bluuebunny/crossref_metadata_2025_split huggingface dataset:
Induction of a φC31 prophage inhibits rRNA transcription in Streptomyces coelicolor A3(2)
Authors: Clayton T. M. , Bibb M. J. 
Published: October 2006

SummaryA lysogen of Streptomyces coelicolor A3(2) containing a thermoinducible mutant of the temperate phage φC31 (φ C31 cts1) was used to obtain synchronous phage development. Filter hybridization experiments indicated a marked reduction in rRNA synthesis after prophage induction. S1 nuclease mapping showed that transcription from each of the four promoters of one rRNA gene set (rrnD) was reduced to approximately the same extent, and that inhibition required protein synthesis. Crude preparations of RNA polymerase from induced lysogens had enhanced transcribing acitivity for φC31 DNA which was lost upon further purification. The purified preparations were unimpaired in their ability to transcribe from the rrnD promoters in vitro and apparently unchanged in poly‐peptide composition. The factor(s) responsible for stimulating phage transcription, and possibly for inhibiting rRNA synthesis, may have been separated from the enzyme during purification.
Characterization of the hlyB gene and its role in the production of the EI Tor haemolysin of Vibrio choierae O1
Authors: Alm R. A. , Manning P. A. 
Published: October 2006

SummaryEI Tor strains of Vibrio cholerae are capable of producing a haemolysin which they actively secrete into the growth medium. This requires translation to produce the protein at the surface of the cytoplasmic membrane and translocation across this membrane, the periplasmic space and the outer membrane. The mechanism by which this occurs is poorly understood.In addition to the structural gene for the haemolysin (hlyA), we have cloned a second adjacent gene, hlyB. By site‐directed mutagenesis, specific hlyB mutants have been constructed. These mutants are defective in the secretion of HlyA in the early to mid‐exponential phase of growth and the haemolysin becomes trapped within the cell and is only released in stationary phase. Nucleotide sequence analysis and cell fractionations reveal HlyB to be a 60.3 kD putative outer membrane‐associated protein.
Surface‐induced swarmer cell differentiation of Vibrio parahaemoiyticus
Authors: McCarter L. , Silverman M. 
Published: October 2006

Summary Vibrio parahaemolyticus distinguishes between life in a liquid environment and life on a surface. Growth on a surface induces differentiation from a swimmer cell to a swarmer cell type. Each cell type is adapted for locomotion under different circumstances. Swimmer cells synthesize a single polar flagellum (Fla) for movement in a liquid medium, and swarmer cells produce an additional distinct flagellar system, the lateral flagella (Laf), for movement across a solid substratum, called swarming. Recognition of surfaces is necessary for swarmer cell differentiation and involves detection of physical signals peculiar to that circumstance and subsequent transduction of information to affect expression of swarmer cell genes (laf). The polar flagellum functions as a tactile sensor controlling swarmer cell differentiation by sensing forces that restrict its movement. Surface recognition also involves a second signal, i.e. nutritional limitation for iron. Studying surface‐induced differentiation could reveal a novel mechanism of gene control and lead to an understanding of the processes of surface colonization by pathogens and other bacteria.
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

SummaryHybrid 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

SummaryThe 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

SummaryUntil 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

SummaryProgrammed 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

Summary 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

SummaryStimulation 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

SummaryThe 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

SummaryThe 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

SummaryThe 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

SummaryThe 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

SummaryThe 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

SummaryWe 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

SummaryWe 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

SummaryThe 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

SummaryThe 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

SummaryTranscription 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.