a recombination site which would have allowed the association of units of separate functions, is compatible with a modular concept for the structure of aminoglycoside‐modifying enzymes.
The transmembrane topology of the sn‐glycerol‐3‐phosphate permease of Escherichia coli analysed by phoA and lacZ protein fusions
Authors: Gött P. , Boss W. 
Published: October 2006

SummaryThe Escherichia coli glpT gene encodes a transport protein that mediates uptake of sn‐glycerol‐3‐phosphate. This permease is a member of a class of bacterial organophosphate permeases which transport substrates by antiport with inorganic phosphate. The glpT gene product, probably an oligomer of a single polypeptide chain, is thought to span the cytoplasmic membrane several times, as predicted by the hydropathic profile. Protein fusions, in which varying lengths of the amino‐terminal end of the permease is attached to alkaline phosphatase (phoA) and to β‐galactosidase (lacZ) were constructed. On the assumption that phoA fusions only exhibit high enzymatic activity when fused to extra‐cytoplasmic regions of the target protein, whereas lacZ fusions will only be active when the β‐galactosidase portion is attached to cytoplasmic domains of the target protein, the activities of the fusions were used to test a two‐dimensional model for the permease. The model proposes that glpT contains 12 transmembrane segments divided by a larger cytoplasmic region. Despite some limitation caused by hot‐spot sites of transpositions, the TnphoA approach was consistent with the model. In contrast, we feel that the enzymatic activity of lacZ fusions is only a limited parameter for studying the topology of a complex membrane protein.
Identification and characterization of TnphoA mutants of Salmonella that are unable to pass through a polarized MDCK epithelial cell monolayer
Authors: Finlay B. B. , Starnbach M. N. , Francis C. L. , Stacker B. A. D. , Chatfield S. , Dougan G. , Falkow S. 
Published: October 2006

SummarySurface protein mutants of the invasive Salmonella species, S. choleraesuis, were generated using the transposon TnphoA. 626 alkaline phosphatase (PhoA+) fusion mutants were identified and screened for their ability to pass through (transcytose) polarized epithelial monolayers of Madin Darby canine kidney (MDCK) cells grown on membrane filters. Forty two mutants were unable to pass through this barrier. All of these transcytosis mutants were unable to adhere to or invade MDCK monolayers, yet these mutations were not in the genes encoding type 1 pili or mannose‐resistant haemagglutination (MRHA). These transcytosis mutants could be grouped into six classes. Class 1 mutants had altered lipopolysaccharide (LPS) O side‐chain structures while Class 2 mutants had defects in their LPS core. Mutants belonging to Classes 5 and 6 did not decrease the transepithelial electrical resistance of polarized MDCK cell mono‐layers, in contrast to the parental strain and the other mutants (Classes 1, 2, 3 and 4). Mutants belonging to Class 1 were less virulent in mice, while Class 2 (defective core) and Classes 4 and 5 (normal LPS) mutant strains were avirulent in mice. Mutants from Classes 3 and 6 were as virulent in mice as S. choleraesuis. These results suggest that the ability to pass through epithelial barriers may be an important virulence characteristic of Salmonella. These data indicate that bacterial adherence, internalization and monolayer transcytosis are closely linked events. It was also demonstrated that a mutant with decreased rates of intracellular replication still passed through the monolayer at rates similar to wild‐type S. choleraesuis.
Nucleotide sequence of the dmsABC operon encoding the anaerobic dimethylsulphoxide reductase of Escherichia coli
Authors: Bilous P. T. , Cole S. T. , Anderson W. F. , Weiner J. H. 
Published: October 2006

SummaryThe nucleotide sequence of a 6.5 kilobasepair chromosomal DNA fragment encoding the anaerobic dimethylsulphoxide (DMSO) reductase operon of Escherichia coli has been determined. The DMSO reductase structural operon was shown to consist of three open reading frames, namely dmsABC, encoding polypeptides with predicted molecular weights of 87350, 23070, and 30789 Daltons, respectively. The DMS A polypeptide displayed a high degree of amino acid sequence homology with the single‐subunit enzyme, biotin sulphoxide reductase (bisC) and with formate dehydrogenase (fdhF), suggesting that the active site and molybdopterin cofactor binding site that is common to these enzymes is located in the DMS A subunit. A comparison of the predicted N‐terminal amino acids of the dmsA gene product to those of the 82600 subunit of purified DMSO reductase indicated that post‐translational processing of a 16 amino acid peptide at the amino terminus of DMS A had occurred. The DMS B polypeptide contains 16 cysteine residues organized in four clusters, two of which are typical of 4Fe–4S binding domains. The DMS C polypeptide is composed of eight segments of hydrophobic amino acids of appropriate length to cross the cytoplasmic membrane, suggesting that this subunit functions to anchor the enzyme to the membrane.
The virulence‐associated gonococcal H.8 gene encodes 14 tandemly repeated pentapeptides
Authors: Baehr W. , Gotschlich E. C. , Hitchcock P. J. 
Published: October 2006

SummaryH.8 is a virulence‐associated, surface‐exposed, immunogenic macromolecule composed of lipid and protein, common to Neisseria gonorrhoeae and Neisseria meningitidis. The H.8 DNA sequence predicted a 6.9 kD peptide comprising 14 tandemly repeated pentameric sequences. Ten were identical: Pro, Ala, Ala, Glu, Ala. Also predicted was a lipoprotein leader consensus sequence which probably specified acylation since the Escherichia coli‐expressed protein was tightly associated with lipid. Lipid appeared to contribute significantly to H.8 antigen's electrophoretic mobility. This is the first description of a prokaryotic outer membrane protein composed solely of tandem repeats. Furthermore, DNA encoding this repeat appears to have been duplicated and translocated into another neisserial gene encoding an azurin.
Compartmentalization of the periplasm at cell division sites in Escherichia coli as shown by fluorescence photobleaching experiments
Authors: Foley M. , Brass J. M. , Birmingham J. , Cook W. R. , Garland P. B. , Higgins C. F. , Rothfield L. I. 
Published: October 2006

SummaryMorphological evidence has previously indicated that the periplasmic space of Escherichia coli is compartmentalized at sites corresponding to future sites of cell division. The borders of these morphological compartments are formed by localized zones of adhesion (periseptal annuli). In the present study, the technique of fluorescence recovery after photo‐bleaching was used to determine whether these structures act as barriers to the free movement of proteins within the periplasm. The recovery of fluorescence in the ftsA filaments was found to be uniformly low over at potential sites of cell division and at the cell poles, indicating that these regions are biochemically sequestered from the remainder of the periplasmic space. Our results provide direct evidence for local compartments within the periplasm, primarily located at the sites of past or future cell divisions. The implications of this finding for cell division and other periplasmic processes are discussed.
Transcription antitermination in an Escherichia coli haemolysin operon is directed progressively by cis‐acting DNA sequences upstream of the promoter region
Authors: Koronakis V. , Cross M. , Hughes C. 
Published: October 2006

SummaryExport of haemolysin protein (HlyA) directed by the Escherichia coli pHly152 hly determinant is dependent upon transcriptional activation, primarily strong intra‐operon transcript antitermination imposed between the haemolysin structural genes hlyC and hlyA and the contiguous downstream export genes hlyB and hlyD. Transcript elongation was dictated by a DNA sequence several kb upstream of the rho‐independent terminator but could not be assigned to a discrete locus; on the contrary, it was progressive, increasing with the addition of up to 3.5kbp of operon‐proximal sequence containing the insertion elements IS2 and IS91. Antitermination was prominent throughout logarithmic growth but absent in stationary phase, and was effective only in cis but not in trans. Primer extension indicated that transcription activation utilized the native transcriptional start sites of the unactivated hly operon.
Relationship between the pel genes of the pelADE cluster in Erwinia chrysanthemi strain B374
Authors: van Gijsegem F. 
Published: October 2006

SummaryIn this paper, we have used filter hybridization and nucleotide sequencing to analyse the relationship between the three genes of the pelADE cluster in the Erwinia chrysanthemi (Ech) strain B374. This cluster encodes for three of the five pectate lyase proteins that are involved in the maceration and soft‐rotting of plant tissue, an important trait in Ech pathogenicity. Southern hybridization revealed homology between each of the three pel genes. A 3560bp DNA fragment containing the pelE and pelD genes was sequenced. These two genes show extensive homology in the coding regions but only low homology in the 5′ and 3′ non‐coding regions. However both genes exhibit sequences homologous to the Escherichia coli CAP‐binding site consensus sequence upstream of the start codon and an inverted repeat sequence which may act as a rho‐independent transcriptional terminator after the translational stop. The pel genes of Ech B374 were also compared with the already sequenced pel genes of EC16, another Ech strain.
Structure and function of hot spots providing signals for site‐directed specific recombination and gene expression in Tn21 transposons
Authors: Schmidt F. R. J. , Nucken E. J. , Henschke R. B. 
Published: October 2006

SummaryTn21‐ and Tn3‐related transposons are widespread and carry various resistance determinants. The insertion points of different resistance genes were precisely defined in Tn2424, Tn1696, Tn2410, Tn4000 and its derivatives and compared to the corresponding sites in Tn7, pSA, R388, R46, Tn2603, Tn1331 and in Tn3‐related elements. Insertional‘hot spots’located at the 3′ end of different genes comprised 55 nucleotides and yielded more than 90% homology to the corresponding consensus sequence, termed hs1. Elements of this class were found to direct recA‐ independent generation of deletions. Flanking the 5’ends, hs2 (CTAAAACAAAGTTA) comprised the terminal nucleotides of hs1. Functional properties of hot spots as recognition sites for site‐specific recombination and regulation of gene expression indicate that they might be involved in transfer, stable inheritance and expression of prokaryotic genes.
Upstream activating sequences that are shared by two divergently transcribed operons mediate cAMP‐CRP regulation of pilus‐adhesin in Escherichia coli
Authors: Göransson M. , Forsman K. , Nilsson P. , Uhlin B. E. 
Published: October 2006

SummaryTranscription of the genes encoding pilus‐adhesin of serotype F13 in digalactoside‐binding Escherichia coli required activation by the cAMP‐CRP complex. Analysis of protein‐DNA interaction in vitro showed that CRP bound in a cAMP‐dependent manner to a sequence located 0.2 kb upstream of the point of transcription initiation of the pilus subunit operon. The cAMP‐CRP activation included, in addition to the main pilus operon, the oppositely oriented operon encoding the Papl regulatory protein. Furthermore, the auto‐regulatory product of the promoter‐proximal gene (papB) in the pilus subunit operon was found to stimulate the papl transcriptional unit. Thus the cAMP‐CRP complex and PapB might act in concert and indirectly promote pili synthesis by stimulating expression of the Papl positive regulator. The results of trans complementation experiments and analyses using lacZ operon fusion derivatives showed that the cAMP‐CRP activation also operated directly in cis on the pilus subunit operon. The region containing the CRP binding site appeared to function as an upstream activating sequence since deletion abolished expression even when the pap regulatory proteins Papl and PapB were supplied in trans. The implications for possible mechanisms of transcriptional activation by the cAMP‐CRP complex at this novel location between the two oppositely oriented operons are discussed.
Single and multiple mutations affecting properties of the regulatory gene nodD of Rhizobium
Authors: Burn J. E. , Hamilton W. D. , Wootton J. C. , Johnston A. W. B. 
Published: October 2006

Summary nodD of Rhizobium leguminosarum has two regulatory properties: it autoregulates and, in cells grown with specific flavonoids, activates other nod genes. We isolated mutations in nodD affecting one or both properties. Those abolishing autoregulation and nod gene induction were at the 5′ end of nodD, as were those which only affected autoregulation. Mutations affecting nod gene activation are at the 3’end of nodD. Eleven mutations in this region of nodD were isolated: some had little effect on the regulatory properties; others reduced activation of other nod genes. 265 bps were removed from the 3’end of nodD: this abolished nodD function. Doubly mutant derivatives of nodD were constructed by making nodD genes with a mutation that conferred the ability to activate transcription of nod genes in the absence of inducers (class IV) plus another that abolished autoregulation and/or flavonoid‐dependent nod gene activation. The behaviour of such double mutants was complex; e.g. in one case, a doubly mutant nodD gene containing the class IV mutation, coupled to one of those that (alone) abolished autoregulation and nod gene induction, was similar in behaviour to the wild type. In other cases, double mutants were similar to one of the parentals.
Genetic transfer systems in Bacteroides: cloning and mapping of the transferable tetracycline‐resistance locus
Authors: Guiney D. G. , Hasegawa P. , Bouic K. , Matthews B. 
Published: October 2006

SummaryConjugation systems that transfer antibiotic resistance in the absence of detectable plasmids are common in Bacteroides, but the mechanism of transfer is poorly understood. We found that linked transfer of tetracycline (ToR) and clindamycin (CIR) resistance by Bacteroides fragilis strain 1126 is induced by growth in either Tc or Cl. We cloned the transferable TcR locus as a 13 kb fragment on the shuttle vector pPH6 in Escherichia coli and showed that this region expresses TcR in Bacteroides but not E. coli. The TcR gene was mapped to a 3 kb region and the CIR gene was shown not to be present in the 13 kb insert. Homologous TcR genes are found in B. fragilis V479 and 1792. Using pulsed‐field electrophoresis, the transferable TcR gene was shown to be physically associated with high molecular‐weight DNA, suggesting that it is located on the chromosome. A new TcR shuttle vector. pPH7δ1.1, was constructed to facilitate use of this selective marker in Bacteroides genetics.
Extracellular and periplasmic isoenzymes of pectate lyase from Erwinia carotovora subspecies carotovora belong to different gene families
Authors: Hinton J. C. D. , Sidebotham J. M. , Gill D. R. , Salmond G. P. C. 
Published: October 2006

SummaryPectate lyase (Pel) plays a crucial role in the maceration of vegetables by soft rot Erwinia spp. We have characterized the four Pel isoenzymes of Erwinia carotovora subspecies carotovora strain SCRI193. In this paper we concentrate on two isoenzymes which have different locations in SCRI193: PLb is periplasmic and PLc is extracellular. Comparison of the gene products and nucleotide sequences of pelB and pelC allowed us to assign them to different gene families. In addition, we have identified a number of conserved amino acid residues that are common to all extracellular Pel isoenzymes.
Pseudo‐allelic relationship between non‐homologous genes concerned with biosynthesis of polyglycerol phosphate and polyglycerol phosphate teichoic acids in Bacillus subtilis strains 168 and W23
Authors: Young M. , Mauël C. , Margot P. , Karamata D. 
Published: October 2006

SummaryA 60 kbp region of the Bacillus subtilis chromosome encompassing the genes concerned with teichoic acid biosynthesis has been subjected to physical analysis. No homo logy was detected by Southern hybridization between DNA segments encoding the tag genes of strain 168, concerned with polyglycerol phosphate (poly(groP)) biosynthesis, and the tar genes of strain W23, concerned with polyribitol phosphate (poly‐(rboP)) biosynthesis. Analysis of 168/W23 interstrain hybrids that incorporate poly(rboP) instead of poly‐(groP) into their cell walls revealed that, in every case, Integral substitution of the W23 tar genes for the 168 tag genes had occurred. Interstrain hybrids of the‘W23‐like’type have inherited larger segments of W23 DNA than interstrain hybrids of the‘mixed’type. The tag and tar genes are located at equivalent positions on the chromosomes of strains 168 and W23, behaving, in genetic crosses, like an allelic pair. They provide the first example of a pseudo‐allelic relationship between non‐homologous genes in B. subtilis.
Secretion, processing and activation of bacterial extracellular proteases
Authors: Wandersman C. 
Published: October 2006

SummaryMany different bacteria secrete proteases into the culture medium. Extracellular proteases produced by Gram‐positive bacteria are secreted by a signal‐peptide‐dependent pathway and have a propeptide located between the signal peptide and the mature protein. Many extracellular proteases synthesized by Gram‐negative bacteria are also produced as precursors with a signal peptide. However, at least two species of Gram‐negative bacteria secrete one or more proteases via a novel signal‐peptide‐independent route. Most proteases secreted by Gram‐negative bacteria also have a propeptide whose length and location vary according to the protease. Specific features of protease secretion pathways and the mechanisms of protease activation are discussed with particular reference to some of the best‐characterized extracellular proteases produced by Gram‐positive and Gram‐negative bacteria.
Characterization and molecular cloning of the PCF8775 CS5 antigen from an enterotoxigenic Escherichia coli 0115:H40 isolated in Central Australia
Authors: Heuzenroeder M. W. , Neal B. L. , Thomas C. J. , Halter R. , Manning P. A. 
Published: October 2006

SummaryThe genes determining the biosynthesis of the colonization factor CS5 have been cloned from Escherichia coli 0115:H40:PCF8775 isolated during an outbreak of diarrhoea among aboriginal children in Central Australia. Electron microscopy has shown purified CS5 to be of semi‐rigid fimbrial type. NH2‐terminal analysis has shown the CS5 determinant to be distinct from other fimbriae, although there is some conservation of certain residues. Expression in minicells of the cloned fimbrial genes encoded on pPM1312 has shown that proteins of 70 and 46.5 kD which co‐purify with the 23kD major fimbrial subunit protein are also co‐expressed along with proteins of 45, 31, 17 and 14kD. The major CS5 subunit is synthesized in precursor form (approximately 26 kD). A synthetic oligonucleotide to the NH2‐terminal amino acid coding sequence of the purified protein has been used in Southern hybridization analyses to define the region on pPM1312 encoding the structural gene for the major pilin subunit