bluuebunny/crossref_metadata_2025 huggingface dataset:
The normally periplasmic enzyme β‐lactamase is specifically and efficiently translocated through the Escherichia coli outer membrane when it is fused to the cell‐surface enzyme pullulanase
Authors: Kornacker M. G. , Pugsley A. P.
Published: October 2006

Hybrid proteins were constructed in which C‐terminal regions of the bacterial cell surface and extracellular protein pullulanase were replaced by the mature forms of the normally periplasmic Escherichia coli proteins β‐lactamase or alkaline phosphatase. In E. coli strains expressing all pullulanase secretion genes, pullulanase‐β‐lactamase hybrid protein molecules containing an N‐terminal 834‐amino‐acid pullulanase segment were efficiently and completely transported to the cell surface. This hybrid protein remained temporarily anchored to the cell surface, presumably via fatty acids attached to the N‐terminal cysteine of the pullulanase segment, and was subsequently specifically released into the medium in a manner indistinguishable from that of pullulanase itself. These results suggest that the C‐terminal extremity of pullulanase lacks signal(s) required for export to the cell surface. When β‐lactamase was replaced by alkaline phosphatase, the resulting hybrid also became exposed at the cell surface, but exposition was less efficient and specific release into the medium was not observed. We conclude that proteins that do not normally cross the outer membrane can be induced to do so when fused to a permissive site near the C‐terminus of pullulanase.
Lipid modification of the 15 kilo Dalton major membrane immunogen of Treponema pallidum
Authors: Purcell B. K. , Swancutt M. A. , Radolf J. D.
Published: October 2006

The 15 kiloDalton major membrane immunogen was included among the Treponema pallidum polypeptides selectively labelled with [3H]‐palmitate. The cloned gene for this immunogen, tpp15, encoded a signal peptide of 17 amino acids, a consensus signal peptidase II cleavage site, and a mature protein of 124 amino acids (13967 Daltons). As predicted by the DNA sequence, the recombinant 15 kiloDalton immunogen labelled selectively with [3H]‐palmitate, and globo‐mycin inhibited processing of the precursor to the mature polypeptide. While the native and recombinant immunogens are amphiphilic, the 15 kiloDalton immunogen synthesized in a cell‐free system was hydrophilic. The covalent attachment of fatty acids appears to be responsible for the amphilicity of the immunogen and its membrane attachment.
New perspectives in tetracycline resistance
Authors: Salyers A. A. , Speer B. S. , Shoemaker N. B.
Published: October 2006

Until recently, tetracycline efflux was thought to be the only mechanism of tetracycline resistance. As studies of tetracycline resistance have shifted to bacteria outside the Enterobacteriaceae, two other mechanisms of resistance have been discovered. The first is ribosomal protection, a type of resistance which is found in mycoplasmas, Gram‐positive and Gram‐negative bacteria and may be the most common type of tetracycline resistance in nature. The second is tetracycline modification, which has been found only in two strains of an obligate anaerobe (Bacteroides). Recent studies have also turned up such anomalies as a tetracycline efflux pump which does not confer resistance to tetracycline and a gene near the replication origin of a tetracycline‐sensitive Bacillus strain which confers resistance when it is amplified.
MicroReview Cascades of sigma factors revisited
Authors: Stragier P. , Losick R.
Published: October 2006

Programmed gene expression during the process of endospore formation in Bacillus subtilis is governed by the successive appearance of five developmental sigma factors. These sigma factors are encoded by genes in which mutations arrest sporulation at a defined stage. These genes are turned on sequentially and depend for their own transcription on the activity of a previously synthesized sigma factor. Superimposed on the regulation of synthesis of the sigma factors are post‐transcriptional control mechanisms that couple the activation of the developmental sigma factors to the course of sporulation. Here we review evidence indicating that these developmental transcription factors comprise a regulatory cascade in the order σH→σF→σE→σG→σK in which the activity of each sigma factor depends on the action of the preceding sigma factor in the cascade.
Developmental‐stage‐specific plasmid supercoiling in Chlamydia trachomatis
Authors: Solbrig M. V. , Wong M. L. , Stephens R. S.
Published: October 2006

Chlamydia trachomatis elementary body (EB) and reticulate body (RB) developmental stages have polymorphic plasmid DNA. Several plasmid forms separated by gel electrophoresis were identified as topoisomers by treatment with topoisomerase I. Among these topoisomers was one form unique to EBs and one form unique to RBs. The unique EB plasmid topoisomer was characterized as highly supercoiled, on the basis of band migrations by gel electrophoresis and its appearance by electron microscopy. The unusual physical state of this topoisomer was probably mediated, in part, by DNA‐specific structural proteins. The unique RB plasmid topoisomer was a supercoiled form of lower superhelical density than the other identified topoisomers. Developmental‐stage‐specific differences in superhelical density of plasmid DNA suggest cause‐and effect relationships between DNA topology and metabolic activity in RBs and metabolic quiescence in EBs.
Promoter‐upstream activator sequences are required for expression of the xylS gene and upper‐pathway operon on the Pseudomonas TOL plasmid
Authors: Holtel A. , Abril M.‐A. , Marques S. , Timmis K. N. , Ramos J. L.
Published: October 2006

Stimulation of transcription from the Pseudomonas TOL plasmid xylS gene promoter (Ps) and the upperpathway operon promoter (Pu) is dependent on the positive regulator protein XylR activated by an effector molecule such as 3‐cholorotoluene, and on RpoN, an RNA polymerase sigma factor. Mutational analysis of the Ps and Pu promoters showed that upstream activator sequences located between ‐110 and ‐218bp upstream of the main transcription initiation point are required for regulated expression from these promoters. A search for homologous nucleotide sequences in the ‐110to ‐218bp region in Pu and Ps revealed conserved sequences that may act as putative recognition sequences for the XylR protein. Ps and Pu exhibit another well‐conserved region at around 50 bp, which is homologous to corresponding sites in other RpoN‐dependent promoters and may constitute a binding site for integration host factor (IHF).
Penicillin‐binding protein 4 of Escherichia coli: molecular cloning of the dacB gene, controlled overexpression, and alterations in murein composition
Authors: Korat B. , Mottl H. , Keck W.
Published: October 2006

The penicillin‐binding protein 4 (PBP4), from Escherichia coli, a DD‐carboxypeptidase/DD‐endopeptidase, was purified in an enzymatically active form to homogeneity by affinity chromatography on 6‐aminopeni‐cillanic acid/Sepharose and heparin/Sepharose. Polyclonal antibodies raised against the pure protein were used to identify and isolate PBP4 overproducing clones from an E. coli expression library, which was established on the basis of a temperature‐inducible runaway replication plasmid. Three positive clones were isolated, one of which carried the intact structural gene dacB that codes for PBP4, on a 1.9kb Smal‐Eco RI fragment, whereas the other two carried truncated forms of this gene. The direction of transcription was determined. The PBP4 overproducing strain, when grown in rich medium, tolerated 160‐fold overexpression. After disrupting cells by sonication, the majority (80%) of the overproduced PBP4 was detected in the 100000 x g supernatant. Southern blotting analysis using the cloned dacB gene as a probe revealed that, in contrast to that described by Takeda et al. (1981), the plasmid pLC18–38 of the Clarke‐Carbon collection does not code for PBP4. The overall composition of murein, synthesized in vitro or in vivo by the PBP4 overproducing strain, as determined by high‐performance liquid chromatography analysis, suggests that PBP4 is not involved in transpeptidation but exclusively catalyses a DD‐carboxypeptidase and DD‐endopeptidase reaction.
The IgA‐binding β antigen of the c protein complex of Group B streptococci: sequence determination of its gene and detection of two binding regions
Authors: Jerlström P. G. , Chhatwal G. S. , Timrnis K. N.
Published: October 2006

The β antigen of the Ibc protein complex of Group B streptococci is a cell‐surface receptor which binds the Fc region of human immunoglobulin A(IgA). Determination of the nucleotide sequence of the β antigen gene shows that it encodes a preprotein having a molecular weight of 130963 daltons and a polypeptide of 1164 amino acid residues that is typical of other Gram‐positive cell‐wall proteins. There is a long signal sequence of 37 amino acids at the N‐terminus. Four of the five C‐terminal amino acid residues are basic and are preceded by a hydrophobic stretch that appears to anchor the C‐terminus in the cell membrane. To the N‐terminal side of this hydrophobic stretch is a putative cell‐wall‐spanning region containing proline‐rich repeated sequences. An unusual feature of these repeated sequences is a three‐residue periodicity, whereby every first residue is a proline, the second residue is alternating positively or negatively charged, and the third residue is uncharged. The IgA‐binding activity was approximately localized by expressing subfragments of the β antigen as fusion proteins. Two distinct but adjacent DNA segments specified pep‐tides that bound IgA, which indicates that the IgA‐binding activity is located in two distinct regions of the protein.
Origin of tetracycline efflux proteins: conclusions from nucleotide sequence analysis
Authors: Sheridan R.P. , Chopra I.
Published: October 2006

The sequences of six tetracycline efflux proteins and three transport proteins which have some resembalance to them were compared. The tetracycline efflux proteins fall into three families: (i) those encoded by pBR322, RP1, and Tn 10 (Escherichia coli); (ii) pT181 (Staphyloccoccus aureus) and pTHT15 (Bacillus subtilis); and (iii) tet347 (Streptomyces rimosus). There is global sequence homology within each of the first two families, but there is none between the families. The pT181/pTHT15 family shares close homology with the N‐terminal half of the methlenomycin A efflux protein (Streptomyces coelicor), while tet347 ressembles the C‐terminal half. Portions of the N‐terminal half of the Tn10‐encoded protein show significant resemblance to portions in the N‐terminal half of the pT181/pTHT15 family, but this sometimes occurs among transport proteins which do not have a common subtrate. Tetracycline efflux proteins, therefore, appear to have arisen on atleast two, or possibly three, separate occasions, probably from other transport proteins.
Intranasal immunization using the B subunit of the Escherichia coli heat‐labile toxin fused to an epitope of the Bordetella pertussis P.69 antigen
Authors: Ljpscombe M. , Charles I. G. , Roberts M. , Dougan G. , Tite J. , Fairweather N. F.
Published: October 2006

The plasmid pBRD026, which directs expression of the B subunrt of the Escherichia coli heat‐labile toxin (LTB), was modified so that DNA encoding epitopes could be inserted at the 3 end of the gene. An oligonucleotide linker containing restriction sites for BglH and Spel was inserted at the Spel site at the 3′ end of the LTB gene to form plasmid pFV1. This linker also encodes the amino acid sequence Gly–Pro–Gly–Pro which we propose acts as a ‘hinge’ between the LTB and the foreign epitope. Oligonucleotides specifying an epitope from the Bordetelia pertussis P.69 outer membrane protein were cloned into pFV1 to form pFV169. The resultant fusion protein (LTB69) was partially purified from the periplasm of E. coli strains in a soluble pentameric form which could bind GM1 gangliosides. Mice immunized intranasally with purified LTB69 produced antibodies against both LTB and the P.69 protein. In addition, ELISPOT assays demonstrated the presence of LTB‐specific and P.69‐specific antibody‐secreting cells in the lungs of immunized mice.
A new system for amplifying 2 μm plasmid copy number in Saccharomyces cerevisiae
Authors: Unternährer S. , Pridmore D. , Hinnen A.
Published: October 2006

The yeast 2 μm plasmid is found in the nucleus of almost all Saccharomyces cerevisiae strains. Its replication is very similar to that of chromosomal DNA. Although the plasmid does not encode essential genes it is stably maintained in the yeast population and exhibits only a small, though detectable, loss rate. This stability is achieved by a plasmid‐encoded copy‐number control system which ensures constant plasmid levels.

For the investigation of 2μm replication, a yeast strain that is absolutely dependent on this plasmid was constructed. This was achieved by disruption of the chromosomal CDC9 gene, coding for DNA ligase and providing this essential gene on a 2μm‐derived plasmid. This plasmid is absolutely stable under all growth conditions tested. Using the temperature‐sensitive mutant allele cdc9‐1 we have developed an artificial control system which allows one to change the copy number of 2μm‐derived plasmids solely by changing the incubation temperature.
Cloning and expression of the rfe–rff gene cluster of Escherichia coli
Authors: Ohta M. , Ina K. , Kusuzaki K. , Kido N. , Arakawa Y. , Kato N.
Published: October 2006

We have cloned a 13 kb Escherichia coli DNA fragment which complemented the rfe mutation to recover the biosynthesis of E. coli O9 polysaccharide. Using Tn5 insertion inactivation, the rfe gene was localized at the 1.5kb Hin dIII–Eco RI region flanking the rho gene. We constructed an rfe‐deficient E. coli K‐12 mutant by site‐directed inactivation using a DNA fragment of the cloned 1.5 kb rfe gene. This also confirmed the presence of the rfe gene in the 1.5 kb region. By simultaneous introduction of both the rfe plasmid and the plasmid of our previously cloned E. coli O9 rfb into this rfe mutant, we succeeded in achieving in vivo rconstitution of O9 polysaccharide biosynthesis. From sequence analysis of the rfe gene, a putative promoter followed by an open reading frame (ORF) was identified downstream of the rho gene. This ORF coincided with the position of the rfe gene determined by Tn5 analysis and site‐directed mutagenesis.

Furthermore, we identified the rff genes in the 10.5kb DNA flanking the rfe gene. We recognized at least two functional domains on this cloned rff region. Region I complemented a newly found K‐12 rff mutant, A238, to synthesize the enterobacterial common antigen (ECA). Deletion of region II resulted in the synthesis of ECAs with shorter sugar chains. When the 10.5kb rff genes of the plasmid were inactivated by either deletion or Tn5 insertion, the plasmid lost its ability to give rise to transformants of the rfe mutants.
Cloning of the rfb gene cluster of a group C2 Salmonella strain: comparison with the rfb regions of groups B and D
Authors: Brown P. K. , Romans L. K. , Reeves P. R.
Published: October 2006

We report the cloning and mapping of the entire rfb gene cluster of a group C2 Salmonella strain. Comparison with the rfb region of group B strain LT2 and group D strain Ty2 reveals an 11.8kb central region of limited similarity flanked by regions of high similarity. The genes from the central region confer a group C2 O‐antigen structure on a Salmonella LT2 partial delete strain. The significance of this region in relation to function and evolutionary origin is discussed. We also report evidence for the existence of an O‐antigen chain‐length determinant in Escherichia coli K12 and propose a model for a possible mechanism by which a preferred chain length is determined.
Closely linked genetic loci required for swarm cell differentiation and multicellular migration by Proteus mirabilis
Authors: Allison C. , Hughes C.
Published: October 2006

The pathogenic bacterium Proteus mirabilis exhibits a form of multicellular behaviour called swarming migration. This involves the differentiation of vegetative cells at the colony margin into swarm cells which are long, aseptate, multinucleate, hyper‐flagellated filaments able to undergo repeated cycles of co‐ordinated population migration and consolidation (reversion to vegetative cells). Transposon mutagenesis of uropathogenic P. mirabilis strain U6450 with Tn5 generated 4860 chromosomal insertions and, of these, 75 (1.6%) caused visibly abnormal swarming behaviour, indicating that at least 45 genes are involved in directing motility, cell differentiation and multicellular behaviour. While about one fifth of the swarm‐defective mutants lacked flagella and were non‐motile non‐swarming (NMNS) the majority were normally flagellated and motile but were unable to form swarm cells (motile non‐swarming, MNS), or were motile and able to form swarm cells but displayed abberant patterns of multicellular migration (dendritic swarming, DS) or consolidation (frequent and infrequent consolidation, FC and IC). Restriction enzyme mapping of representative mutant DNAs by Southern hybridization with transposon DNA probes identified eight different mutated genetic loci within the five phenotypic classes. Subsequent Southern analysis of large restriction fragments separated by pulsed‐field electrophoresis showed that these eight mutated loci required for motility, cell differentiation and multicellular migration were clustered on a region of DNA spanning approximately 8% of the 4.2mbp P. mirabilis chromosome. Further linkage analysis showed that the DS locus involved in the ordered migration of the swarm cell population mapped separately from two main clusters of swarm loci, one cluster containing, within 112kbp, genetic determinants of motility (NMNS) and also differentiation into swarm cells (MNS1, MNS2), and second within aneighbouring 95kbp DNA sequence containing three loci involved in the control of consolidation (FC, IC1, IC2).
Pseudomonas aeruginosa LasA: a second elastase under the transcriptional control of lasR
Authors: Toder D. S. , Gambello M. J. , Iglewski B. H.
Published: October 2006

The full elastolytic phenotype of Pseudomonas aeruginosa requires lasB, the structural gene for elastase, its transcriptional activator lasR, and lasA. The lasB gene was insertionally inactivated with the omega fragment and this mutated gene introduced into the P. aeruginosa chromosome. Replacement of the wild‐type gene with the inactivated gene was verified by Southern analysis and confirmed by lack of elastase antigen on Western blots and lack of activity in liquid assays. The mutant did, however, retain elastolytic activity on elastin plates. This residual activity was abolished by inactivation of lasB in PAO‐E64, a lasA‐deficient mutant, demonstrating that it was due to the lasA gene product. Northern analysis demonstrated that, like lasB, lasA is transcriptionally controlled by the lasR gene product.
Transcription of the Vibrio cholerae haemolysin gene, hlyA, and cloning of a positive regulatory locus, hlyU
Authors: Williams S. G. , Manning P. A.
Published: October 2006

Transcription of the Vibrio cholerae hlyA gene, which encodes a cytotoxic haemolysin, has been investigated. The hlyA transcript initiates 430 nucleotides (nt) upstream of the translational start site, hlyA–cat transcriptional fusion constructs were active in V. cholerae but not in Escherichia coli. An hlyA–cat fusion was used to select, from a V. cholerae O17 plasmid library, a clone that could activate the hlyA promoter in E coli. This regulatory locus has been designated hlyU. hlyU appears to be distinct from the previously described hlyR locus.
A recombinant C‐terminal toxin fragment provides evidence that membrane insertion is important for Clostridium perfringens enterotoxin cytotoxicity
Authors: Hanna P. C. , McClane B. A.
Published: October 2006

Clostridium perfringens enterotoxin (CPE) is believed to be involved in several important gastrointestinal illnesses. Recent studies have identified a number of distinct molecular events which occur after CPE treatment of eukaryotic cells or isolated membranes. Additional studies are underway to determine the temporal order and intrinsic importance of each CPE event for cytotoxicity. We now demonstrate that a truncated CPE fragment binds to membranes, but is unable to Insert into membranes or cause any other subsequent post‐insertion event. This is the first experimental evidence supporting the importance of membrane insertion for CPE cytotoxicity. Binding of the CPE fragment is also shown to be irreversible, strongly suggesting that the irreversible binding of wild‐type CPE is not due solely to insertion of CPE into membranes.
ymoA, a Yersinia enterocolitica chromosomal gene modulating the expression of virulence functions
Authors: Cornells G. R. , Sluiters C. , Delor I. , Geib D. , Kaniga K. , de Rouvroit C. Lambert , Sory M.‐P. , Vanooteghem J.‐C. , Michiels T.
Published: October 2006

The virulence functions of Yersinia enterocolitica include the pYV‐encoded Yop proteins and YadA adhesin as well as the chromosome‐encoded enterotoxin, Yst. The yop and yadA genes form a temperature‐activated regulon controlled by the transcriptional activator VirF. Gene virF, also localized on pYV, is itself thermoinduced in the absence of other pYV genes. The enterotoxin ysf gene is silent in some collection strains including strain W22703. This paper describes two Tn5‐Tc1 chromosomal insertion mutants of W22703 transcribing virF, and hence the yop and yadA genes, at low temperature. These mutants also resumed their production of Yst, with its typical temperature dependence. Both mutations were insertions in the same gene called ymoA for‘Yersinia modulator’The cloned ymoA gene fully complemented the two mutations. Several properties of the mutants suggest that ymoA encodes a histone‐like protein. According to the nucleic acid sequence, the product of ymoA is an 8064 Da protein rich in aspartic acid (9%), glutamic acid (9%) and lysine (10.5%), but the predicted amino acid sequence shows no similarity with any described histone‐like protein. This work supports recent reports which propose a role for DNA topology and bacterial chromatin structure in thermoregulation of virulence functions.
A pyruvate‐stimulated adenylate cyclase has a sequence related to the fes/fps oncogenes and to eukaryotic cyclases
Authors: Peters E. P. , Wilderspin A. F. , Wood S. P. , Zvelebil M. J. J. M. , Sezer O. , Danchin A.
Published: October 2006

The pyruvate‐stimulated adenylate cyclase from Brevibacterium liquefaciens produces up to 450μM cyclic AMP in the culture medium when the bacterium is grown on glucose and alanine. In this paper we report the cloning, expression and sequencing of the gene for this enzyme. Residues were identified, within the C‐terminal domain, which are conserved in adenylate and guanylate cyclase sequences from eukaryotes and in the adenylate cyclase of the prokaryote Rhizobium meliloti. We have also identified a sequence of 30 residues near the N‐terminus of the protein which is homologous to part of the regulatory domain of the cellular homologues of the oncogenes fes and fps; this sequence is also present in the avian Fujinami sarcoma virus fps gene.
Escherichia coli chromosomal mutations that permit direct cloning of the Bacteroides fragiiis metallo‐β‐lactamase gene, ccrA
Authors: Rasmussen B. A. , Gluzman Y. , Tally F. P.
Published: October 2006

The class B, metallo‐β‐lactamase genes ccrA (carbapenem‐ and cephamycin resistance) from three Bacteroides fragilis isolates — QMCN3, QMCN4, and TAL3636 — were cloned and expressed in Escherichia coli. Cloning of the genes, by selecting for ampicillin resistance, was facilitated by two classes of Escherichia coli chromosomal mutations which resulted in at least a 5‐10‐fold increase in metallo‐β‐lactamase enzymatic activity. The observed increase in enzymatic activity is due to either increased translation of the ccrA gene or an effect on localization or stability of the protein. Comparison of the DNA sequences of the three ccrA genes revealed that their protein‐coding sequences shared greater than 97% DNA sequence identity. However, the 5’upstream sequence for the TAL3636 ccrA gene was unrelated to that of the other two genes.
Evidence for a common molecular origin of the capsule gene loci in Gram‐negative bacteria expressing group II capsular polysaccharides
Authors: Frosch M. , Edwards U. , Bousset K. , Krauße B. , Weisgerber C.
Published: October 2006

Capsular polysaccharides of Gram‐negative bacteria contribute to a large extent to the pathogenicity of these organisms. We show here that the molecular organization of the capsule gene loci in different serogroups of Neisseria meningitidis is similar to that of Haemophilus influenzae and Escherichia coli. A common molecular origin of the mechanisms of encapsulation is indicated by strong homology of the genes involved in transport of the capsular polysaccharides to the cell surface in all these organisms. The proteins involved in capsular polysaccharide transport fit the characteristics of ABC (ATP‐binding cassette) transporters. Furthermore, by sequence comparison of the sialyltransferases of N. meningitidis B and E. coli K1, the capsule of which is composed of α2,8‐linked polyneuraminic acid, a significant degree of homology was observed, indicating that the capsular polysaccharide type itself has the same evolutionary origin in these two pathogens.
The hha gene modulates haemolysin expression in Escherichia coli
Authors: Nieto J. M. , Carmona M. , Bolland S. , Jubete Y. , de la Cruz F. , Juárez A.
Published: October 2006

A mutation in the hha allele results in a large increase in the production of intracellular as well as extracellular haemolysin in Escherichia coli cells harbouring the haemolytic recombinant plasmid pANN202‐312. This single gene mutation was located between 490 and 491.6kb on the physical map of the E. coli chromosome. From the DNA sequence of hha a small polypeptide of 8629 Da was predicted and was expressed in minicells. The deduced polypeptide sequence did not show significant similarities to other characterized proteins related to the regulation of gene expression in E. coli, although it was shown that the hha mutation increases cyloplasmic synthesis of haemolysin.
TyrR protein of Escherichia coli and its role as repressor and activator
Authors: Pittard A. J. , Davidson B. E.
Published: October 2006

The TyrR protein regulates the expression of eight transcriptional units that comprise the TyrR regulon. In all but one case, regulation is by repression, while in two cases activation of expression can occur. Notwithstanding the fact that the TyrR protein contains an ATP‐binding domain and a helix‐turn‐helix DNA‐binding domain which are structurally homologous to domains of similar functions in proteins such as NifA, NtrC, DctD and XylR, it differs from them in a number of respects. It is not a part of a two‐protein component system and it lacks the amino‐terminal domain that is present on NtrC and DctD. It activates transcription from‘Eσ70’promoters but not from‘Eσ54’promoters. ATP binding seems to be essential for tyrosine‐mediated repression but not for activation. In addition, the activity of the TyrR protein is modulated by the binding of one or more of the aromatic amino acids. The consensus sequence for TyrR‐binding sites in DNA, referred to as TyrR boxes, is TGTAAAN6TTTACA. Tyrosine‐mediated repression occurs at operators containing a pair of adjacent boxes. These have unequal affinities for the TyrR protein. The box that overlaps the RNA polymerase binding site is only bound by TyrR in the presence of both ATP and tyrosine, and binding appears to involve co‐operativity between two TyrR protein dimers.

In contrast, activation of expression by TyrR appears to require phenylalanine but not ATP. It occurs as a consequence of the binding of TyrR protein to a single upstream TyrR box, and appears to involve interactions between the bound TyrR molecule and a molecule of RNA polymerase bound to the promoter. The spacing between the box and the promoter is critical for effective activation.
Substitutions at a single amino acid residue in the nitrogen‐regulated activator protein NTRC differentially influence its activity in response to phosphorylation
Authors: Dixon R. , Eydmann T. , Henderson N. , Austin S.
Published: October 2006

Four substitutions at serine residue 160 which increase the activity of the σ54‐dependent activator protein NTRC in the absence of NTRB have been analysed in detail. Mutagenesis of the putative phosphoacceptor site of NTRC and analysis of double mutants indicate that the positive control function of the S160W and S160C mutants is phosphorylation‐dependent, whereas the activity of the S160Y and S160F mutants is phosphorylation‐independent. This was confirmed with two purified mutant proteins in vitro. Occupancy of tandem NTRC‐binding sites upstream of the Klebsiella pneumoniae nifL promoter by S160W protein is also phosphorylation‐dependent in contrast to occupancy by S160F protein, confirming that both the DNA‐binding and activator functions of NTRC are influenced by phosphorylation. The S160W and S160C mutants are apparently more responsive than wild‐type protein to ‘cross‐talk’ by other members of the histidine protein kinase family but are less responsive to phosphorylation and dephosphorylation mediated by NTRB.
Characterization of the oriT region of the IncFV plasmid pED208
Authors: Di Laurenzio L. , Frost L. S. , Finlay B. B. , Paranchych W.
Published: October 2006

DNA sequence analysis of a 2.2kb EcoRI‐Hin dIII fragment from pED208, the derepressed form of the IncFV plasmid Folac, revealed sequences highly homologous to the oriT region, traM, and traJ genes of other IncF plasm ids. The TraM protein was purified and immunoblots of fractionated cells containing pED208 or Folac showed that TraM was predominantly in the cytoplasm. Using DNA retardation assays and the DNase I footprinting technique, the TraM protein was found to bind to three large motifs in the oriT region: (I) an inverted repeat, (II) two direct repeats, and (III) the traM promoter region. These three footprint regions contained a HinfI‐like sequence (GANTC) that appeared 16 times, spaced 11–12bp (or multiples thereof) apart, suggesting that TraM protein binds in a complex manner over this entire region.
Site‐directed mutagenesis at histidines of aerolysin from Aeromonas hydrophila: a lipid planar bilayer study
Authors: Wilmsen H. U. , Buckley J. T. , Pattus F.
Published: October 2006

The role of histidine residues in the formation of channels by the cytolytic toxin aerolysin has been studied in planar lipid bilayers by substituting each of the six histidines in the native protein with asparagine. His341 or His186 mutants had the same channel‐forming ability as native toxin, whereas the His332 and His121 mutants were less active. Mutations at His132 and His107, which interfere with the oligomerization of the toxin, drastically reduce pore formation. These findings support the conclusion that oligomerization of the toxin must precede channel formation, and that at least two of the six histidine residues are essential for this to occur. The aerolysin channel is a water‐filled pore with an approximate diameter of 9.3 ± 0.4 Å.
Three in‐frame N‐terminally different proteins are produced from the repressor locus of the Streptomyces bacteriophage φC31
Authors: Smith M. C. M. , Owen C. E.
Published: October 2006

The sequence of the repressor locus, c, of the Streptomyces temperate phage, φC31, was shown previously to contain an open reading frame encoding a 74 kDa protein. Further analysis of the transcriptional and translational products of the c gene shows a more complex pattern of expression. A nest of three in‐frame N‐terminally different, C‐terminally identical proteins of 74, 54 and 42 kDa were found to be expressed from a corresponding nest of transcripts. The repressor proteins were produced in Escherichia coli and the 42 kDa protein was purified, verified by N‐terminal sequencing, and used to raise antibody. The antibody cross‐reacted in Western blots with the 74, 54 and 42 kDa proteins expressed in E. coli and Streptomyces lividans and from Streptomyces coelicolorφC31 lysogens. Analysis of transcription of the c gene by S1 mapping and primer extension showed that the nest of transcripts encoding the repressor protein were induced after heat treatment of the cts locus (Sinclair and Bibb, 1989; this paper). Correspondingly, all three of the repressor proteins were induced. In addition to a promoter, cp1, which lies upstream of the 74 kDa open reading frame, the c locus contained at least one internal promoter, cp2, which transcribes DNA encoding the 54 and 42 kDa proteins. Transcripts initiating from cp3 were observed in RNA preparations from S. lividans containing the c gene deleted for cp1 and cp2, but gene fusions using DNA which should contain any putative promoting activity from this region transcriptionally fused to the xylE gene showed very low levels of expression of catechol 2,3 dioxygenase in S. lividans. The 74 kDa protein was not necessary for super‐infection immunity. Data described here and current knowledge of the nature of other‘dual start’genes suggest a model for the regulation of lysis versus lysogeny in φC31.
Identification of Treponema pallidum subspecies pallidum genes encoding signal peptides and membrane‐spanning sequences using a novel alkaline phosphatase expression vector
Authors: Blanco D. R. , Giladi M. , Champion C. I. , Haake D. A. , Chikami G. K. , Miller J. N. , Lovett M. A.
Published: October 2006

Treponema pallidum subspecies pallidum is a pathogenic spirochaete for which there are no systems of genetic exchange. In order to provide a system for the identification of T. pallidum surface proteins and potential virulence factors, we have developed a novel expression vector which confers the utility of TnphoA transposition. The relevant features of this plasmid vector, termed pMG, include an inducible tac promoter, a polylinker with multiple cloning sites in three reading frames, and an alkaline phosphatase (AP) gene lacking the signal sequence‐encoding region. Library construction with Sau3A‐ digested T. pallidum genomic DNA resulted in the creation of functional T. pallidum‐AP fusion proteins. Analysis of fusion proteins and their corresponding DNA and deduced amino acid sequences demonstrated that they could be grouped into three categories: (i) those with signal peptides containing leader peptidase I cleavage sites, (ii) those with signal peptides containing leader peptidase II cleavage sites, and (iii) those with non‐cleavable hydrophobic membrane‐spanning sequences. Triton X‐114 detergent phase partitioning of individual T. pallidum‐AP fusions revealed several clones whose AP activity partitioned preferentially into the hydrophobic detergent phase. Several of these fusion proteins were subsequently shown to be acylated by Escherichia coli following [3H]‐palmitate labelling, indicating their lipoproteinaceous nature. DNA and amino acid sequence analysis of one acylated fusion protein, Tp75, confirmed the presence of a hydrophobic N‐terminal signal sequence containing a consensus
Environmentally regulated algD promoter is responsive to the cAMP receptor protein in Escherichia coli
Authors: DeVault J. D. , Hendrickson W. , Kato J. , Chakrabarty A. M.
Published: October 2006

The environmentally activated algD promoter of Pseudomonas aeruginosa has been shown to be influenced by DNA supercoiling. It is believed that protein‐induced bending or looping is required for this activation. We studied the role of Escherichia coli cAMP‐CRP on algD promoter activation in E. coli and show that a functional CRP is required for this activation. We also demonstrate that the algD promoter is sensitive to glucose repression both in E. coli and P. aeruginosa. Deletion of a putative consensus CRP binding sequence upstream of the algD promoter renders the promoter non‐responsive to glucose repression. The involvement of c AM P‐CRP complex in the activation of the afgD promoter in E. coU has been demonstrated directly through binding of a 255 base pair DNA fragment containing the putative consensus CRP binding sequence. Other fragments, upstream or downstream but without any consensus CRP binding sequence, did not show any binding with CRP. A CRP‐like analogue, similar to that in Xanthomonas campestris, but capable of activating genes without forming a complex with cAMP, is believed to allow glucose repression in P. aeruginosa.
The hydrogenase structural operon in Rhodobacter capsulatus contains a third gene, hupM, necessary for the formation of a physiologically competent hydrogenase
Authors: Cauvin B. , Colbeau A. , Vignais P. M.
Published: October 2006

The hupM gene, previously called ORFX, found downstream from and contiguous with the structural hydrogenase genes hupS and hupL in Rhodobacter capsulatus, is shown here to form a single hupSLM transcription unit with the two other genes. The hupM gene was Inactivated by interposon mutagenesis. The two selected mutants, BCX1 and BCX2, which contained the kanamycin‐resistance gene in opposite orientation, still exhibited hydrogenase activity when assayed with the artificial electron acceptors benzyl‐viologen and methylene blue. However, the hydrogenase was not physiologically active in these mutants, which could not grow autotrophically and were unable to recycle electrons to nitrogenase or to respire on H2. The hupM gene starts nine base pairs downstream from the TGA stop codon of hupL gene, which encodes the large subunit of the [NiFe]hydrogenase of Rhodobacter capsulatus. The three contiguous genes hupS, hupL and hupM were subcloned downstream from the promoter of hupSL, either with the promoter in the correct orientation (plasmid pBC8) or with the promoter in the opposite orientation (plasmid pBC9), then the constructs were introduced into the mutant strains. Only plasmid pBC8 could restore the formation of a competent hydrogenase in mutants BCX1 and BCX2, indicating that the hupM gene is expressed only from the hupSL promoter.
Construction of a physical map of the chromosome of Shigella flexneri 2a and the direct assignment of nine virulence‐associated loci identified by Tn5 insertions
Authors: Okada N. , Sasakawa C. , Tobe T. , Talukder K. A. , Komatsu K. , Yoshikawa M.
Published: October 2006

To establish the molecular basis of the chromosomal virulence genes of Shigella flexneri 2a (YSH6000), a Notl restriction map of the chromosome was constructed by exploiting Notl‐linking clones, partial Notl digestion and DNA probes from various genes of Escherichia coli K‐12. The map revealed at least three local differences in the placements of genes between YSH6000 and E. coli K‐12. Using the additional Notl sites introduced by Tn5 insertion, nine virulence loci Identified previously by random Tn5 insertions were physically mapped on the chromosome. To demonstrate the versatility of the Notl map in direct assignment of the virulence loci tagged by Tn5 to a known genetic region in E. coli K‐12, the major class of avirulent mutants defective in the core structure of lipopolysaccharide (LPS) was examined for the sites of Tn5 insertions. The two Notl segments created by the Tn5 insertion in the Notl fragment were analysed by Southern blotting with two DNA probes for the 5′ and 3’flanking regions of the rfa region, and shown to hybridize separately with each of them, confirming the sites of Tn5 in the rfa locus. This approach will facilitate direct comparison of genetically mapped Tn5 insertion mutations of S. flexneri with genes physically determined in E. coli K‐12.
Separate promoters direct expression of phoAIII, a member of the Bacillus subtilis alkaline phosphatase multigene family, during phosphate starvation and sporulation
Authors: Chesnut R. S. , Bookstein C. , Hulett F. M.
Published: October 2006

Alkaline phosphatase (APase) expression can be induced in Bacillus subtilis by phosphate starvation or by sporulation. We have recently shown that there are multiple APase structural genes contributing to the total alkaline phosphatase expression in B. subtilis. The expression of the alkaline phosphatase III gene (phoAIII) was analysed under both phosphate‐starvation induction and sporulation induction conditions. phoAIII is transcribed from two promoter regions, Pv and Ps The Pv promoter initiated transcription 37 bp before the translation initiation codon and was used to transcribe phoAIII during phosphate‐starvation induction in vegetative cells. The Ps promoter initiated transcription 119 bp before the translation initiation codon and was used during sporulation induction. Genes which have previously been shown to affect total vegetative APase, pho regulon genes phoP, phoR and phoS, affected expression of phoAIII during phosphate starvation. Genes known to affect expression of total sporulation APase, i.e. spoIIE, spoIIG and spoIIE, affected phoAIII expression during sporulation induction. Our data show that one member of the APase multigene family, phoAIII, contributes to the total APase expression both during phosphate‐starvation induction and sporulation induction, and that the mechanism of regulation includes two promoters, each requiring different regulatory genes.
Horizontal transfer of multiple penicillin‐binding protein genes, and capsular biosynthetic genes, in natural populations of Streptococcus pneumoniae
Authors: Coffey T. J. , Dowson C. G. , Daniels M. , Zhou J. , Martin C. , Spratt B. G. , Musser J. M.
Published: October 2006

Multiply antibiotic‐resistant serotype 23F isolates of Streptococcus pneumoniae are prevalent in Spain and have also been recovered recently in the United Kingdom and the United States. Analysis of populations of these isolates by multilocus enzyme electrophoresis, and restriction endonuclease cleavage electrophoretic profiling of penicillin‐binding protein (PBP) genes, has demonstrated that these isolates are a single clone (Muñoz et al., 1991). Here we report studies of non‐serotype 23F penicillin‐resistant pneumococci isolated in Spain and the United Kingdom. One of the isolates expressed serotype 19 capsule but was otherwise indistinguishable from the serotype 23F clone on the basis of multilocus enzyme electrophoresis, antibiotic resistance profiling, and restriction endonuclease patterns of genes encoding PBP1A, PBP2B and PBP2X, a result which suggests that horizontal transfer of capsular biosynthesis genes had occurred. These same techniques revealed that six other resistant isolates, all expressing serotype 9 polysaccharide capsule, represent a clone. Interestingly, the chromosomal lineage of this clone is not closely related to the 23F clone; however, the serotype 9 and 23F clones harbour apparently identical PBP1 A, ‐2B and ‐2X genes. To explain these data, we favour the interpretation that horizontal gene transfer in natural populations has distributed genes encoding altered forms of PBP1A, ‐2B and ‐2X to distinct evolutionary lineages of S. pneumoniae.
Pullulanase secretion in Escherichia coli K‐12 requires a cytoplasmic protein and a putative polytopic cytoplasmic membrane protein
Authors: Possot O. , D'Enfert C. , Reyss I. , Pugsley A. P.
Published: October 2006

The previously uncharacterized third and fourth genes (pulE and pulF) of the pullulanase secretion gene operon of Klebsiella oxytoca strain UNF5023 are, respectively, predicted to encode a 55 kDa polypeptide with a putative nucleotide‐binding site, and a highly hydrophobic 44 kDa polypeptide that probably spans the cytoplasmic membrane several times. Expression of pulE in minicells or under the control of a strong bacteriophage T7 promoter resulted in the production of a c. 58 kDa cytopLasmic protein. A representative PulE‐β‐galactosidase hybrid protein created by Tnlac mutagenesis was also found mainly in the cytoplasm. These results are in line with the predicted absence from PulE of a region of sufficient hydrophobicity to function as a signal sequence. The PulF polypeptide could not be detected either in minicells or when the gene was transcribed from the T7 promoter, but the acquirement of three pulF‐lacZ gene fusions that encoded hybrid proteins with relatively high levels of β‐galactosidase activity indicates that this gene can be transcribed and translated. Gene disruption experiments indicated that both pulE and pulF are required for pullulanase secretion in Escherichia coli K‐12. Both proteins exhibit considerable homology throughout their entire lengths with other proteins involved in protein secretion, pilin assembly, conjugation and transformation competence in a variety of bacteria. In addition, PulE protein has consensus sequences found in a wide variety of nucleotide‐binding proteins. This study completes the initial characterization of the pullulanase secretion gene operon, which comprises 13 genes that are all essential for the transport of pullulanase across the outer membrane.
Cytotoxic activity of a recombinant chimaeric protein between Pseudomonas aeruginosa exotoxin A and Corynebacterium diphtheriae diphtheria toxin
Authors: Guidi‐Rontani Chantal
Published: October 2006

A segment of the exotoxin A gene of Pseudomonas aeruginosa, coding for the N‐terminal end of domain I and domain II of the toxin (ETA), was genetically fused to the diphtheria toxin gene of Corynebacterium diphtheriae, coding for the N‐terminal end of A fragment of diphtheria toxin (DT). The resulting hybrid protein (termed CED1) was produced in large amounts and exported to the periplasm in Escherichia coli. This chimaeric protein reacted with both anti‐ETA and anti‐DT antisera. Furthermore, the chimaeric protein displayed ADP‐ribosylation activity and exhibited cytotoxicity to mouse 3T6 fibroblasts. These results demonstrated that the chimaeric protein is cytotoxic, and that the toxic potential of DTA can be selectively internalized and translocated via domains I and II of exotoxin A, which are thus sufficient to direct and translocate an enzymatically active heterologous polypeptide segment into the cytosol of sensitive cells.
Transcriptional control of sex‐pheromone‐inducible genes on plasmid pAD1 of Enterococcus faecalis and sequence analysis of a third structural gene for (pPD1 ‐encoded) aggregation substance
Authors: Galli D. , Friesenegger A. , Wirth R.
Published: October 2006

The expression of several neighbouring genes on plasmid pAD1 that are necessary for conjugation depend on induction with sex pheromone CAD1. Analyses of transcripts by Northern blot hybridization demonstrated that the genes seal (encoding surface exclusion protein) and asa 1 (encoding aggregation substance) are transcribed independently. Both genes are organized in different operons together with neighbouring open reading frames of unknown function. Several transcripts could be identified for seal and asa1. Their transcriptional start sites were determined by primer extension experiments, confirming the results of the Northern blot experiments. We also could identify seal‐ and iad‐ (encoding an inhibitory peptide counteracting sex pheromone CAD1) specific transcripts which are expressed constitutively, but to a lower extent relative to induced conditions. In addition, we localized the asp1 gene coding for aggregation substance of sex pheromone plasmid pPD1 and determined its DNA sequence, which was found to be highly homologous to asa1 (aggregation substance gene of pAD1) and prgB (aggregation substance gene of pCFiO). The structural genes were found to be organized more or less identically on the three sex‐pheromone plasmids pAD1, pCF10, and pPD1, and to be highly conserved. Regions supposed to be of crucial importance for regulatory functions, however, were found to differ. We also could identify some conserved DNA motifs which might be potential target sites for transcriptional regulators. In combination these data allowed us to formulate a model for the regulation of sex‐pheromone‐induclble genes of plasmid pAD1. Its main statement is that only in the presence of cAD1 can the gene traE1 be transcribed. The positive regulatory factor TraE1 then can trigger expression of the structural genes seal and asa1.