bluuebunny/crossref_metadata_2025 huggingface dataset:
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

The 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

H.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

Morphological 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

Export 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

In 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

Tn21‐ 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

Transcription 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

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

Conjugation 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

Pectate 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

A 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

Many 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

The 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
The O75X adhesin of uropathogenic Escherichia coli is a type IV collagen‐binding protein
Authors: Westerlund B. , Kuusela P. , Risteli J. , Risteli L. , Vartio T. , Rauvala H. , Virkola R. , Korhonen T. K.
Published: October 2006

Interaction of the basement‐membrane binding O75X adhesin of uropathogenic Escherichia coli with various extracellular matrix proteins was studied. The adhesin showed strong binding to type IV collagen immobilized on microtitre plates, whereas other collagens, laminin and fibronectin, were only weakly recognized. Similarly, specific binding of [125I]‐labelled type IV collagen to 075X‐positive bacteria was shown. Interaction of the two proteins was also demonstrated by affinity chromatography of the O75X adhesin on immobilized type IV collagen. The adhesin bound strongly to the immobilized N‐terminal 7S domain of type IV collagen, and the binding of [125I]‐labelled type IV collagen to O75X‐positive bacteria was inhibited by the soluble 7S domain. Binding of O75X to type IV collagen and to its 7S domain was specifically inhibited by chloramphenicol but was not affected by periodate or endoglycosidase‐H treatment of the glycoproteins. Our results show that the 7S domain of type IV collagen is the basement membrane receptor for the O75X adhesin and suggest an interaction based on protein‐protein recognition. Inhibition of the interaction by chloramphenicol favours the supposition that a modified tyrosine is involved in the binding site.
Regulation of exotoxin A synthesis in Pseudomonas aeruginosa: characterization of toxA‐lacZ fusions in wild‐type and mutant strains
Authors: Vasil M. L. , Grant C. C. R. , Prince R. W.
Published: October 2006

A mobilizable plasmid which carries the promoter for the exotoxin A (ETA) structural gene fused to lacZ was integrated into the chromosome of wild‐type and mutant strains of Pseudomonas aeruginosa at the toxA locus by homologous recombination. β‐galactosidase synthesis in the strains (cointegrates) carrying the tox4‐lacZ fusions was regulated like ETA synthesis is in P. aeruginosa.

Two multicopy plasmids carrying a positive regulatory gene designated toxR were constructed which are identical except with respect to the orientation of toxR to the lacZ promoter on the plasmid. These plasmids were then introduced into P. aeruginosa cointegrate strains. When toxR was using its own promoter, synthesis of β‐galactosidase in the cointegrate strains was increased but the pattern of iron regulation was not altered. In contrast, when the lacZ promoter was directing synthesis of the toxR product in the cointegrate strains, iron regulation of β‐galacto‐sidase and ETA synthesis were abolished.
virG of Agrobacterium tumefaciens plasmid pTiC58 encodes a DNA‐binding protein
Authors: Powell B. S. , Rogowsky P. M. , Kado C. I.
Published: October 2006

Virulence genes of the Agrobacterium tumefaciens Ti plasmid are positively regulated by the products of virA and virG. To study the DNA‐binding properties of the VirG protein, a translational fusion between virG and the frpEgene of Escherichia coli was constructed, and antiserum was raised against the encoded fusion protein. Using this antiserum, a protein of Mr= 29000, a size similar to that calculated from the virG nucleotide sequence, was detected in an E. coli strain harbouring a virG expression vector. Both the VirG protein and the fusion protein were found, by filter‐binding and gel retardation analyses, to bind DNA nonspecifically. These data support an existing model for the two‐component regulatory systems of bacteria.
Cloning and analysis of the gene for the major outer membrane lipoprotein from Pseudomonas aeruginosa
Authors: Cornelis P. , Bouia A. , Belarbi A. , Guyonvarch A. , Kammerer B. , Hannaert V. , Hubert J. C.
Published: October 2006

The gene for the Pseudomonas aeruginosa outer membrane lipoprotein I was isolated from a genomic library in the phage λ EMBL3 vector and subsequently subcloned in the low copy‐number, wide host‐range plasmid vector, pKT240. The cloned gene was highly expressed, resulting in the production of a low molecular‐weight protein (8kD) that was found to be associated with the outer membrane. Sequence anatysis showed an open reading frame of 83 amino acids with a putative N‐terminal hydrophobic signal peptide of 19 residues immediately followed by the lipoprotein consensus sequence, GLY‐CYS‐SER‐SER (residues 19–22). The predicted amino acid composition of the mature polypeptide and that of the purified lipoprotein I of P. aeruginosa (Mizuno and Kageyama, 1979) were identical. In contrast with other Gram‐negative outer membrane lipoproteins, conformation predictions suggested that the mature protein was a single alpha helix.
Nucleoprotein structures at positively regulated bacterial promoters: homology with replication origins and some hypotheses on the quaternary structure of the activator proteins in these complexes
Authors: Rajbaud O.
Published: October 2006

The quaternary structure of regulatory proteins undoubtedly plays an important role in the initiation of transcription and DNA replication. To date, the best‐characterized regulatory proteins are oligomers in which protomers are bound together by isologous interactions. From the examples presented in this article, it appears that the formation of certain nucleoprotein complexes implicated in transcription initiation might involve heterologous rather than isologous interactions, allowing differentiation between two classes of transcription activators.
The high‐affinity K+‐translocating ATPase complex from Bacillus acidocaldarius consists of three subunits
Authors: Hafer J. , Siebers A. , Bakker E. P.
Published: October 2006

Cells of the thermoacidophilic bacterium Bacillus acidocaldarius express a high‐affinity K+‐uptake system when grown at low external K+. A vanadate‐sensitive, K+ ‐ and Mg2+ ‐stimulated ATPase was partially purified from membranes of these cells by solubilization with a non‐ionic detergent followed by ion‐exchange chromatography of the extract. Combinations of non‐denaturing and denaturing electrophoretic separation methods revealed that the ATPase complex consisted of three subunits with molecular weights almost identical to those of the KdpA, B and C proteins, which together form the Kdp high‐affinity, K+ ‐translocating ATPase complex of Escherichia coli. The affinity of the partially purified ATPase from B. acidocaldarius for its substrates K+ (Km 2–3 μM) and ATP (Km 80 μM), its stimulation by various divalent cations, and its inhibition by vanadate (Ki 1–2 μM), bafilomycin A1 (Ki, 20 μM), DCCD (Ki 200 μM) or Ca2+ were also similar to those of the E coli enzyme, indicating that the two K+ ‐translocating ATPases have almost identical properties.
Role of cysteine residues and of metal ions in the regulatory functioning of FNR, the transcriptional regulator of anaerobic respiration in Escherichia coli
Authors: Trageser M. , Unden G.
Published: October 2006

FNR, the transcriptional regulator of gene expression of anaerobic respiration in Escherichia coli, contains a cluster of cysteine residues at the amino terminus which resembles the metal‐binding domains of metal‐binding proteins. It is possible, therefore, (i) that FNR binds metals with the cysteines as ligands and (ii) that this property is related to the regulatory function of FNR. These questions were investigated, with the following results.

Approximately 2.4 of the 4 cysteine residues of FNR can be alkylated with iodoacetate in permeabilized aerobic or anaerobic bacteria without the addition of reducing agents. The time required for half‐maximal labelling of the cysteines was 50 min in anaerobic bacteria and 6 min in aerobic bacteria. The difference in the reactivity was specific for the cysteines of FNR. These cysteine residues were also highly reactive in anaerobically grown bacteria, when the growth medium contained chelating agents such as 1,10‐phe‐nanthroline (15 μM). The effect of the chelating agents was reversed by an excess of divalent metal ions such as Fe(11) or Cu(11) in the medium. The presence of 1,10‐phenanthroline (10 μM) also inhibits the expression of fumarate reductase, an FNR‐dependent enzyme. These results suggest that FNR exists in two different forms which differ in terms of the reactivity of their cysteine residues to iodoacetate. The interconversion of both forms appears to be regulated by the availability of O2and by the binding of metal ions. The two forms of FNR may be involved in the regulation of O2‐dependent gene expression.
A bacterial c‐type cytochrome can be translocated to the periplasm as an apo form; the biosynthesis of cytochrome cd1 (nitrite reductase) from Paracoccus denitrificans
Authors: Page M. D. , Ferguson S. J.
Published: October 2006

An apo form of cytochrome cd1, (nitrite reductase) of Paracoccus denitrificans has been detected immunologically in the periplasm of a mutant that lacks all c‐type cytochromes. A method for the preparation of apo‐nitrite reductase (lacking both c‐ and d‐type haem) from the holoenzyme of wild‐type cells has been developed. The apoprotein synthesized by the mutant is indistinguishable from the chemically prepared apoprotein in respect of: (i) subunit molecular weight; (ii) formation of a homodimer; (iii) properties on anion exchange chromatography. The holoenzyme has similar properties in respect of (i) and (ii) but behaves differently during anion exchange. A suggested mode of assembly of cytochrome cd1, is trans‐location into the periplasm of a precursor polypeptide, maturation by a signal peptidase to give an apoprotein identical to that prepared chemically from the holoenzyme, followed by insertion of c‐type and d‐type haem in an as yet unknown order.
Cloning and characterization of beta‐lactam biosynthetic genes
Authors: Miller J. R. , Ingolia T. D.
Published: October 2006

Seven genes coding for two different enzymes of the penicillin/cephalosporin biosynthetic pathway have been cloned from fungal and bacterial sources. Using amino acid sequences derived from the purified enzymes, oligonucleotide probes were designed to hybridize to their cognate genes in a genomic library. The high degree of similarity (57%) between enzymes of bacterial and fungal origin suggests a horizontal transfer of a primordial beta‐lactam pathway, probably from a bacterial cell to a fungal cell. Overproduction of the proteins in Escherichia coli has allowed further study of the mechanism of action of these important enzymes.
Identification, molecular cloning and sequence analysis of a gene cluster encoding the Class II fructose 1,6‐bisphosphate aldolase, 3‐phosphoglycerate kinase and a putative second glyceraldehyde 3‐phosphate dehydrogenase of Escherichia coli
Authors: Alefounder P. R. , Perham R. N.
Published: October 2006

To investigate a possible chromosomal clustering of glycolytic enzyme genes, the complete nucleotide sequence of the 8029 bp insert of Escherichia coli DNA in the ColE1 plasmid pLC33‐5 of the Clarke and Carbon collection (Clark and Carbon, 1976) was determined. Genes (pgk, fda) encoding the phosphoglycerate kinase and Class II fructose 1,6‐bisphosphate aldolase, respectively, of E. coli were identified. The phosphoglycerate kinase was found to be highly homologous in primary structure to the same enzyme from eukaryotic organisms. A further large open reading frame, designated gapB, was also identified, which on the basis of sequence homology, appears to encode another glycolytic enzyme, glyceraldehyde 3‐phosphate dehydrogenase. This putative gene differs significantly from that (designated gapA) already identified as coding for this enzyme in E. coli and which maps elsewhere on the chromosome. The products, if any, of several other open reading frames remain to be identified.
The Rhizobium meliloti host range nodQ gene encodes a protein which shares homology with translation elongation and initiation factors.
Authors: Cervantes E. , Sharma S. B. , Maillet F. , Vasse J. , Truchet G. , Rosenberg C.
Published: October 2006

The Rhizobium meliloti nod region lib is involved in host‐range determination: (i)the presence of region lib is necessary for transfer of alfalfa root hair curling ability to Rhizobium legumlnosarum biovar trifolii; (ii) a mutation in region llb extends the R. meliloti infection host range to Vicia sativa nigra; (iii) dominance of R. meliloti nod genes over R. leguminosarum biovar viciae nod genes is abolished by mutations in region llb. The nucleotide sequence of this region has been determined. Genes corresponding to the two open reading frames identified are designated nodP and nodQ. The predicted amino acid sequence of the NodQ protein shows homology with translation initiation and elongation factors. The consensus sequence involved in the GTP‐binding domain is conserved.
Notes The ribonucleoside diphosphate reductase gene (nrdA) of Escherichia coli carries a repetitive extragenic palindromic (REP) sequence in its 3'structural terminus
Authors: Merino E. , Bolivar F.
Published: October 2006

A computer search for repeated sequences led us to identify five REP (repetitive extragenic palindromic) sequences in the 3′ ‐terminal region of the Escherichia coli ribonucleoside diphosphate reductase gene (nrdA). These REP sequences are located within a putative duplicated DNA region, the first of them being part of the carboxy‐terminal coding region of the nrdA gene. This is the first report of a REP sequence within a structural gene and also the first example of a REP sequence apparently generated by DNA duplication.
Molecular genetic analysis of FNR‐dependent promoters
Authors: Eiglmeier K. , Honoré N. , Iuchi S. , Lin E. C. C. , Cole S. T.
Published: October 2006

In enteric bacteria, the expression of many genes encoding various anaerobic electron transfer functions is controlled by FNR, the product of the autoregulated fnr gene. FNR is structurally and functionally homologous to CAP, the catabolite gene activator protein, and increased FNR production strongly stimulates transcription of its target genes. By analysis of RNA produced in vivo the promoters of four FNR‐dependent genes were localized and shown to display a common arrangement. A 22bp dyad symmetry was found about 30 nucleotides upstream of the transcriptional startpoints and a similar sequence was shown to overlap the site of transcription initiation in the negatively controlled fnr gene. The consensus sequence for the half site recognized by FNR (AAA‐TTGAT) is only slightly different from that of CAP (AA‐TGTGA). Studies with two mutant frd promoters from Escherichia coli, displaying altered regulation and FNR response, provided additional evidence for recognition of this sequence by FNR.
Assembly of colicin genes from a few DNA fragments. Nucleotide sequence of colicin D
Authors: Roos U. , Harkness R. E. , Braun V.
Published: October 2006

The nucleotide sequence of a 2.4 kb Dral‐EcoRV fragment of pColD‐CA23 DNA was determined. The segment of DNA contained the colicin D structural gene (cda) and the colicin D immunity gene (cdi). From the nucleotide sequence it was deduced that colicin D had a molecular weight of 74683D and that the immunity protein had a molecular weight of 10057D. The amino‐terminal portion of colicin D was found to be 96% homologous with the same region of colicin B. Both colicins share the same cell‐surface receptor, FepA, and require the TonB protein for uptake. A putative TonB box pentapeptide sequence was identified in the amino terminus of the colicin D protein sequence. Since colicin D inhibits protein synthesis, it was unexpected that no homology was found between the carboxy‐terminal part of this colicin and that of the protein synthesis inhibiting colicin E3 and cloacin DF13. This could indicate that colicin D does not function in the same manner as the latter two bacteriocins. The observed homology with colicin B supports the domain structure concept of colicin organization. The structural organization of the colicin operon is discussed. The extensive amino‐terminal homology between colicins D and B, and the strong carboxy‐terminal homology between colicins B, A, and N suggest an evolutionary assembly of colicin genes from a few DNA fragments which encode the functional domains responsible for colicin activity and uptake.
Cloing and characterization of an Escherichia coli gene, pcnB, affecting plasmid copy number
Authors: March J. B. , Colloms M. D. , Hart‐Davis D. , Oliver I. R. , Masters M.
Published: October 2006

A gene, pcnB, affecting the copy number of ColE1‐related plasmids has been cloned and mapped to 3.6 min on the Escherichia coli chromosome between panD and fhu. The gene encodes a previously un‐described 48 kD protein. Several independently isolated mutants exhibiting the same phenotye, reduced copy number, have been shown to be pcnB.
Cloning and analysis of a deletable tetracycline‐resistance determinant of Streptomyces lividans 1326
Authors: Kessler A. , Dittrich W. , Betzler M. , Schrempf H.
Published: October 2006

An unstable determinant encoding resistance to tetracycline and minocycline has been cloned and characterized. It could be demonstrated that this determinant shares extensive homology with the otrA gene identified in Streptomyces rimosus. Tetracycline‐sensitive variants of Streptomyces lividans derive mostly by deletion of this resistance determinant and neighbouring DNA regions.
Extensive sequence homology between IgA receptor and M proteins in Streptococcus pyogenes
Authors: Frithz E. , Hedén L.‐O. , Lindahl G.
Published: October 2006

Many strains of Streptococcus pyogenes are known to express a receptor for IgA. The complete nucleotide sequence of the gene for such a receptor, protein Arp4, has been determined. The deduced amino acid sequence of 386 residues includes a signal sequence of 41 amino acids and a putative membrane anchor region, both of which are homologous to similar regions in other streptococcal surface proteins. The processed form of the IgA receptor has a length of 345 amino acids and a calculated molecular weight of 39544. The N‐terninal sequence of the processed form is different from that previously found for a similar IgA receptor isolated from a S. pyogenes strain of type M60. The sequence of protein Arp4 shows extensive homology to the C‐terminal half of streptococcal M proteins, but not to the streptococcal IgG receptor protein G or staphlyococcal protein A. Apart from the membrane anchor, this homology includes a sequence of 119 amino acid residues containing three repeated units and a 54‐residue sequence without repeats. The protein expressed in Escherichia coli is found in the periplasmic space, in which it constitutes the major protein. Protein Arp4 is the first example of a surface protein that has both immunoglobulin‐binding capacity and structural features characteristic of M proteins. And structural features characteristic of MJ proteins.
The role of the cell surface in social and adventurous behaviour of myxobacteria
Authors: Shimkets L. J.
Published: October 2006

The myxobacteria are an unusually social group of prokaryotic organisms that form fruiting bodies containing dormant myxospores in response to nutritional stress. Social behaviour is controlled by a multigene system known as ‘S’ and by a series of intercellular signals that are released during development. The genes controlling these communication systems have been identified by mutational analysis and current research is directed toward examining the functions of these genes. S− mutants are generally nondevelopmental and noncohesive. They lack pili, a Congo red receptor, and 50‐nm‐wide fibrils which extend outward from the ceil surface. Changes in the architecture of the cell surface have been studied by means of surface labelling and with monoclonal antibodies directed against cell‐surface antigens. The cell surface undergoes dramatic changes during the course of development. Most vegetative antigens decrease in concentration or disappear completely while new development‐specific antigens appear.
Fine‐tuning of nif and fix gene expression by upstream activator sequences in Bradyrhizobium japonicum
Authors: Gubler M.
Published: October 2006

The significance of Bradyrhizobium japonicum upstream activator sequences (UASs) for differential NifA‐mediated fix and nif gene expression was investigated by two means: (i) hybrid fixA‐ and fixB‐lacZ fusions were constructed by transposing a nifH‐UAS cartridge in front of their promoters; and (ii) B. japonicum mutants were generated carrying specific chromosomal deletions or UAS cartridge insertions within the fixA, fixB or nifH promoter‐upstream regions. Expression of fixA was not affected, and expression of fixB decreased only to 42%, when the respective fixA and fixB promoter‐upstream DNAs were deleted. This shows that in B. japonicum the Njf A‐dependent activation of at least the fixA promoter does not require the presence of a closely adjacent UAS. Deletion of the UASs in front of the nifH gene not only reduced the expression of nifH down to 2.5% but, surprisingly, also resulted in a reduction of the fixB mRNA level to less than 20%. This suggests that the nifH‐UASs may exert a long‐range effect on the expression of the 3‐kb‐distant fixBCX operon in nif cluster I of B. japonicum. Artificial transposition of the nif‐UASs in front of the fixA and fixB promoters strongly enhanced fixA and fixB expression.
Characterization of a cis regulatory DNA element necessary for formate induction of the formate dehydrogenase gene (fdhF) of Escherichia coli
Authors: Birkmann A. , Bock A.
Published: October 2006

The fdhF gene, encoding the selenopolypeptide of formate dehydrogenase (FDHH). has a ‐12/‐24 nif‐type consensus promoter. A cis‐acting DNA element, which is required for the regulation of the promoter by formate under anaerobic conditions, has been identified. This regulatory sequence of about 25bp in length is located 110 bp upstream of the transcription start site. By analysing a variety of mutant constructs in this region (5’deletions, internal deletions and point mutations) we were able to identify a hexanucleotide sequence ‐GTCACG‐, which is important for the formate regulation of the fdhF promoter. The data also indicate that this element has many of the properties characteristic of eukaryotic enhancers.
Molecular cloning and characterization of chromosomal virulence region kcpA of Shigella flexneri
Authors: Yamada M. , Sasakawa C. , Okada N. , Makino S.‐I. , Yoshikawa M.
Published: October 2006

In Shigella flexneri, in addition to several well‐recognized plasmid‐borne virulence loci, at least three genetic loci implicated in pathogenesis have been recognized on the chromosome. To understand more about the pathogenesis of bacillary dysentery at a molecular level, the genetically recognized but previously unidentified KcpA region (one of the chromosomal regions near purE) was cloned and sequenced. A single translatable open reading frame encoding a 12310 Dalton protein corresponding to the minicell product was found. Immunofluorescence microscopy, as well as optical and electron microscopic comparison of tissue‐cultured cells and guinea‐pigs’eyes infected with wild‐type or kcpA mutant bacteria, revealed that the kcpA product is required by invading bacteria for spread into adjacent cells.
Mistranslation induces the heat‐shock response in the yeast Saccharomyces cerevisiae
Authors: Grant C. M. , Firoozan M. , Tuite M. F.
Published: October 2006

The synthesis of heat‐shock proteins can be triggered by a variety of stress‐inducing conditions. Here we show that translational misreading caused by growth in the presence of the aminoglycoside antibiotic paromomycin will induce the heat‐shock response in the yeast Saccharomyces cerevisiae. This was demonstrated (i) by the acquisition of thermotolerance, and (ii) by elevated levels of expression of the heat‐shock protein, hsp70. In addition, transcription of the ubiquitin gene (UBI4) was increased in paromomycin‐grown cells. Control experiments with the protein synthesis inhibitor cycloheximide (which does not induce translational misreading) demonstrated that the response was not due to inhibition of protein synthesis per se. These observations strongly suggest that the synthesis of abnormally high levels of aberrant proteins is the trigger of the heat‐shock response in this simple eukaryote.
Nucleotide sequence and analysis of the N‐terminal coding region of the Spodoptera‐activeδ‐endotoxin gene of Bacillus thuringiensis aizawai 7.29
Authors: Sanchis V. , Lereclus D. , Menou G. , Chaufaux J. , Guo S. , Lecadet M.‐M.
Published: October 2006

The nucleotide sequence of a 2711 bp DNA segment which contains the N‐terminal coding sequence and the 5’flanking region of a crystal protein gene (bta) from Bacillus thuringiensis subsp. aizawai 7.29 has been determined. The coding region encodes an 824 amino‐acid polypeptide corresponding to a carboxy‐terminally truncated δ‐endotoxin specifically active against the cotton leaf worm Spodoptera littoralis. Comparison of the deduced amino acid sequence of the bta gene with that of the 4.5, 5.3 and 6.6 kb classes of lepidopteran‐active β‐endotoxins revealed that the Bta sequence contains a very high level of amino acid substitutions in the N‐terminal part of the protoxin molecule. The substitutions are grouped in several highly variable segments separated by highly conserved regions. These conserved domains are also present in the dipteran‐ and coleopteran‐active δ‐endotoxins. The control region of the bta gene shows considerable DNA identity with the control regions of the other lepidopteran‐active genes. Deletions of the 3’region of the gene were carried out and the toxic fraction of the btaδ‐endotoxin was identified with the N‐terminal half of the molecule.
β‐lactamase as a probe of membrane protein assembly and protein export
Authors: Broome‐Smith J. K. , Tadayyon M. , Zhang Y.
Published: October 2006

The enzyme TEM β‐lactamase constitutes a versatile gene‐fusion marker for studies on membrane proteins and protein export in bacteria. The mature form of this normally periplasmic enzyme displays readily detectable and distinctly different phenotypes when localized to the bacterial cytoplasm versus the periplasm, and thus provides a useful alternative to alkaline phosphatase for probing the topology of cytoplasmic membrane proteins. Celts producing translocated forms of β‐lactamase can be directly selected as ampicillin‐resistant colonies, and consequently a β‐lactamase fusion approach can be used for positive selection for export signals, and for rapid assessment of whether any protein expressed in Escherichia coli inserts into the bacterial cytoplasmic membrane. The level of ampicillin resistance conferred on a cell by an extracytoplasmic β‐lactamase derivative depends on its level of expression, and therefore a β‐lactamase fusion approach can be used to directly select for increased yields of any periplasmic or membrane‐bound gene products expressed in E. coli.
HeLa cell invasion by a strain of enteropathogenic Escherichia coli that lacks the O‐antigenic polysaccharide
Authors: Riley L. W. , Junio L. N. , Schoolnik G. K.
Published: October 2006

The interaction with HeLa cells of an enteropathogenic Escherichia coli (EPEC) strain and its plasmid‐cured derivative strain was examined. An O111:NM EPEC strain B171 harbours a 54 megadalton plasmid (pYR111) necessary for the expression of both localized adherence (LA) to HeLa cells and the 0‐repeating side chain of the lipopolysaccharide. Under light microscopy, the plasmid‐cured derivative strain B171‐4 was observed to interact with HeLa cells in a pattern distinct from LA. Transmission electron microscopy showed that the bacteria were internalized by HeLa cells. In contrast, strain B171 induced pedestal‐like projections and invaginations of the plasma membrane, but was never completely internalized. A quantitative assay to determine the number of internalized bacteria revealed that strain B171‐4 was internalized at levels 30–70‐fold higher than those of aviruient E. coli strains. Cytochalasin B reduced the levels of internalization of both strain B171‐4 and an entero‐invasive E. coli strain (E11), but did not affect LA by strain B171. These results suggest that EPEC strain B171 may carry a specific chromosomally determined surface factor needed to initiate internalization by HeLa cells. However, a plasmid‐determined factor alters the nature of this interaction; the combined effects of the chromosomal and plasmid determinants lead to the characteristic attachment of the bacteria in clusters on the surface of the eukaryotic cell.
Isolation and characterization of the gene encoding a novel, thermostable serine proteinase from the mould Tritirachium album Limber
Authors: Samal B. B. , Karan B. , Boone T. C. , Osslund T. D. , Chen K. K. , Stabinsky Y.
Published: October 2006

A number of proteinases are induced and secreted into the culture medium of Tritirachium album Limber when the nitrogen source is limited to exogenous proteins. We have constructed a cDNA library using the polyadenylated RNA isolated during the nutritional induction with bovine serum albumin. A full‐length clone of a gene for a new proteinase (named proteinase R) was identified from this library. This clone contained sequences coding for the 108‐amino‐acid prepro‐leader as well as for the 279‐amino‐acid mature preoteinase. Proteinase R apparently belongs to the subtilisin group of serine proteases that contains disulphide bonds. Homology between proteinase R and proteinase K was found to be about 87% at the nucleotide as well as at the amino acid level. The Brookhaven Protein Data Base co‐ordinate file of proteinase K was used as a template to study the proteinase R substitutions in three‐dimensional space. The majority of the substitutions of proteinase R with respect to proteinase K were found to be on the exterior of the protein model.
Prolongation of the yeast life span by the v‐Ha‐RAS oncogene
Authors: Chen J. B. , Sun J. , Jazwinski S. M.
Published: October 2006

The budding yeast Saccharomyces cerevisiae has a finite life span that is defined by the number of times the cell divides. The patterns of expression of certain genes change in a specific manner during the life span, implying that at least some of the manifestations of the ageing process are subject to gene regulation. It has now been determined that the controlled expression of the RAS oncogene in yeast increases the longevity of this organism, indicating that, conversely, a defined alteration in the activity of a single gene can extend this organism's life span. The results suggest that there is a balance between life‐span extension and growth arrest when RAS is expressed. Inasmuch as the homologues of RAS in yeast function to integrate cell metabolism with the cell cycle, these studies raise the possibility that this integrative function may also apply to the co‐ordination of successive cell cycles during the life span.
Identification of developmentally regulated Giardia lamblia cyst antigens using GCSA‐1, a cyst‐specific monoclonal antibody
Authors: Ward H. D. , Kane A. V. , Ortega‐Barria E. , Keusch G. T. , Pereira M. E. A.
Published: October 2006

GCSA‐1, a monoclonal antibody raised against cysts generated in vitro was shown to be Giardia cyst‐specific by immunoblot analysis and immunofluorescence. GCSA‐1 recognized four polypeptides ranging from 29–45kD present in the cyst wall. These antigens appeared within eight hours of exposure of trophozoites to encystation medium and were shown to be synthesized by encysting parasites by means of metabolic labelling with [35S]‐cysteine. Trophozoites were not stained by the antibody. GCSA‐1 also reacted with in vivo cysts obtained from faeces of infected humans, gerbils and mice. These data demonstrate that the determinants recognized by GCSA‐1 are early cyst antigens which are developmentally regulated and conserved components of the cyst wall. The actual role of the antigens detected by GCSA‐1 in encystation are unknown, but they represent a potential target for strategies directed at inhibiting this process.