bluuebunny/crossref_metadata_2025 huggingface dataset:
Pro‐subtilisin E: purification and characterization of its autoprocessing to active subtilisin E in vitro
Authors: Ohta Y. , Inouye M.
Published: October 2006

The formation of active subtilisin E from pro‐subtilisin E requires the removal of the N‐terminal pro‐sequence of 77 residues. Pro‐subtilisin E produced in Escherichia coli using a pINIII‐ompA vector was first extracted with 6M guanidine‐HCl and 5M urea and purified to homogeneity in the presence of 5M urea. Upon drop dialysis against 0.2M sodium phosphate buffer (pH 6.2), the purified pro‐subtilisin in 5M urea was processed to active subtilisin of which the N‐terminal sequence and migration in SDS‐polyacrylamide gel electrophoresis were identical to those of authentic active subtilisin E. This process was found to be very sensitive to the ionic strengths and anions used. Under the optimum conditions (dialysis against 0.5 M (NH4)2SO4 and 1 mM CaCl2 in 10mM Tris‐HCl buffer (pH 7.0) at 4°C for 1 h), approximately 20% of pro‐subtitisin E was converted to active subtilisin E. The activation process was not inhibited by Streptomyces subtilisin inhibitor, and pro‐subtiiisin E in which the active site was mutated (Asp32 to Asn) was unable to be processed under the optimum conditions. These results confirmed the previous hypothesis that the processing of pro‐subtilisin occurs by an intramolecular, autoprocessing mechanism.
Transcriptional analysis of the gene encoding pyruvate formate‐lyase‐activating enzyme of Escherichia coli
Authors: Sauter M. , Sawers R. G.
Published: October 2006

The act gene of Escherichia coli encodes the pyruvate formate‐lyase‐activating enzyme which is necessary for the post‐translational modification of pyruvate formate‐lyase. The gene is located 191 bp downstream from the pfl structural gene. Northern blot analysis revealed that the act transcript is monocistronic and that transcription is independent of pfl gene expression. Through mapping of the 5′ and 3′ ends of the act transcript, sequences could be identified showing similarity to both an Escherichia coliσ70 promoter and to a rho‐independent transcription terminator. Expression of the act gene was analysed with the aid of chromosomally integrated transcriptional and translational lacZ fusions. The results verified that the act gene is transcribed from its own promoter and that expression of the gene is essentially constitutive. Anaerobiosis led only to a two‐fold increase in expression over that observed in aerobically grown cells and this elevated expression was independent of the transcriptional regulator, Fnr. Moreover, effectors such as pyruvate and nitrate, which substantially influence anaerobic transcription of the pfl gene, did not affect act gene expression.
Purine biosynthesis in Escherichia coli K12: structure and DNA sequence studies of the purHD locus
Authors: Flannigan K. A. , Hennigan S. H. , Vogelbacker H. H. , Gots J. S. , Smith J. M.
Published: October 2006

The de novo purine biosynthetic enzymes 5‐amino‐4‐imidazolecarboxamide‐ribonucleotide (AICAR) transformylase (EC 2.1.2.3), IMP cyclohydrolase (EC 3.5.4.10) and glycineamide‐ribonucleotide (GAR) synthetase (EC 2.1.2.2) are encoded by the purHD locus of Escherichia coli. The DNA sequence of this locus revealed two open reading frames encoding polypeptides of Mr57335 and 45945 (GAR synthetase), respectively, that formed an operon. The DNA sequence, maxicell and complementation analyses all supported the concept that the Mr 57335 polypeptide is the product of the purH gene and encodes a bifunctional protein containing both AICAR trans‐formylase and IMP cyclohydrolase activities. The 5′ end of the purHD mRNA was determined by primer extension mapping and contains two regions of dyad symmetry capable of forming ‘hairpin’ loops where the formation of the one would prevent the formation of the other but not vice versa. Regulation by the purR gene product was explained by the discovery of a purR binding site in the purHD control region.
The coagulase of Staphylococcus aureus 8325‐4. Sequence analysis and virulence of site‐specific coagulase‐deficient mutants
Authors: Phonimdaeng P. , O'Reilly M. , Nowlan P. , Bramley A. J. , Foster T. J.
Published: October 2006

The sequence of the coagulase gene (coa) from Staphylococcus aureus strain 8325‐4 is reported. The deduced amino acid sequence of the coagulase protein is compared with previously reported sequences of coagulases from strains 213 and BB. The secreted mature forms of coagulase proteins are composed of three distinct segments: (i) the N‐terminal 150–270 residues, which are c. 50% identical, (ii) a central region with high (>90%) residue identities, and (iii) a C‐terminal region composed of repeated 27‐amino‐acid residue sequences. The variable N‐terminal sequences are probably responsible for antigenic differences among coagulases of different serotype. The region of coagulase which binds to prothrombin and activates it to form staphylothrombin is also located in the N‐terminal half of the protein.

A site‐specific substitution mutation in the coa gene, which abolished plasma clotting activity, was isolated by recombinational allele‐replacement in strains 8325‐4 and M60. The Coa− mutants did not show diminished virulence in subcutaneous and intramammary infections of mice. No evidence for a role for coagulase in virulence of toxigenic or non‐toxigenic strains was obtained. This contradicts findings of several groups using Coa− mutants generated by chemical mutagenesis and suggests that the earlier results were obtained with strains that had suffered additional mutations in virulence‐related genes.
Activation potassium efflux from Escherichia coli by glutathione metabolites
Authors: Elmore M. J. , Lamb A. J. , Ritchie G. Y. , Douglas R. M. , Munro A. , Gajewska A. , Booth I. R.
Published: October 2006

The mechanism by which N‐ethylmaleimide (NEM) elicits potassium efflux from Escherichia coli has been investigated. The critical factor is the formation of specific glutathione metabolites that activate transport systems encoded by the kefB and kefC gene products. Formation of N‐ethyl‐succinimido‐S‐glutathione (ESG) leads to the activation of potassium efflux via these transport systems. The addition of dithiothreitol and other reducing agents to cells reverses this process by causing the breakdown of ESG and thus removing the activator of the systems. Chlorodinitrobenzene, p‐chloromercuribenzoate and phenylmaleimide provoke similar effects to NEM. Iodoacetate, which leads to the formation of S‐carboxymethyl‐glutathione, does not activate the systems but does prevent the action of NEM. It is concluded that the KefB and KefC systems are gated by glutathione metabolites and that the degree to which they are activated is dependent upon the nature of the substituent on the sulphydryl group.
Comparison and cross‐species expression of the acetyl‐CoA synthetase genes of the ascomycete fungi, Aspergillus nidulans and Neurospora crassa
Authors: Connerton I. F. , Fincham J. R. S. , Sandeman R. A. , Hynes M. J.
Published: October 2006

The genes encoding the acetate‐inducible enzyme acetyl‐coenzyme A synthetase from Neurospora crassa and Aspergillus nidulans (acu‐5 and facA, respectively) have been cloned and their sequences compared. The predicted amino acid sequence of the Aspergillus enzyme has 670 amino acid residues and that of the Neurospora enzyme either 626 or 606 residues, depending upon which of the two possible initiation codons is used. The amino acid sequences following the second alternative AUG show 86% homology between the two species; the extended N‐terminal sequences show no homology. The Neurospora protein is characterized by the appearance of the S(T)PXX sequence motif where the amino acid homologies break down. The codon usage is biased in both genes, with a marked deficiency, especially in Neurospora, of codons with A in the third position.

The facA transcribed sequence contains six introns: one in the long leader sequence, one in the 5′ coding sequence not homologous with acu‐5, and four within the sequence that is largely similar to that of acu‐5. Only one intron, corresponding in size and position to the furthest downstream of the facA introns, is found in acu‐5. The evolution of introns during the divergence of these two Ascomycete fungi is discussed. Each of the two genes has been transferred by transformation into the other species. Each species is evidently able to splice out the other's introns. Most transformants have normal acetate‐induction of acetyl‐CoA synthetase, emptying that the two genes respond to transcriptional control signals common to both species, in spite of the striking divergence of their 5′ ends.
The life‐cycle proteins RodA of Escherichia coli and SpoVE of Bacillus subtilis have very similar primary structures
Authors: Joris B. , Dive G. , Henriques A. , Piggot P. J. , Ghuysen J. M.
Published: October 2006

Comparison of the predicted amino acid sequence of the cell‐cycle RodA protein with the National Research Foundation protein sequence database shows that the 370‐amino‐acid RodA, a protein that is essential for wall elongation in Escherichia coli and maintenance of the rod shape of the cell, is highly analogous, in terms of primary structure, with the Bacillus subtilis SpoVE protein involved in stage V of sporulation.
Structure and dynamics of the colicin E1 channel
Authors: Cramer W. A. , Cohen F. S. , Merrill A. R. , Song H. Y.
Published: October 2006

The toxin‐like and bactericidal colicin E1 molecule is of interest for problems of toxin action, polypeptide translocation across membranes, voltage‐gated channels, and receptor function. Colicin E1 binds to a receptor in the outer membrane and is translocated across the cell envelope to the inner membrane. Import of the colicin channel‐forming domain into the inner membrane involves a translocation‐competent intermediate state and a membrane potential‐dependent movement of one third to one half of the channel peptide into the membrane bilayer. The voltage‐gated channel has a conductance sufficiently large to depolarize the Escherichia coli cytoplasmic membrane. Amino acid residues that affect the channel ion selectivity have been identified by site‐directed mutagenesis. The colicin E1 channel is one of a few membrane proteins whose secondary structures in the membrane, predominantly α‐helix, have been determined by physico‐chemical techniques. Hypotheses for the identity of the trans‐membrane helices, and the mechanism of binding to the membrane, are influenced by the solved crystal structure of the soluble colicin A channel peptide. The protective action of immunity protein is a unique aspect of the colicin problem, and information has been obtained, by genetic techniques, about the probable membrane topography of the imm gene product.
Analysis of the structure‐function relationship of Pseudomonas aeruginosa exotoxin A
Authors: Wick M. J. , Hamood A. N. , Iglewski B. H.
Published: October 2006

Biochemical and genetic techniques have provided considerable insight into the structure‐function relationship of one of the ADP‐ribosyl transferases produced by Pseudomonas aeruginosa, exotoxin A. Exotoxin A contains a typical prokaryotic signal sequence which, in combination with the first 30 amino‐terminal amino acids of the mature protein, is sufficient for exotoxin A secretion from P. aeruginosa. Determination of the nucleotide sequence and crystalline structure of this prokaryotic toxin allowed a molecular model to be constructed. The model reveals three structural domains of exotoxin A. Analysis of the identified domains shows that the amino‐terminal domain (domain I) is involved in recognition of eukaryotic target cells. Furthermore, the central domain (domain II) is involved in secretion of exotoxin A into the periplasm of Escherichia coli. Evidence also implicates the role of domain II in translocation of exotoxin A from the eukaryotic vesicle which contains the toxin after it becomes internalized into susceptible eukaryotic cells via receptor‐mediated endocytosis. The carboxy‐terminal portion of exotoxin A (domain III) encodes the enzymatic activity of the molecule. The structure of this domain includes a cleft which is hypothesized to be the catalytic site of the enzyme. Several residues within domain III have been identified as having a direct role in catalysis, while others are hypothesized to play an important structural role.
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

Regions 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

The 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

The 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

Sites 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

The 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

Two 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

Protein 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

We 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

All 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

A 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

Capsule‐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

The 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

The 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

The 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

The 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

FNR, 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

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

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.
Nucleotide sequence and organization of the upstream region of the Corynebacterium glutamicum lysA gene
Authors: Marcel T. , Archer J. A. C. , Mengin‐Lecreulx D. , Sinskey A. J.
Published: October 2006

Maximum expression of the Corynebacterium glutamicum lysA gene is dependent upon the presence of a 2.3 kb region immediately 5’of the lysA reading frame. Subcloning and functional analysis of the upstream region implied that this region contained the lysA promoter. Sequence determination of the upstream region revealed a single open reading frame, orfX, in the same orientation as lysA. The orfX coding sequence exhibited all the sequence characteristics of a gene with the potential for a 550‐amino‐acid polypeptide product. Expression of lysA is coupled to that of orfX via a common promoter located immediately 5 of orfX. The RNA start site has been determined by S1 nuclease mapping. Both the orfX and the lysA gene are expressed as a single 3.0kb RNA transcript. These data indicate that orfX and lysA are genes within a two‐gene operon. Expression of the lysA gene is not subject to regulation by lysine. The orfX gene product was shown not to be directly linked to the lysine biosynthetic pathway, nor is it the enzyme incorporating DAP into the peptidoglycan precursor.
Instability of bacteriophage Mu transposase and the role of host Hfl protein
Authors: Gama M.‐J. , Toussaint A. , Pato M. L.
Published: October 2006

The activity of the transposase of bacteriophage Mu is unstable, requiring the protein to be synthesized throughout the lytic cycle (Pato and Reich, 1982). Using Western blot analysis, we analysed the stability of the transposase protein during the lytic cycle and found that it, too, is unstable. The instability of the protein is observed both in the presence and the absence of Mu ONA replication, and is independent of other Mu‐encoded proteins and the transposase binding sites at the Mu genome ends. Stability of the protein is enhanced in host strains mutated at the hfl locus; however, stability of the transposase activity is not enhanced in these strains, suggesting that functional inactivation of the protein is not simply a result of its proteolysis.
Role of the nodD and syrM genes in the activation of the regulatory gene nodD3, and of the common and host‐specific nod genes of Rhizobium meliloti
Authors: Maillet F. , Debellé F. , Dénarié J.
Published: October 2006

To analyse the regulation of the nodulation (nod) genes of Rhizobium meliloti RCR2011 we have isolated lacZ gene fusions to a number of common, host‐range and regulatory nod genes, using the mini‐MU‐lac bacteriophage transposon Mud II1734. Common (nodA, nodC, nod region IIa) and host‐range (nodE, nodG, nodH) genes were found to be regulated similarly. They were activated (i) by the regulatory nodD1 gene in the presence of flavones such as chrysoeriol, luteolin and 7,3′,4′‐trihydroxyflavone, (ii) by nodD2 in the presence of alfalfa root exudate but not with the NodD1‐activating flavones, and (iii) by the regulatory genes syrM‐nodD3 even in the absence of plant inducers. Thus common and host‐range nod genes belong to the same regulon. In contrast to the nodD1 gene, the regulatory nodD3 gene was not expressed constitutively and exhibited a complex regulation. It required syrM for expression, was activated by nodD1 in the presence of luteolin and was positively autoregulated.
Towards an understanding of the structural basis of ‘forbidden’ transport pathways in the Escherichia coli lactose carrier: mutations probing the energy barriers to uncoupled transport
Authors: King S. C. , Wilson T. H.
Published: October 2006

Recent progress in the analysis of mutants of the Eschenchia coli lactose carrier function is reviewed, with special emphasis on the structural basis for energy barriers which prevent ‘forbidden’ con‐formational changes. Mutations which break down the barriers to forbidden isomerizations involving the binary carrier:sugar (CS) and carrier:proton (CH) complexes have been obtained in several laboratories. These mutants allow uncoupled transport of H+ or galactoside in the lactose carrier which normally couples cation and sugar movement in a 1:1 stoichiometry. These uncoupled mutants appear to be associated with changes in both sugar and cation recognition, suggesting that the physical interactions forming the basis for co‐substrate recognition and uncoupling are not independently variable.

By postulating that translocation involves transformation of the stable intermediate of the co‐transport cycle to unstable transition state conformations of the carrier, it is possible to consider the consequences of mutagenesis in terms of transition state theory. Consistent with several experimental observations, the analysis predicts in each mutant the occurrence of more than one abnormality in the transport cycle (such as changes in sugar recognition, cation recognition or the coupling reaction). We have called the general phenomenon a ‘mutational double‐effect’ because any mutation which alters the Gibbs free energy change of one reaction in the transport
How Escherichia coli RNA polymerase can negatively regulate transcription from a constitutive promoter
Authors: Duval‐Valentin G. , Reiss C.
Published: October 2006

We previously described the structures and functions of specific complexes between the bla promoter from Tn3(present in pBR322) and RNA polymerase (RNAP), showing that, at excess RNAP, complexes can form in which one or two RNAPs bind to the same promoter (1:1 and 2:1 complexes) (Duval‐Valentin and Ehrlich, 1988). We report here that the 2:1 complex cannot be detected below 25° C; above that temperature, a 1:1 complex forms at a rate one order of magnitude faster than that of the 2:1 complex, and above 30° C, the amounts of both species become equal for RNAP/promoter ratio r30≤r≤70. The 2:1 complex decays back to a 1:1 complex losing the last RNAP at a rate about three times that of the 1:1 complex decay. Functional assays of the complexes formed at excess RNAP show that both 1:1 and 2:1 complexes are immediately and permanently inhibited, even when the promoters are pre‐incubated with ribonucleotide selections potentially enabling entrance into abortive cycling or formation of a stressed complex. We conclude that the inhibition step probably takes place in the complex formation pathway between RPi and RPo, at a novel stable intermediate isomer, RPj, formed above 25° C. A possible mechanism of formation of the 2:1 complex is outlined. In vivo studies, in which r was modified by varying the bacterial growth rate, show a reduction of bla expression as r values are upshifted, specific to the bla promoter from Tn3.
Import‐defective colicin B derivatives mutated in the TonB box
Authors: Mende J. , Braun V.
Published: October 2006

The pore‐forming colicin B is taken up into Escherichia coli by a receptor and TonB‐dependent process. The receptor and colicin B both contain a similar amino acid sequence, close to the N‐terminal end, termed the TonB box. Point mutations were introduced into the TonB‐box region of the colicin B structural gene cba resulting in colicin B derivatives which were partially or totally Inactive against E. coli cells. All derivatives still bound to the receptor. An inactive derivative killed cells when translocated across the outer membrane by osmotic shock treatment, and formed pores in planar lipid bilayer membranes identical to the wild‐type colicin. Some of the mutations were partially suppressed by mutations in the tonB structural gene. It was concluded that the TonB‐box mutations define a region that is involved in the uptake of colicin B across the outer membrane.
Pore‐forming cytolysins of Gram‐negative bacteria
Authors: Welch R. A.
Published: October 2006

A great deal is known about the structure, function and metabolic effects of enzymatic bacterial toxins such as the diphtheria, pertussis and cholera toxins. By comparison, our understanding of the pore‐forming, cytolytic toxins, particularly those produced by Gram‐negative bacterial pathogens, is far less complete. The genetics and biochemistry of a large, newly discovered family of calcium‐dependent, pore‐forming cytotoxins (RTX toxins) produced by different genera of the Enterobacteriaceae and Pasteurellaceae are discussed in this review. This toxin family is especially noteworthy because the individual toxins often exhibit different cell‐ and host‐specificity. A brief review is also included of two ancestrally unrelated groups of calcium‐independent, pore‐forming toxins, the haemolysins produced by Proteus mirabilis and Serratia marcescens and the aerolysins secreted by species of Aeromonas. Their structure and function are contrasted with those of the RTX family members. Emerging questions about the role of cytolysins in pathogenesis are presented. Perhaps the most important issue raised is whether or not less attention should be paid to the the lytic capacity of these cytotoxins, with more energy being devoted to the understanding of their non‐lytic Inhibitory activities against host cells.
Organization of the fimbrial gene region of Bacteroides nodosus: class I and class II strains
Authors: Hobbs M. , Dalrymple B. P. , Cox P. T. , Livingstone S. P. , Delaney S. F. , Mattick J. S.
Published: October 2006

The fimbrial subunit genes of Bacteroides nodosus may be divided into two distinct classes, based on the sequence of the major subunit gene fimA (accompanying paper — Mattick et al., 1991). The genetic organization of the fimbrial gene region in these two classes is also distinct. Upstream of fimA in both classes in opposite transcriptional orientation is the gene aroA which encodes amino acid biosynthetic enzyme 5‐enolpyruvylshikimate‐3‐phosphate synthase. However, downstream of fimA the two classes are quite different until homology is restored at a bidirectional transcription termination signal separating the fimbrial operon from a gene clpB, which appears to encode the regulatory subunit of an ATP‐dependent protease. Between aroA and clpB class I strains contain, apart from fimA, only one other gene (fimB). Sequence and polymerase chain reaction analyses indicate that fimB does not have a separate promoter but rather is co‐transcribed with fimA at a level attenuated by the strength of the transcription termination signal in the intergenic region. In class II strains fimA is followed by a more extended region containing three genes, which appear to have the same transcriptional arrangement as fimB. The second of these genes (fimD) may represent a functional analogue of fimB although there is no close sequence homology. The first gene (fimC) has no obvious similarity to either fimB or fimD. Beyond fimO, at the 3 end of the class ll‐specific region, is a variant fimbrial subunit gene (fimZ) which is virtually identical in serogroups 0 and H and which appears to represent a duplicate, possibly redundant, gene closely related to the progenitor of the more divergent structural subunit fimA gene found in these strains. Comparisons of the predicted ftmZ product with those of fimA in class I and class II strains, as well as of the boundaries of the class‐specific regions, suggest that the class II sequences evolved in another type 4 fimbriate species and were subsequently substituted in the 8. nodosus genome by lateral transfer. Analysis of the sequences flanking fimA in different strains indicates that recombinational exchange of both fimA and the entire operon has also occurred between strains, and is possibly a mechanism for disseminating structural diversity in the population.
Ionizing groups in lipopolysaccharides of Pseudomonas cepacia in relation to antibiotic resistance
Authors: Cox A. D. , Wilkinson S. G.
Published: October 2006

Contrary to previous reports, lipopolysaccharides from Pseudomonas cepacia contain a 3‐deoxyoct‐2‐ulosonic acid (probably a single residue). The lipopolysaccharides contain only two phosphate residues, one of which apparently forms a phosphodiester bridge between 4‐amino‐4‐deoxyarabinose and a glucosamine residue in lipid A. The second, unlocated phosphate residue occurs mainly as a monoester in some lipopolysaccharides, and mainly as a diester in others. All lipopolysaccharides lack pyrophosphate residues. The results support the view that the resistance of P. cepacia to cationic antibiotics stems from ineffective binding to the outer membrane, as a consequence of the low number of phosphate and carboxylate groups in the lipopolysaccharide, and the presence of the protonated aminodeoxypentose.