bluuebunny/crossref_metadata_2025 huggingface dataset:
Electrophoretic karyotype and gene assignment to resolved chromosomes of Trichoderma spp.
Authors: Herrera‐Estrella A. , Goldman G. H. , Van Montagu M. , Geremia R. A.
Published: October 2006

A molecular karyotype for three different Trichoderma species (T. harzianum, T. viride, and T. reesei) was determined by using two different systems: countour‐clamped electric‐field and rotating‐electrode electrophoresis. Six chromosomal DNA bands were observed in T. harzianum and T. reesei and five in T. viride. The sizes of these molecules were estimated by their mobility relative to the Schizosaccharomyces pombe chromosomes and ranged between 2.2 and 7.4 megabase pairs (mbp). The estimated genome sizes range from 31 to 39 mbp. A number of genes were located in the different chromosomes by means of Southern analysis. The implications of these findings are discussed.
The RpoS Sigma factor relieves H‐NS‐mediated transcriptional repression of csgA, the subunit gene of fibronectin‐binding curli in Escherichia coli
Authors: Olsén Arne , Arnqvist Anna , Hammar Måing;rten , Sukupolvi Soila , Normark Staffan
Published: October 2006

Curli encoded by the curlin subunit gene, csgA, are fibronectin‐ and laminin‐binding fibres expressed by many natural Escherichia coli and E. coli K‐12 strains in response to low temperature, low osmolarity and stationary‐phase growth conditions. Curli expression is dependent on RpoS, a sigma factor that controls many stationary phase‐inducible genes. Many commonly used K‐12 strains carry an amber mutation in rpoS. Strains able to form Curli carry an amber suppressor whereas curli‐negative E. coli K‐12 strains, in general, are sup°. Introduction of supD, supE, or supF suppressors into sup0 strains resulted in expression of temperature‐regulated curli. In curli‐deficient, RpoS−E. coli K‐12 strains, csgA is transcriptionally activated by mutations in hns, which encodes the histone‐like protein H‐NS. Curli expression, fibronectin binding, and csgA transcription remain temperature‐ and osmoregulated in such double mutants. Our data suggest that RpoS+ strains, and hence curli‐proficient strains of E. coli K‐12, are relieved for the transcriptional repression mediated by the H‐NS protein upon accumulating RpoS as cells reach stationary phase.
Regulation of pyelonephritis‐associated pili phase‐variation in Escherichia coli: binding of the Papl and the Lrp regulatory proteins is controlled by DNA methylation
Authors: Nou Xiangwu , Skinner Brett , Braaten Bruce , Blyn Lawrence , Hirsch Dwight , Low David
Published: October 2006

Expression of pyelonephritis‐associated pili (Pap) in Escherichia coli is under a phase‐variation control mechanism in which individual cells alternate between pili+ (ON) and pili− (OFF) states through a process involving DNA methylation by deoxyadenosine methylase (Dam). Methylation of two GATC sites (GATC1028 and GATC1130) within the pap regulatory region is differentially inhibited in phase ON and phase OFF cells. The GATC1028 site of phase ON cells is non‐methylated and the GATC1130 site is fully methylated. Conversely, in phase OFF cells the GATC1028 site is fully methylated whereas the GATC1130 site is non‐methylated. Two transcriptional activators, Papl and Lrp (leucine‐responsive regulatory protein), are required for this specific methylation inhibition. DNA footprint analysis using non‐methylated pap DNAs indicates that Lrp binds to a region surrounding the GATC1130 site, whereas Papl does not appear to bind to pap regulatory DNA. However, addition of Lrp and Papl together results in an additional DNasel footprint around the GATC1028 site. Moreover, Dam methylation inhibits binding of Lrp/Papl near the GATC1028 site and alters binding of Lrp at the GATC1130 site. Our results support a model in which Dam and Lrp/Papl compete for binding near the GATC1028 site, regulating the methylation state of this GATC site and, consequently, the pap transcription state.
Cloning and characterization of the bundle‐forming pilin gene of enteropathogenic Escherichia coli and its distribution in Salmonella serotypes
Authors: Sohel Indira , Puente Jose Luis , Murray William J. , Vuopio‐Varkila Jaana , Schoolnik Gary K.
Published: October 2006

bfp, the structural gene of the major repeating bundle‐forming pilus (BFP) subunit, was cloned from the enteroadherent factor (EAF) plasmid of enteropathogenic Escherichia coli (EPEC) strain B171 (0111:NM). The bfp open reading frame encoded a 193‐amlno‐acid protein; comparison of this sequence with the biochemically determined N‐terminal amino acid sequence showed that the mature pilin protein is comprised of 180 amino acids, that this sequence is similar to other members of the type IV pilin family, and that it is preceded by a 13‐amino‐acid signal peptide. Expression of the cloned bfp structural gene in an EPEC strain that had been cured of the EAF plasmid yielded a 21000 dalton protein that co‐migrated with the BFP precursor protein. Thus, other genes, probably carried by the EAF plasmid, are required for the maturation of the bfp product and for the production of extracellular pilus filaments. Use of bfp as a hybridization probe showed that homologous sequences are present in all tested EPEC strains and in 13 of 16 tested Salmonelia serotypes. Fifty per cent of these bfp probe‐sensitive salmonellae exhibited the localized‐adherence (LA) phenotype when incubated with tissue culture cell monolayers, a trait previously associated with EAF plasmid‐containing EPEC strains. Scanning electron micrographs of a bfp probe‐positive, LA‐positive Salmonella dublin strain showed that it grows as adherent colonies on infected monolayers and that within these colonies, BFP‐like fibres form inter‐bacterial linkages. For EAF plasmid‐containing EPEC strains and for severai Salmonella serotypes, BFP expression may lead to the development of adherent colonies on epithelial surfaces early in the infective process.
Identification and characterization of IS 1138, a transposable element from Mycoplasma pulmonis that belongs to the IS3 family
Authors: Bhugra B. , Dybvig K.
Published: October 2006

Insertion sequence (IS) elements are mobile genetic elements found in prokaryotes. We have identified a repetitive element from Mycoplasma pulmonis, a murine pathogen, that is similar to eubacterial IS elements. By subcloning a single strain of M. pulmonis, we isolated a variant clone in which the IS element had undergone an apparent transposition event. The nucleotide sequences of the element, designated IS 1138, and the target site into which it inserted were determined. IS1138 consists of 1288bp with 18bp perfect terminal inverted repeats. Sequence analysis of the target site before and after insertion of IS1138 identified a 3bp duplication of target DNA flanking the element. The predicted amino acids encoded by the major open reading frame of IS 1138 share significant similarity with the transposases of the IS3 family. Southern hybridization analysis indicates that repetitive sequences similar to IS 1138 are present in most, if not all, strains of M. pulmonis, but Is1138–like sequences were not detected in other mycoplasmal species.
Characterization of the diphtheria toxin receptor‐binding domain
Authors: Rolf John M. , Eidels Leon
Published: October 2006

The carboxyl‐terminal region of diphtheria toxin (DT) has been analysed in order to determine regions of receptor recognition. Biochemical cleavage of the toxin with hydroxylamine (HA) was used to generate the peptides HA9DT (residues 454–535), HA6DT (residues 482–535), and HA3DT (residues 454–461). Characterization of HA6DT demonstrated that the final 54 amino acids of DT are sufficient to constitute the receptor‐binding domain of the toxin. Within HA9DT, the region encompassing HA3DT and containing the highly cationic polyphosphate‐binding site did not contribute to the binding ability of HA6DT. Consistent with this observation, HA3DT itself did not compete for binding of radiolabelled DT to Vero cells. A 30‐amino acid synthetic peptide composed of residues 506–535 did not block receptor binding of DT, indicating that residues toward the amino‐terminus of HA6DT, or the entire HA6DT region, are required for receptor recognition.
Characterization of mutations in divlB of Bacillus subtilis and cellular localization of the DivlB protein
Authors: Harry E. J. , Stewart B. J. , Wake R. G.
Published: October 2006

Four temperature‐sensitive mutations in the divlB gene of Bacillus subtilis have been localized to the region corresponding to the C‐terminal half of the 263‐residue DivlB protein. Antiserum was raised to the 80%C‐terminal portion lying on one side of a putative transmembrane (hydrophobic) segment, and used to examine aspects of the nature and localization of the DivIB protein in the cell. A single DivIB species of a size equal to the full‐length protein encoded by the divIB gene was detected in wild‐type cells. Cell fractionation studies established that DivIB is associated preferentially with the cell envelope (membrane plus cell wall), with approximately 50% being released into solution upon treatment of cells with lysozyme under conditions that yield protoplasts. Of the remaining 50%, approximately half remained firmly associated with the membrane fraction. On the basis of the‘positive‐inside rule’of von Heijne (1986) it is suggested that the topology of membrane‐bound DivlB is such that the long C‐terminal portion is directed to the outside and the smaller N‐terminal portion to the inside of the cell. DivlB in protoplasts was rapidly degraded by proteinase K under conditions where there was no general proteolysis of the cytoplasmic proteins. This is consistent with its absence from the cytoplasm, and with the predicted membrane topology.

Septum positioning in a divIB null mutant, which grows as filaments at temperatures of 30°C and below, was found to be normal. It appears that DivlB is needed for achieving the appropriate rate of initiation of septum formation at normal division sites. It is proposed that the C‐terminal portion of DivlB, localized on the exterior surface of the membrane and in juxtaposition to the peptidoglycan, normally interacts with another protein (or proteins) to initiate septum formation.
Homologous overexpression of a light‐driven anion pump in an archaebacterium
Authors: Heymann Jürgen A. W. , Havelka Wendy A. , Oesterhelt Dieter
Published: October 2006

The retinal protein halorhodopsin (HR), a light‐driven chloride pump from Halobacterium halobium, was homologously overexpressed in this archaebacterium. Two DNA expression systems differing in their promoter region were investigated. The halo‐opsin, hop, promoter coupled to the hop gene gave an increased level of HR synthesis. However, the extent of expression was driven by the copy number of the shuttle vector and did not reach the magnitude of the bacterio‐opsin, bop, promoter system. Employing a gene fusion approach, the promoter for the bop gene was used to drive expression of the hop gene. A shuttle vector containing a bop‐hop‐cartridge was transformed into a HR‐deficient strain and blueish‐coloured transformants were obtained. The bop promoter expressed HR to an extent where a specific membrane fraction resembled the crystalline purple membrane of BR in terms of the lipid to protein ratio. HR could, therefore, be easily isolated in a natural membrane‐bound state. This allows for direct use in biophysical studies without the application of deter‐gents. This was the first successful overexpression of a 7‐helical transmembrane protein and may be extended to other proteins of this family.
Regulation of peptide antibiotic production in Bacillus
Authors: Marahier M. A. , Nakano M. M. , Zuber P.
Published: October 2006

In Bacillus species, starvation leads to the activation of a number of processes that affect the ability to survive during periods of nutritional stress. Activities that are induced include the development of genetic competence, sporulation, the synthesis of degradative enzymes, motility, and antibiotic production. The genes that function in these processes are activated during the transition from exponential to stationary phase and are controlled by mechanisms that operate primarily at the level of transcription initiation. One class of genes functions in the synthesis of special metabolites such as the peptide antibiotics tyrocidine and gramicidin S as well as the cyclic lipopeptide surfactin. These genes include the grs and tyc operons in Bacillus brevis, which encode gramicidin S synthetase and tyrocidine synthetase, respectively, and the srfA operon of Bacillus subtilis which encodes the enzymes of the surfactin synthetase complex. Peptide antibiotic biosynthesis genes are regulated by factors as diverse as the early sporulation gene product Spo0A, the transition‐state regulator AbrB, and gene products (ComA, ComP, and ComQ) required for the initiation of the competence developmental pathway.
Strategies in pathogenesis: mechanistic specificity in the detection of generic signals
Authors: Duban M. Eugene , Lee Kyunghee , Lynn David G.
Published: October 2006

The virulence genes of the plant pathogen Agrobacterium tumefaciens are induced by more than 40 low‐molecular‐weight phenolic compounds. The prevailing opinion is that (i) wound‐derived phenols produced on breach of the integrity of the cell wall act as the initiating signal in a series of events which results in host cell transformation, and (ii) a classical membrane receptor, putatively VirA, is responsible for the recognition of all such phenolic inducers. Here, we argue that the discovery of the subset of inducers that are relatives of the dehydrodiconiferyl alcohol glucoside (DCG) growth factors redirects our attention to work on the plant wound as a site of cell division, and suggests that we further explore the implications of early work on the relationship between transformation efficiency and the status of the cell cycle of the host. In addition, we argue that the significant structural diversity allowed in the para position of the phenol ring of inducers suggests that a receptor–ligand interaction based solely on structural recognition is insufficient, but that recognition followed by a specific proton transfer event may be sufficient to explain vir induction activity. Hence, the specificity of the response of A. tumefaciens may be a consequence of the features required for a chemical reaction to occur on the receptor surface. Finally, we review affinity labelling studies which exploit this phenol detection mechanism and which provide evidence that the phenol receptor may be other than VirA, the sensory kinase of the two component regulatory system implicated in Agrobacterium virulence. Taken together, these hypotheses form a model which has predictive and therefore experimental value, and which may be of broader interest in that it calls attention to the possibility of an intricate co‐evolution of signalling pathways of host and pathogen.
In situ hybridization analysis of developmental stages of Pneumocystis carinii that are transcriptionally active for a major surface glycoprotein gene
Authors: Haidaris Patricia J. , Wright Terry W. , Gigliotti Francis , Fallon Margaret A. , Whitbeck April A. , Haidaris Constantine G.
Published: October 2006

An abundant glycoprotein on the surface of Pneumocystis carinii, termed gpA or gp120, is thought to play a role in the interaction of this opportunistic pathogen with its host. Using RNA:RNA hybridization techniques, the in situ expression of gpA mRNA in developmental forms of the organism was investigated in a ferret model of P. carinii pneumonia. The results suggested that the relative abundance of gpA‐specific mRNA was variable in different developmental stages of ferret P. carinii. P. carinii localized along the epithelial lining of alveoli were transcriptionally active. Immunocytochemical detection of gpA and Giemsa staining suggested that many of these organisms were trophic forms of P. carinii. While no detectable gpA mRNA signal was found in the majority of P. carinii cysts, a portion of identifiable cysts co‐localized with significant levels of gpA mRNA signal. Differential staining of the cyst wall with Gomori's methenamine silver suggested that the transcriptionally active P. carinii cysts were the intermediate or precyst forms of the organism, while the cysts with no detectable mRNA signal were either mature or empty (excysted). Alveolar macrophages were observed surrounded by transcriptionally active organisms; however, no gpA‐transcriptional activity was detected within macrophages. Taken together, the results suggest that transcription of gpA occurs in forms of P. carinii that are actively replicating, and in close proximity or contact with, alveolar epithelial cells.
Molecular analysis of a cytotoxin‐converting phage, φCTX, of Pseudomonas aeruginosa: structure of the attP–cos–ctx region and integration into the serine tRNA gene
Authors: Hayashi Tetsuya , Matsumoto Hideki , Ohnishi Makoto , Terawaki Yoshiro
Published: October 2006

The Pseudomonas aeruginosa ctx gene encoding cytotoxin is carried by a temperate phage φCTX. The genome of φCTX is a 35.5 kb double‐stranded DNA with cohesive ends (cos). It is unique in that the ctx gene and attP site of φCTX exist very close to the respective cohesive ends. In this study, we determined the structure of this attP–cos–ctx region. The termini of φCTX are 21‐base 5′ extended‐single‐stranded DNAs. The ctxgene is located 361 bp downstream of the left end (cosL). The attP core sequence of 30 bp exists only 647 bp apart from the right end (cosR). The attP–cos–ctx region contains six kinds of repeats and integration host factor‐binding sequences and showed sequence‐directed static bends, suggesting its potential to form a highly ordered structure. In addition, φCTX was found to integrate into the serine tRNA gene which was mapped to the 43–45 min region on the P. aeruginosachromosome.
Deletion analysis of the SUP35 gene of the yeast Saccharomyces cerevisiae reveals two non‐overlapping functional regions in the encoded protein
Authors: Ter‐Avanesyan Michael D. , Kushnirov Vitaly V. , Dagkesamanskaya Adilya R. , Didichenko Svetlana A. , Chernoff Yury O. , Inge‐Vechtomov Sergey G. , Smirnov Vladimir N.
Published: October 2006

SUP35is an omnipotent suppressor gene of Saccharomyces cerevisiae coding for a protein consisting of a C‐terminal part similar to the elongation factor EF‐1α and a unique N‐terminal sequence of 253 amino acids. Twelve truncated versions of the SUP35 gene were generated by the deletion of fragments internal to the coding sequence. Functional studies of these deletion mutants showed that: (i) only the EF‐1α‐like C‐terminal part of the Sup35 protein is essential for the cell viability; (ii) overexpression of either the N‐terminal part of the Sup35 protein or the full‐length Sup35 protein decreases translational fidelity, resulting in omnipotent suppression and reduced growth of [psi+] strains; (iii) expression of the C‐terminal part of the Sup35 protein generates an antisuppressor phenotype; and (iv) both the N‐ or C‐terminal segments of the Sup35 protein can bind to 80S ribosomes. Thus, the data obtained define two domains within the Sup35 protein which are responsible for different functions.
The virA promoter is a host‐range determinant in Agrobacterium tumefaciens
Authors: Turk Stefan C. H. J. , Nester Eugene W. , Hooykaas Paul J. J.
Published: October 2006

The limited host range (LHR) Agrobacterium tumefaciens strain Ag162 is an isolate with a narrow host range. Introduction of the wide host range (WHR) virA gene is essential for extending the host range to Kalanchoë daigremontiana. In this report we show that the region upstream of the ATG start codon is responsible for the LHR phenomenon and that this is probably due to the non‐inducibility of the LHR virA promoter. By comparing the characteristics of the LHR and WHR VirA receptor proteins, it was found that the LHR VirA protein is able to activate the WHR VirG protein in the presence of acetosyringone and that this acetosyringone‐dependent vir‐induction is enhanced by the presence of d‐glucose, as in the case of WHR VirA proteins. These results indicate that the domains, acting as receptors for sugars and phenolic signals, must be conserved between the LHR and WHR VirA receptor proteins.
Repeat unit polysaccharides of bacteria: a model for polymerization resembling that of ribosomes and fatty acid synthetase, with a novel mechanism for determining chain length
Authors: Bastin David A. , Stevenson Gordon , Brown Peter K. , Haase Antje , Reeves Peter R.
Published: October 2006

We report the identification and sequence from Escherichia coli and Salmonella enterica strains of the cld gene, encoding the chain‐length determinant (CLD) which confers a modal distribution of chain length on the O‐antigen component of lipopolysaccharide (LPS). The distribution of chain lengths in the absence of this gene fits a model in which as the chain is extended there is a constant probability of 0.165 of transfer of growing chain to LPS core, with termination of chain extension. The data for E. coli 0111 fit a model in which the CLD reduces this probability for short chains and increases it to 0.4 for longer chains, leading to a reduced number of short chain molecules but an increase in numbers of longer molecules and transfer of essentially all molecules by chain length 21. We put forward a model for O‐antigen polymerase which resembles the ribosome and fatty acid synthetase in having two sites, with the growing chain being transferred from a D site onto the new unit at the R site to extend the chain and then back to the D site to repeat the process. It is proposed that the CLD protein and polymerase form a complex which has two states:‘E’facilitating extension and T facilitating transfer to core. The complex is postulated to enter the E state as O‐antigen polymerization starts, and to shift to the T state after a predetermined time, the CLD acting as a molecular clock. The CLD is not O‐antigen or species‐specific but the modal value does depend on the source of the cld gene.
Bacillus subtilis FlhA: a flagellar protein related to a new family of signal‐transducing receptors
Authors: Carpenter Phillip B. , Ordal Georges W.
Published: October 2006

The Bacillus subtilis flhA gene lies in the major che/fla operon, a transcription unit that spans 26 kilobases (kb) of DNA. flhA encodes a 677‐amino‐acid polypeptide that is a strong candidate for an integral membrane protein. The sequence of FlhA displays substantial homology to a newly identified family of putative signal‐transducing receptors that have been implicated in diverse cellular processes. FlhA is the first member of this family to be described from a Gram‐positive bacterium. We demonstrate that flhA is a flagellar gene and that FlhA is required in trans for the formation of products from some, but not all, B. subtilis motility‐related operons that are regulated by the αD form of RNA polymerase. We suggest that FlhA is a component of a signalling system that is involved with the formation of some flagellar gene products during the biosynthesis of the flagellum.
Structural analysis and demonstration of the 29 kDa antigen of pathogenic Entamoeba histolytica as the major accessible free thiol‐containing surface protein
Authors: Flores Becky M. , Batzer Mark A. , Stein Murry A. , Petersen Carolyn , Diedrich Dana L. , Torian Bruce E.
Published: October 2006

The 29 kDa protein of pathogenic Entamoeba histolytica is a cysteine‐rich surface antigen which we recently characterized by cDNA sequencing and by using monoclonal antibodies which differentiated between pathogenic and non‐pathogenic clinical isolates. To determine the structure and biochemical attributes of this protein, a repertoire of immunologcal techniques using monoclonal antibodies, and radiolabelling were employed. We demonstrated that the 29 kDa protein forms a 60 kDa dimer and a high‐molecular‐mass oligomer(s) on the surface of the organism through disulphide bonds, and is the major accessible free thiol‐containing surface protein of E. histolytica. The deduced amino acid sequence encoding the 29 kDa protein was found to share a common amino acid domain with sequences reported for Helicobacter pylori, Salmonella typhimurium, MER5 gene expressed in murine erythroleukemia cells, Clostridium pasteurianum, and a Bacillus spp.
Cloning and identification of a two‐component signal‐transducing regulatory system from Bacteroides fragilis
Authors: Rasmussen Beth A. , Kovacs Endre
Published: October 2006

A DNA fragment was cloned from Bacteroides fragilis that bestowed low‐level tetracycline resistance to Escherichia coli strains harbouring the cloned fragment on a multicopy plasmid. The tetracycline resistance determinant was localized to a 4.3kb BglII–Pstl subfragment of the original clone. DNA sequence analysis of this fragment revealed that it contained an operon encoding two proteins: one of 519 amino acids, RprX, and a second of 236 amino acids, RprY. Protein sequence analysis revealed that the two proteins shared sequence identity with a family of multicomponent signal‐transducing regulatory proteins identified from many diverse bacterial genera. RprX shared identity with the first component of the regulatory system, the histidine protein kinase receptor (for example EnvZ, PhoR, CheA, and VirA). RprY shared identity with the second member of the regulatory protein pair, the regulatory response protein (for example OmpR, PhoB, CheY, and VirG). Expression of these proteins from a multicopy plasmid vector in E. coli resulted in a decrease in the level of the outer membrane porin protein OmpF and an increase in the level of the outer membrane porin protein OmpC. The decrease in OmpF levels correlates with, and may be the cause of, the increased tetracycline resistance. Regulation of the levels of OmpF and OmpC is normally controlled by a multicomponent signal‐transducing regulatory pair of proteins, EnvZ and OmpR. The effect RprX and RprY have on OmpF expression is mediated at the level of transcription. Thus, RprX and RprY may be interfering with the normal regulation of OmpF by OmpR and EnvZ.
Chlamydia trachomatis Mip‐like protein has peptidylprolyl cis/trans isomerase activity that is inhibited by FK506 and rapamycin and is implicated in initiation of chlamydial infection
Authors: Lundemose Anker G. , Kay John E. , Pearce John H.
Published: October 2006

The Mip‐like protein of Chlamydia trachomatis has sequence similarity with both the Mip protein of Legionella pneumophila, a virulence factor necessary for optimal intracellular infection, and FK506‐binding proteins (FKBPs) of both prokaryotic and eukaryotic origin. FKBPs contain a site for peptidyl‐prolyl cis/trans isomerase activity, which is blocked upon binding of the drugs, FK506 or rapamycin. In this paper we report that the recombinant chlamydial Mip‐like protein exhibits a peptidyl‐prolyl cis/trans isomerase activity which is inhibited by either rapamycin or FK506. To assess the role of the Mip‐like protein in chlamydial infection, rapamycin or FK506 (25μM), were used in either treatment of chlamydial organisms prior to inoculation, or were present at different intervals through the infection. Pretreatment of organisms alone reduced infectivity for McCoy cells by 30%, with inhibition rising to 80% on more prolonged exposure from 0 to 8h and 8 to 16 h post‐inoculation and declining thereafter. When drug was present during the developmental cycle at intervals from 0 to 24h post‐inoculation abnormal chlamydiae were induced in residual inclusions. The results suggest that inhibition of the isomerase of the Mip‐like protein interferes with one or more early events in the infective process that determine productive intracellular infection.
Molecular analysis of spv virulence genes of the salmonella virulence plasmids
Authors: Gulig Paul A. , Danbara Hirofumi , Guiney Donald G. , Lax Alistair J. , Norel Françoise , Rhen Mikael
Published: October 2006

Genes on an 8 kb region common to the virulence plasmids of several serovars of Salmonella are sufficient to replace the entire plasmid in enabling systemic infection in animal models. This virulence region encompasses five genes which previously have been designated with different names from each investigating laboratory. A common nomenclature has been devised for the five genes, i.e. spv for salmonella plasmid virulence. The first gene, spvR, encodes a positive activator for the following four genes, spvABCD. DNA sequence analysis of the spv genes from Salmonella typhimurium. Salmonella dublin, and Salmonella choleraesuis demonstrated extremely high conservation of the DNA and amino acid sequences. The spv genes are induced at stationary phase and in carbon‐poor media, and optimal expression is dependent on the katF locus. The cirulence functions of the spv genes are not known, but these genes may increase the growth rate of salmonellae in host cells and affect the interaction of salmonellae with the host immune system.
The Rad3 protein from Saccharomyces cerevisiae: a DNA and DNA:RNA helicase with putative RNA helicase activity
Authors: Deschavanne Patrick J. , Harosh Itzik
Published: October 2006

The Rad3 protein from Saccharomyces cerevisiae is a DNA helicase which participates in the repair of ultraviolet‐irradiated DNA and is Inhibited in the presence of DNA containing thymine dimers. This protein is also involved in mitotic recombination and spontaneous mutagenesis and is essential for cell viability in the absence of DNA damage. Furthermore, the Rad3 protein also exhibits a DNA:RNA helicase activity in which there is a significant preference for a partial DNA:RNA hybrid rather than a partial duplex DNA substrate, which suggests that this protein might be involved in DNA repair within transcriptionally active genes. Finally, the Rad3 protein contains the DEAH motif and shares high amino acid sequence similarity with the DEAD family of RNA helicase proteins, suggesting that it might also possess an RNA helicase activity.
Involvement of the RNA polymerase α subunit C‐terminal region in co‐operative interaction and transcriptional activation with OxyR protein
Authors: Tao Kazuyuki , Fujita Nobuyuki , Ishihama Akira
Published: October 2006

The role of the α subunit of Escherichia coli RNA polymerase in transcription activation by the OxyR protein was investigated using in‐vitro‐reconstituted RNA polymerase containing α subunits carrying C‐terminal truncations or an amino acid substitution. Mutant RNA polymerases failed to respond to transcription activation of the E. coli OxyR‐dependent promoters. DNase I footprinting analysis indicates that the OxyR protein exerts a co‐operative effect on the binding of wild‐type RNA polymerase, but not the mutant RNA polymerases, to the katG promoter. Together, these results suggest that direct protein‐protein contact between the OxyR protein and the C‐terminal contact site I region of the RNA polymerase α subunit plays an essential role in transcription activation at the OxyR‐dependent promoters.
Antisense RNA mediates transcriptional processing in an archaebacterium, indicating a novel kind of RNase activity
Authors: Stolt Pelte , Zillig Wolfram
Published: October 2006

Strains of the extremely halophilic archaebacterium Halobacterium salinarium that are lysogenic for the phage φH produce an antisense RNA transcript complementary to the first 151 nucleotides (nt) of the early lytic phage transcript T1. This is the first case of antisense control of gene expression in an archaebacterium. We show through transformation of H. salinarium that the antisense RNA functions in trans, rendering the early lytic phage transcript T1 susceptible to specific cleavage by an unidentified RNase of unique endonucleolytic activity. The single‐stranded ends of RNA are cut off at the ends of the 151 nt RNA duplex, removing the ribosomal binding sites from the first open reading frame of transcript T1 but without concominant digestion of the products.
Organization of Lrp‐binding sites upstream of ilvlH in Salmonella typhimurium
Authors: Wang Qing , Sacco Margherita , Ricca Ezio , Lago Carmine T. , DeFelice Maurilio , Calvo Joseph M.
Published: October 2006

Lrp, a major regulatory protein in Escherichia coli, controls the expression of numerous operons, including ilvlH. Lrp binds to six sites upstream of ilvlH, and Lrp binding is required for ilvlH expression. We show here that an Lrp‐like protein is also present in Salmonella typhimurium. This protein can bind both E. coli and S. typhimurium ilvlH DNA, as can E. coli Lrp. Methidiumpropyl‐EDTA footprinting studies were performed with purified E. coli Lrp and S. typhimurium ilvlH DNA. Six binding sites were defined, three of them being similar to corresponding sites in E. coli, and three being organized differently. A consensus derived from six S. typhimurium sites is compatible with that derived from a similar analysis of E. coli sequences.
Molecular characterization of the enniatin synthetase gene encoding a multifunctional enzyme catalysing N‐methyldepsipeptide formation in Fusarium scirpi
Authors: Haese Angela , Schubert Monika , Herrmann Matthias , Zocher Rainer
Published: October 2006

The gene encoding the multifunctional enzyme enniatin synthetase from Fusarium scirpi (esyn1) was isolated and characterized by transcriptional mapping and expression studies in Escherichia coli. This is the first example of a gene encoding an N‐methyl peptide synthetase. The nucleotide sequence revealed an open reading frame of 9393 bp encoding a protein of 3131 amino acids (Mr 346 900). Two domains designated EA and EB within the protein were identified which share similarity to each other and to microbial peptide synthetase domains. In contrast to the N‐terminal domain EA, the carboxyl terminal domain EB is interrupted by a 434‐amino‐acid portion which shows local similarity to a motif apparently conserved within adenine and cytosine RNA and DNA methyltransferases and therefore seems to harbour the N‐methyl‐transferase function of the multienzyme.
The conjugative plasmid SLP2 of Streptomyces lividans is a 50 kb linear molecule
Authors: Chen Carton W. , Yu Tin‐Wein , Lin Yi‐Shing , Kieser Helen M. , Hopwood David A.
Published: October 2006

The SLP2 plasmid had previously been demonstrated genetically to exist In Streptomyces lividans by its ability to promote conjugation and to elicit‘pocks’on recipient (SLP2−) cultures, but it had not been physically detected. Using pulsed‐field gel electrophoresis, a 50kb linear DNA was isolated from SLP2+ but not SLP2− strains of S. lividans, and from Streptomyces coelicolor and Streptomyces parvulus strains to which SLP2 had been transferred by conjugation or transformation. We conclude that this linear DNA is SLP2. The terminal fragments of SLP2 were cloned. The determined sequences revealed a 44 bp imperfect terminal inverted repeat. The terminal 12 bp sequence of SLP2 was identical to those of two other Streptomyces linear plasmids, pSLA2 and pSCL, and similar to the terminal sequences of another Streptomyces linear plasmid, SCP1. The termini of SLP2 DNA were resistant to digestion by λ exonuclease and ExoIII. A truncated (probably crippled) copy of Tn4811 is present on the plasmid. While the SLP2 plasmid exists as a tree form in the host, a 15.7 kb sequence corresponding to the segment of SLP2 from Tn4811 to the right terminus is also present (at a copy number similar to the free form) elsewhere in the genome of S. lividans. Furthermore, SLP2 is partially homologous to a newly discovered 650 kb linear plasmid in S. parvulus.
Recombination‐deficient mutants of Salmonella typhimurium are avirulent and sensitive to the oxidative burst of macrophages
Authors: Buchmeier Nancy A. , Lipps Craig J. , So Magdalene Y. H. , Heffron Fred
Published: October 2006

Mutations in the genes recA and recBC were constructed in the virulent Salmonella typhimurium strain 14028s. Both the recA and recBC mutants were attenuated in mice. The mutants were also sensitive to killing by macrophages in vitro. The recombination mutants were no longer macrophage sensitive in a variant line of J774 macrophage‐like cells that fail to generate superoxide. This suggests that repair of DNA damage by Salmonella is necessary for full virulence in vivo and that the oxidative burst of phagocytes is one source of such DNA damage.
In vitro transcription in Chlamydia psittaci and Chlamydia trachomatis
Authors: Mathews Sarah A. , Douglas Annemarie , Sriprakash K. S. , Hatch Thomas P.
Published: October 2006

Extracts of Chlamydia psittaci and Chlamydia trachomatis were used to transcribe molecularly cloned chlamydial genes in vitro. The extracts were prepared by Iysing reticulate bodies, obtaining the 10000 ×g centrifugation pellet, and eluting RNA polymerase from the pellet by treatment with 2M KCI to yield a fraction designated SS2. Some in vitro transcription was initiated from non‐chlamydial promoters and a small amount of transcription was from endogenous DNA template in SS2. However, optimal transcription from exogenous templates required chlamydial promoter sequences, and primer extension analysis indicated that chlamydia promoter‐specific in vitro transcription was initiated from the same start sites recognized in vivo. A monoclonal antibody that was generated against Escherichia coliσ;70 and which immunologically cross‐reacts with C. trachomatisσ;66 inhibited in vitro transcription of vector and cloned chlamydial DNA, suggesting that transcriptional initiation in the SS2 fraction is mediated by CT66. An in vitro transcription assay based on detection of transcripts of specific lengths was applied to the chlamydial system; this assay and others described here should be useful in defining chlamydial promoters and other transcriptional regulatory elements.
Expression of the pspA gene stimulates efficient protein export in Escherichia coli
Authors: Kleerebezem Michiel , Tommassen Jan
Published: October 2006

Expression of several mutant forms of outer membrane protein PhoE of Escherichia coli, which are disturbed in normal biogenesis, resulted in high expression of a 26kDa protein. This 26kDa protein fractionated as a peripherally bound inner membrane protein. It appeared to be identical to a previously identified protein (PspA = phage shock protein A) of unknown function that is induced upon infection of E. coli with filamentous phages. PspA was not expressed upon synthesis of mutant PhoE proteins in a secB mutant, nor upon expression of a PhoE mutant that lacks the signal sequence, suggesting that entrance into the export pathway of prePhoE is essential for induction. PspA synthesis was also induced under other conditions that are known to block the export apparatus, i.e. in secA, secD and secF mutants when grown at their non‐permissive temperature or upon induction of the synthesis of MalE‐LacZ or LamB‐LacZ hybrid proteins. The inducing conditions for PspA synthesis suggested a rote for this protein in export. In vivo pulse‐chase experiments showed that the translocation of (mutant) prePhoE and of the precursors of other exported proteins was retarded in a pspA mutant strain. Also, in in vitro translocation assays, a role for PspA in protein transport could be demonstrated.
Two genes present on a transposon‐like structure in Lactococcus lactis are involved in a Clp‐family proteolytic activity
Authors: Huang Dao Chao , Huang Xian Fang , Novel Georges , Novel Madeleine
Published: October 2006

The lactose‐protease plasmid pUCL22 of Lactococcus lactis subsp. lactis strain CNRZ270 contained two inverted copies of IS 1076 flanking a region of 3.7 kb. This internal region was sequenced and found to contain two large open reading frames, ORF1 and ORFP in opposite orientations. ORF1 consists of 2289 bp; the deduced 763‐amino‐acid sequence is similar to the ATPases of the ClpA family. It contains two well‐conserved consensus ATP‐binding sites. It was named ClpL. ORFP consists of 930 bp encoding a protein of 310 amino acids. No similarity with any known protein was found in GenBank data for ORFP. Increased ATP‐dependent proteolytic activity was detected in extracts from Escherichia coli cells expressing the clpL and ORFP genes.
A positive feedback loop controls transcription of the spo0F gene, a component of the sporulation phosphorelay In Bacillus subtilis
Authors: Strauch Mark A. , Wu Jiunn‐Jong , Jonas Robert H. , Hoch James A.
Published: October 2006

The spo0Fgene of Bacillus subtilisencodes a protein that functions as a secondary messenger in a phosphorelay system controlling the initiation of sporulation. Transcription of the spo0F gene was known to be dependent on an intact gene for the transcription regulator Spo0A. In vitro footprint analysis revealed that Spo0A protein bound to two locations in the spo0F promoter region. Deletion of a 40 bp region upstream of one of the promoters (P2) abolished the activation of spo0Fexpression that occurs at the onset of stationary phase and sporulation. This 40 bp region contains a Spo0A‐binding site. These observations are consistent with a hypothesis that Spo0A binding to this region is responsible for activating spo0F transcription. Additionally, Spo0A binding at a downstream site could modulate the level of this activation. Since Spo0F protein is required for the formation of Spo0A∼P (the form needed for transcriptional activation) a positive feedback loop controls transcription of spo0F.
Translation across the 5′‐splice site interferes with autocatalytic splicing
Authors: Öhman‐Hedén Marie , Åhgren‐Stálhandske Agneta , Hahne Solveig , Sjöberg Britt‐Marie
Published: October 2006

The bacteriophage T4 nrdB gene, encoding the ribonucleotide reductase small subunit, contains a self‐splicing group IA2 intron with an ochre codon in frame with the preceding exon sequence. The stop codon was changed to an amino acid codon and splicing efficiency was compared with that of the wild type in the presence and absence of translation. In vivo the mutant has a much tower efficiency for producing a mature transcript than the wild type. Also, the relative production of the full‐length translation product is correspondingly lower in the mutant than in the wild type. These results confirm the importance of the stop codon, which spans the splice site of the nrdB intron.

The occurrence of stop codons in 56 group I introns in protein‐encoding genes was investigated. In 33 of those translation is terminated upstream of the first common elements of the catalytic core, of group I introns. In the rest translation is terminated in intron regions outside the heart of the catalytic core, with one exception.

Our observations suggest that in situations where transcription and translation are coupled events there has been an evolutionary pressure to preserve stop codons in the 5′‐region of these introns or to prevent translational termination from occurring in vital parts of the introns.
Ribosome activity and modification of 16S RNA are influenced by deletion of ribosomal protein S20
Authors: Rydén‐Aulin Monica , Shaoping Zhang , Kylsten Per , Isaksson Leif A.
Published: October 2006

A spontaneous mutant of Escherichia coli K‐12 was isolated that shows an increased misreading ability of all three nonsense codons together with an inability to grow at 42° C. It is demonstrated that the mutation is a deletion of the gene rpsT, coding for ribosomal protein S20. The loss of this protein not only influences the decoding properties of the ribosome; the modification pattern of 16S ribosomal RNA is also changed. This leads to a deficiency in the ability of the mutant to associate its 30S subunits with 50S subunits to form 70S ribosomes. It is suggested that two modified bases, m5C and m62A, are directly or indirectly essential for association of subunits to functional ribosomes in the rpsT mutant strain. Two other modifications were also studied; m2G which is not affected at all and m3U which is undermodified in both active and inactive subunits and, therefore, not involved in subunit association.
Mitochondrial transcription: is a pattern emerging?
Authors: Jaehning Judith A.
Published: October 2006

Despite the striking similarities of RNA polymerases and transcription signals shared by eubacteria, archaebacteria and eukaryotes, there has been little indication that transcription in mitochondria is related to any previously characterized model. Only in yeast has the subunit structure of the mitochondrial RNA polymerase been determined. The yeast enzyme is composed of a core related to polymerases from bacteriophage T7 and T3, and a promoter recognition factor similar to bacterial sigma factors. Soluble systems for studying mitochondrial transcript initiation in vitro have been described from several organisms, and used to determine consensus sequences at or near transcription start sites. Comparison of these sequences from fungi, plants, and amphibians with the T7/T3 promoter suggests some intriguing similarities. Mammalian mitochondrial promoters do not fit this pattern but instead appear to utilize upstream sites, the target of a transcriptional stimulatory factor, to position the RNA polymerase. The recent identification of a possible homologue of the mammalian upstream factor in yeast mitochondria may indicate that a pattern will eventually be revealed relating the transcriptional machineries of all eukaryotic mitochondria.
Influence of pH on bacterial gene expression
Authors: Olson Eric R.
Published: October 2006

Bacteria respond to changes in internal and external pH by adjusting the activity and synthesis of proteins associated with many different processes, including proton translocation, amino acid degradation, adaptation to acidic or basic conditions and virulence. While, for many of these examples, the physiological and biological consequence of the pH‐induced response is clear, the mechanism by which the transcription/translation machinery is signalled is not. These examples are discussed along with several others in which the function of the gene or protein remains a mystery.
Organization of the genes necessary for hydrogenase expression in Rhodobacter capsulatus. Sequence analysis and identification of two hyp regulatory mutants
Authors: Colbeau Annette , Richaud Pierre , Toussaint Bertrand , Caballero F. Javier , Elster Christine , Delphin Christian , Smith Russell L. , Chabert Jacqueline , Vignais Paulette M.
Published: October 2006

A 25kbp DNA fragment from the chromosome of Rhodobacter capsulatus B10 carrying hydrogenase (hup) determinants was completely sequenced. Coding regions corresponding to 20 open reading frames were identified. The R. capsulatus hydrogenase‐specific gene (hup and hyp) products bear significant structural identity to hydrogenase gene products from Escherichia coli (13), from Rhizobium liguminosarum (16), from Azotobacter vinelandii (10) and from Alcaligenes eutrophus (11). The sequential arrangement of the R. capsulatus genes is: hupR2‐hupU‐hypF‐hupS‐hupL‐hupM‐hupD‐hupF‐hupG‐hupH‐huoJ‐hupK‐hypA‐hypB‐hupR1‐hypC‐hypD‐hypE‐ORF19‐ORF20, all contiguous and transcribed from the same DNA strand. The last two potential genes do not encode products that are related to identified hydrogenase‐specific gene products in other species. The sequence of the 12 R. capsulatus genes underlined above is presented. The mutation site in two of the Hup− mutants used in this study, RS13 and RCC12, was identified in the hypF gene (deletion of one G) and in the hypD qene (deletion of 54 bp), respectively. The hypF gene product shares 45% identity with the product of hydA from E. coli and the product of hypF from R. leguminosarum. Those products present at their N‐terminus a Cys arrangement typical of zinc‐finger proteins. The G deletion in the C‐terminal region of hypF in the RS13 mutant