bluuebunny/crossref_metadata_2025_split huggingface dataset:
Constraints in chromosomal inversions in Escherichia coli are not explained by replication pausing at inverted terminator‐like sequences
Authors: François V. , Louarn J. , Patte J. , Rebollo J.E. , Louarn J.‐M. 
Published: October 2006

SummaryRegions close to the replication terminus of the Escherichia coli chromosome are strongly refractory to genomic inversions (Rebollo et al., 1988). Since these regions also harbour polar replication terminator‐like sequences or pause sites (François et al., 1989), we have investigated the possibility that slowing of replication as a result of pausing at inverted pause sites is responsible for inability to isolate stable inversions affecting these regions. A mutation in the tus gene is known to abolish replication pausing at terminators (Hill et al., 1988). We show here that the distribution of invertible and noninvertible segments along the chromosome is not affected by tus mutations. This observation eliminates replication pausing as a cause for the reduced fitness of bacteria harbouring certain chromosomal inversions.
Molybdenum cofactor requirement for in vitro activation of apo‐molybdoenzymes of Escherichia coli
Authors: Giordano G. , Boxer D. H. , Pommier J. 
Published: October 2006

SummaryThe apo‐nitrate reductase precursor in an Escherichia coli chiB mutant preparation obtained following growth in the presence of tungstate is activated by incubation with protein FA and a heat‐treated preparation from an E. coli crude extract. We show that the requirement for heat‐treated E. coli crude extract can be fulfilled by material obtained from either of two heat–denatured purified E. coli molybdoenzymes, namely nitrate reductase or trimethylamine N‐oxide reductase. Apo‐trimethylamine N‐oxide reductase precursor in the tungstate‐grown chIB preparation can be activated in a similar manner with material from either heat‐denatured molybdoenzyme. The active component in the denatured molybdoenzyme preparations is shown to be the molybdenum cofactor by Neurospora crassa nit1 molybdenum cofactor assay, size estimation and fluorimetric analysis. The direct demonstration of the requirement for molybdenum cofactor in the E. coli tungstate‐grown chIB complementation system is an important step towards the molecular definition of the activation process and an understanding of the mechanism of cofactor acquisition during molybdoenzyme biosynthesis.
The N‐terminal region of an endoglucanase from Pseudomonas fluorescens subspecies cellulosa constitutes a cellulose‐binding domain that is distinct from the catalytic centre
Authors: Gilbert H. J. , Hall J. , Hazlewood G. P. , Ferreira L. M. A. 
Published: October 2006

SummaryThe substrate specificity of an endoglucanase (EGB) from Pseudomonas fluorescens subspecies cellulosa was determined. The enzyme was most active against barley β‐glucan, but showed significant activity against amorphous and crystalline cellulose. EGB was purified to homogeneity by affinity chromatography with crystalline cellulose (Avicel). The Mr of the purified enzyme was 50000, which is in good agreement with the size of EGB deduced from the nucleotide sequence of the celB gene, coding for EGB. The N‐terminal region of the mature form of EGB showed strong homology to another endoglucanase and to a xylanase expressed by the same organism; homologous sequences included highly conserved serine‐rich regions. Truncated forms of celB, in which the gene sequence encoding the conserved domain had been deleted, directed the synthesis of a functional endoglucanase that did not bind to crystalline cellulose. This indicates that the conserved region of endoglucanases and xylanases expressed by P. fluorescens subsp. cellulosa constitutes a cellulose‐binding domain, which is distinct from the active centre. The possible role of this substrate‐binding region is discussed.
Chromosome‐specific identification and quantification of S1 nuclease‐sensitive sites in yeast chromatin by pulsed‐field gel electrophoresis
Authors: Geigl E.M. , Eckardt‐Schupp F. 
Published: October 2006

SummarySites that are sensitive to the single‐strand‐specific endonuclease S1 ('S1‐sensitive sites', SSS) occur in native chromatin and, like DNA double‐stranded breaks (DSB), they are induced by DNA‐damaging agents, such as ionizing radiation. We have developed a method to quantify SSS and DSB in yeast chromatin by using pulsed‐field gel electrophoresis (PFGE) to separate the intact chromosomal‐length DNA molecules from the lower molecular‐weight broken ones. Direct evaluation of the photonegatives of the ethidium bromide‐stained gels by laser densitometry enabled us to calculate the numbers of DSB and SSS per DNA molecule. These numbers were determined from the bulk of the non‐separated genomic DNA of yeast, corresponding to a single band in the PFGE (pulse time 10 seconds), and in each of the eight largest yeast chromosomes, corresponding to distinct bands in the PFGE gels (pulse time 50 seconds), which were not superimposed by the smear of the broken, low molecular‐weight DNA. Furthermore, the induction of DSB and SSS in a specific chromosome (circular chromosome III) was determined by Southern hybridization of the PFGE gels with a suitable centromere probe, followed by densitometry of the autoradiographs. Our method allows the chromosome‐specific monitoring of DSB and all those DNA structures that are processed either in vivo or in vitro into DSB and which may not be distributed randomly within the genome.
Sequence and relatedness in other bacteria of the Pseudomonas aeruginosa oprP gene coding for the phosphate‐specific porin P
Authors: Siehnel R. , Martin N. L , Hancock R. E. W. 
Published: October 2006

SummaryThe oprP gene encoding the Pseudomonas aeruginosa phosphate‐specific outer membrane porin protein OprP was sequenced. Comparison of the derived amino acid sequence with the known sequences of other bacterial porins demonstrated that OprP could be no better aligned to these porin sequences than it could to the periplasmic phosphate‐binding protein PhoS of Escherichia coli. Southern hybridization and restriction mapping of the oprP gene in 37 clinical isolates and the 17 serotype strains of P. aeruginosa revealed that restriction sites in the vicinity of the oprP gene were highly conserved. Several species from the Pseudomonas fluorescens rRNA homology group contained DNA that hybridized to an oprP gene probe.
Novobiocin‐resistance sequences from the novobiocin‐producing strain Streptomyces niveus
Authors: Mitchell J. I. , Logan P. G. , Cushing K. E. , Ritchie D. A. 
Published: October 2006

SummaryTwo distinct DNA sequences expressing novobiocin resistance In Streptomyces lividans were cloned from the novobiocin‐producing species Streptomyces niveus. Clone pGL 101 (5kb) conferred resistance to 510μg ml−1 novobiocin, whereas clones pGL 102 and pGL 103, which carry the same 6.5kb insert but in opposite orientations, expressed resistance to 150μg ml−1. The cloned inserts from pGL 101 and pGL 103 failed to hybridize with each other or with the cloned novobiocin‐resistant gyrB sequence from Streptomyces sphaeroides. Both probes hybridized strongly with DNA from the novobiocin‐producing species S. niveus and S. sphaeroides but no hybridization (pGL 103) or very weak hybridization (pGL 101) was detected with DNA from the non‐producing species S. lividans, Streptomyces griseus and Streptomyces antibioticus. S. niveus contains at least three novobiocin‐resistance determinants with the pGL 101 and pGL 103 cloned sequences specific for novobiocin‐producing strains of Streptomyces.
Construction of isogenic gonococci with variable porin structure: effects on susceptibility to human serum and antibiotics
Authors: Carbonetti N. , Simnad V. , Elkins C. , Sparling P. F. 
Published: October 2006

SummaryProtein I (PI) is the most abundant protein on the gonococcal cell surface and besides its porin function it may have important properties contributing to pathogenicity. By allelic exchange using cloned PI genes from FA19 (PIA) and MS11 (PIB) and a selectable marker introduced closely downstream of these genes, we constructed sets of isogenic gonococcal strains that differ only in their PI gene. Analysis revealed that PI has a major effect on stable resistance to normal human serum, and a slight effect on low‐level resistance to antibiotics. All PIA/B hybrids were hypersusceptible to serum, suggesting a possible explanation for why such hybrids do not occur in nature.
Translation initiation in Escherichia coli: old and new questions
Authors: Jacques N. , Dreyfus M. 
Published: October 2006

SummaryWe discuss the features of Escherichia coli mRNAs which determine where and how efficiently translation is initiated. We have shown that DNA fragments comprising 60–80 nucleotides that bracket the initiation codon of real genes generally promote translation when inserted within a foreign mRNA, while those not corresponding to an authentic gene start do not do so even if they include a Shine‐Dalgarno‐like element followed by AUG or GUG. Therefore, the information that pinpoints the correct start sites, while extending beyond the mere presence of these elements, remains essentially local. The possible nature of this information is discussed. Next, we point out that, in order to remain accessible, translational starts must escape long‐range base‐pairing within large mRNAs, and we argue that the tight coupling between translation and transcription plays an important role in achieving this. Finally, we discuss two intriguing situations in which the initiation frequency should be dependent upon the rate of translation elongation.
Identification and characterization of a porcine‐specific transferrin receptor in Actinobacillus pleuropneumoniae
Authors: Gonzalez G. C. , Caamano D. L. , Schryvers A. S. 
Published: October 2006

SummaryAll six strains of Actinobacillus pleuropneumoniae screened for the ability to use different transferrins as a source of iron for growth were capable of using porcine but not human, bovine, or avian transferrins. A specific binding activity for porcine transferrin (pTf) was expressed in cells grown in the presence of specific iron‐chelators and was repressed by addition of excess iron. Two iron‐repressible outer‐membrane proteins of 105 and 56kD were specifically isolated from serotype 1, 2 and 7 strains of A. pleuropneumoniae by an affinity‐isolation method using biotinylated porcine transferrin and streptavidin‐agarose.
Inducible erythromycin resistance in staphlyococci is encoded by a member of the ATP‐binding transport super‐gene family
Authors: Ross J. I. , Eady E. A. , Cove J. H. , Cunliffe W. J. , Baumberg S. , Wootton J. C. 
Published: October 2006

SummaryA Staphylococcus epidermidis plasmid conferring inducible resistance to 14‐membered ring macrolides and type B streptogramins has been analysed and the DNA sequence of the gene responsible for resistance determined. A single open reading frame of 1.464kbp, preceded by a complex control region containing a promoter and two ribosomal binding sites, was identified. The deduced sequence of the 488‐amino‐acid protein (MsrA) revealed the presence of two ATP‐binding motifs homologous to those of a family of transport‐related proteins from Gram‐negative bacteria and eukaryotic cells, including the P‐glycoprotein responsible for multidrug resistance. In MsrA, but not these other proteins, the two potential ATP‐binding domains are separated by a Q‐linker of exceptional length. Q‐linkers comprise a class of flexible inter‐domain fusion junctions that are typically rich in glutamine and other hydrophilic amino acids and have a characteristic spacing of hydrophobic amino acids, as found in the MsrA sequence. Unlike the other transport‐related proteins, which act in concert with one or more hydrophobic membrane proteins, MsrA appears to function independently when cloned in a heterologous host (Staphylococcus aureus RN4220). MsrA might, therefore, interact with and confer antibiotic specificity upon other transmembrane efflux complexes of staphylococcal cells. The active efflux of [14C]‐erythromycin from cells of S. aureus RN4220 containing msrA has been demonstrated.
Generation of capsule‐deficient Neisseria meningitidis strains by homologous recombination
Authors: Frosch M. , Schultz E. , Glenn‐Calvol E. , Meyer T. F. 
Published: October 2006

SummaryCapsule‐deficient mutants of Neisseria meningitidis serogroup B strain B 1940 were constructed by allelic replacement using the plasmids pMF120 and pMF121, which contain the flanking regions of the gene locus for the biosynthesis pathway of the group B meningococcal capsular polysaccharide. Southern blot analysis of chromosomal DNA of the capsule‐deficient meningococcal strains confirmed the generation of large deletions in the chromosomal cps gene complex. The same strategy proved useful in constructing meningococcal strains with capsular types A, C., W 135, Y and Z.
The TraT lipoprotein as a vehicle for the transport of foreign antigenic determinants to the cell surface of Escherichia coli K12: structure–function relationships in the TraT protein
Authors: Taylor I. M. , Harrison J. L. , Timmis K. N. , O'Connor C. D. 
Published: October 2006

SummaryThe TraT protein is a surface‐exposed lipoprotein, specified by plasmids of the IncF group, that mediates serum resistance and surface exclusion. The structure and function of the TraT protein determined by plasmid R6‐5 was probed by genetic insertion of a foreign antigenic determinant, the C3 epitope of polio virus, at residues 61, 125, 180, 200 or 216 of the protein. The chimaeric proteins were transported to the outer membrane and, in three cases, immunoassays with an anti‐C3 monoclonal antibody indicated that the C3 epitope was exposed on the cell surface. Three of the hybrids, with insertions at residues 125, 180 and 200, assembled into the trypsin‐resistant oligomeric form characteristic of the wild‐type protein, which suggested that these regions are not involved in TraT subunit:subunit interactions. Additionally, the hybrid protein carrying the C3 epitope at position 180 functioned in a genetic suppression assay and retained partial surface‐exclusion activity. Thus, its localization, folding and organization does not appear to be grossly altered from that of the wild‐type protein. Applications of the protein for the transport of foreign antigenic determinants to the cell surface are discussed.
Nucleotide sequence and codon usage of the elongation factor Tu(EF‐Tu) gene from Mycoplasma pneumoniae
Authors: Yogev D. , Sela S. , Bercovier H. , Razin S. 
Published: October 2006

SummaryThe Mycoplasma pneumoniae tuf gene, encoding the elongation factor protein Tu, was cloned and sequenced. The nucleotide sequence of the mycoplasmal gene showed about 60% homology to the sequences of tuf genes of other prokaryotes, yeast mitochondria and Euglena gracilis chloroplasts, and about 75% similarity was found when comparing the deduced amino acid sequences of the various Tu proteins. The relatively low G+C content (40%) of the M. pneumoniae DNA was reflected in a low G+C content (44.6%) of the tuf gene, and in a preferential use of adenine and uracil at the third position of codons, yet codon usage analysis revealed the presence of almost all of the codons of the genetic code in the mycoplasmal gene. Southern blot hybridization of digested DNAs of 11 Mollicutes species with the entire M. pneumoniae tuf gene and with its 5′ part suggested the presence of one copy only of this gene in the representative species of the Mollicutes. In this respect, the Mollicutes resemble Gram‐positive bacteria and differ from the Gram‐negative bacteria, which carry two copies of the tuf gene.
The mobilization and origin of transfer regions of a Thiobacillus ferrooxidans plasmid: relatedness to plasmids RSF1010 and pSC101
Authors: Drolet M. , Zanga P. , Lau P. C. K. 
Published: October 2006

SummaryThe components for the mobilization function of a plasmid DNA during conjugation include a cis‐acting sequence (the origin of transfer, oriT) and a transacting sequence coding for mobilization (Mob) proteins. By genetic and deletion analysis, we have located the mobilization region of pTF1, a cryptic plasmid previously isolated from a Thiobacillus ferrooxidans strain. Within a 2797 base‐pair sequenced region, several open reading frames (ORFs) were predicted; two of the ORFs are divergently transcribed and they encode proteins of calculated molecular masses, 42.6kD (ORF2) and 11.4kD (ORF6). Surprisingly, these protein sequences are substantially similar to two of the previously characterized mobilization proteins of the Escherichia coli IncQ plasmid, RSF1010. Moreover, the pTF1 ORF2 (now designated MobL) sequence is also found to be similar to a presumed mobilization protein of plasmid pSC101. Regions of sequence identity of plasmids pTF1, RSF1010 and pSC101 include their oriT sites. By alkaline agarose gel electrophoresis and DNA sequencing, we have established the location of the relaxation complex nick site within the oriT of pTF1. An identical nick site, which is adjacent to a characteristic 10 base‐pair inverted repeat sequence, is also found for plasmid RSF1010. A recombinant plasmid containing a 42 base‐pair synthetic piece of DNA encompassing the pTF1 inverted repeat and nick sequence was shown to be oriT‐active.
The benzoylarginine peptidase from Treponema denticola (strain ASLM), a human oral spirochaete: evidence for active‐site carboxyl groups
Authors: Mäkinen K. K. , Chen C.‐Y. , Mäkinen P.‐L. , Ohta K. , Loesche W. J. 
Published: October 2006

SummaryThe benzoylarginine peptidase of Treponema denticola (strain ASLM; a human oral spirochaete) was progressively and irreversibly inactivated by 1‐(ethoxycarbonyl)‐2‐ethoxy‐1, 2‐dihydroquinoline, a carboxyl‐group reagent. At acidic pH values, reaction of one mole of the modifier per active site of the enzyme resulted in total inactivation of the enzyme. Assuming that this modifier is a specific carboxyl reagent, the data suggest that the inactivation of the T. denticola benzoylarginine peptidase was caused by the modification of one carboxyl group located close to the active site of the enzyme. Results obtained with Woodward's reagent K (N‐ethyl‐5‐phenylisoxazolium 3’‐sulphonate) supported these findings. Carbethoxylation with diethylpyrocarbonate effectively inactivated the enzyme, and addition of hydroxylamine at pH 7.0 restored the activity almost totally, suggesting that the pyrocarbonate had reacted with tyrosyl or histidyl residues.
Isolation of intact FNR protein (Mr 30 000) of Escherichia coli
Authors: Trageser M. , Spiro S. , Duchêne A. , Kojro E. , Fahrenholz F. , Guest J. R. , Unden G. 
Published: October 2006

SummaryFNR, the activator of anaerobic respiratory genes of Escherichia coli, has previously only been isolated as a protein of Mr, 29 000, which lacks nine N‐terminal amino acid residues. The underlying proteolytic events have been studied with the aim of isolating intact FNR and determining whether cleavage is the result of a physiologically significant intracellular processing mechanism or proteolytic degradation during isolation.The FNR protein was present in aerobically and anaerobically grown bacteria as the intact protein (Mr, 30 000). Proteolysis only occurred during and shortly after disruption of the bacteria. The production of FNR (Mr, 29 000) must therefore be regarded as an isolation artefact. The proteolysis was caused by a protease which is located outside the cytoplasmic membrane or activated upon disruption of the membrane. Protease inhibitors directed against serine, cysteine or metalloproteases failed to prevent cleavage of FNR. In E. coli strain CAG627, proteolysis was greatly reduced making it possible to isolate FNR of Mr, 30 000. The N‐terminal sequence of FNR (Mr, 30 000) was identical to that predicted from the fnr gene starting with the initiating methionine residue and including a four‐cysteine cluster (16)Cys–X3–Cys–X2–Cys–X5–Cys(29).
Engineering upstream transcriptional and translational signals of Bordetella pertussis serotype 2 fimbrial subunit protein for efficient expression in Escherichia coli: in vitro autoassembly of the expressed product into filamentous structures
Authors: Walker M. J. , Rohde M. , Brownlie R. M. , Timmis K. N. 
Published: October 2006

Summary Escherichia coli containing a cloned gene encoding the Bordetella pertussis serotype 2 fimbrial subunit failed to produce detectable levels of the gene product in whole‐cell extracts. To engineer plasmids capable of directing the expression in E. coli of high levels of this product, both as a pre‐protein and as a methionylated mature form the upstream signals of the fimbrial subunit gene were replaced by the lambda PL and PR promoters and the E. coli atpE translational initiation region. These constructs did not result in the expression of fimbrial subunit at detectable levels in several E. coli strains including DH5. However, they did in E. coli CAG629, which is Ion protease and heat shock protein deficient. Both pre‐protein and methionylated mature protein had molecular weights of 25.0 kD, which indicated that correct processing of the leader sequence had occurred and thus that it was transposed across the inner membrane. Electron microscopic investigation of the cell surface of E. coli cells expressing either form of the fimbrial gene failed to detect the presence of filamentous structures. The methionytated mature form of the recombinant fimbrial subunit was purified to apparent homogeneity. After dialysis in appropriate conditions it was seen to autoassemble into protein polymers. Antibodies raised against polymerized recombinant subunit reacted weakly with whole B. pertussis serotype 2 fimbriae in immunodot blot assays. However, such antibodies reacted in Western blots equally well with the recombinant and wild‐type form of the fimbrial subunit. These results suggest that the assembly in E.
Tn 7 inserts in both orientations at a single chromosomal location and apparently forms cointegrates in Pasteurella multocida
Authors: Nnalue N. A. 
Published: October 2006

Summary Pasteurella multocida transconjugants isolated after mating with Escherichia coli strains that carry one or the other of two Tn7‐containing suicide plasmids, pRKTV5 and pUW964 (pRKTV5::Tn5), were analysed. These plasmids have the C0IEI replication origin and were thus expected to deliver transposons but not be maintained as free replicons in Pasteurella. Five out of six transconjugants selected for acquisition of Tn7 from E. coli (pRKTV5) had simple insertions of the transposon, in either orientation, at a single chromosomal location, while the sixth had pFtKTV5 integrated at the same location. By contrast, all of 27 transconjugants selected for acquisition of either Tn7 or Tn5 from E. coli (pUW964) maintained pUW964. Of seven subsequently examined at the molecular level, all had pUW964 (in one case, a deletion derivative) integrated at the same location as the Tn 7 insertions obtained with pRKTV5. A copy of Tn7 was present at each boundary between the integrated plasmids (pRKTV5 or pUW964) and the chromosome in each strain. The two copies of Tn7 at either end of an integrated plasmid were either in the same (six cases) or in opposite (two cases) orientations with respect to each other. These seem to be products of replicative transposition by Tn7but can also derive from conservative mechanisms.