bluuebunny/crossref_metadata_2025 huggingface dataset:
Mobile gene cassettes and integrons: capture and spread of genes by site‐specific recombination
Authors: Hall Ruth M. , Collis Christina M.
Published: October 2006

An integron is a genetic unit that includes the determinants of the components of a site‐specific recombination system capable of capturing and mobilizing genes that are contained in mobile elements called gene cassettes. An integron also provides a promoter for expression of the cassette genes, and integrons thus act both as natural cloning systems and as expression vectors. The essential components of an integron are an int gene encoding a site‐specific recombinase belonging to the integrase family, an adjacent site, attl, that is recognized by the integrase and is the receptor site for the cassettes, and a promoter suitably oriented for expression of the cassette‐encoded genes. The cassettes are mobile elements that include a gene (most commonly an antibiotic‐resistance gene) and an integrase‐specific recombination site that is a member of a family of sites known as 59‐base elements. Cassettes can exist either free in a circularized form or integrated at the attl site, and only when integrated is a cassette formally part of an integron. A single site‐specific recombination event involving the integron‐associated attl site and a cassette‐associated 59‐base element leads to insertion of a free circular cassette into a recipient integron. Multiple cassette insertions can occur, and integrons containing several cassettes have been found in the wild. The integrase also catalyses excisive recombination events that can lead to loss of cassettes from an integron and generate free circular cassettes. Due to their ability to acquire new genes, integrons have a clear role in the evolution of the genomes of the plasmids and transposons that contain them. However, a more general role in evolution is also likely. Events involving recombination between a specific 59‐base‐element site and a nonspecific secondary site have recently been shown to occur. Such events should lead either to the insertion of cassettes at non‐specific sites or to the formation of stable cointegrates between different plasmid molecules, and a cassette situated outside the integron context has recently been identified.
The twisted ‘life’ of DNA in the cell: bacterial topoisomerases
Authors: Luttinger Amy
Published: October 2006

DNA topoisomerases are essential to the cell for the regulation of DNA supercoiling levels and for chromosome decatenation. The proposed mechanisms for these reactions are essentially the same, except that a change in supercoiling is due to an intramolecular event, while decatenation requires an intermolecular event. The characterized bacterial topoisomerases appear capable of both types of reaction in vitro. Four DNA topoisomerases have been identified in Escherichia coli. Topoisomerase I, gyrase, and topoisomerase IV normally appear to have distinct essential functions within the cell, Gyrase and topoisomerase I are responsible for the regulation of DNA supercoiling. Both gyrase and topoisomerase IV are necessary for chromosomal decatenation. Multiple topoisomerases with distinct functions may give the cell more precise control over DNA topology by allowing tighter regulation of the principal enzymatic activities of these different proteins.
Chlamydial envelope components and pathogen‐host cell interactions
Authors: Raulston Jane E.
Published: October 2006

Few bacterial pathogens are as widespread in nature or as capable of eliciting such a diversity of disease syndromes as are the chlamydiae. As obligate intra‐cellular organisms, they pose a special research challenge in defining the molecular components and mechanisms for productive growth within host cells and the overall progress of infection throughout host tissue. Although a comprehensive view of chlamydial envelope composition and respective functions in pathogenesis is far from complete, ongoing investigations continue to expose new and intriguing avenues for exploration.
The enterotoxin gene (cpe) of Clostridium perfringens can be chromosomal or plasmid‐borne
Authors: Cornillot Emmanuel , Saint‐Joanis Brigitte , Daube Georges , Katayama Sei‐ichi , Granum Per Einar , Canard Bruno , Cole Stewart T.
Published: October 2006

The location of the cpe gene, encoding the enterotoxin responsible for food poisoning in humans, has been studied in a series of enterotoxigenic Ciostridium perfringens strains by means of pulsed field gel electrophoresis of genomic DNA. The cpe gene was found at the same chromosomal locus in strains associated with food poisoning in humans and was shown to be linked to a repetitive sequence, the Hin dlll repeat, and an open reading frame, ORF3, that may be part of an insertion sequence. In contrast, when the strains originated from domesticated livestock cpe was located on a large episome where it was often close to a copy of the transposable element IS 1151. In these cases, the Hin dlll repeat was not linked to the cpe gene although this was generally preceded by ORF3.
Protective antigen‐binding domain of anthrax lethal factor mediates translocation of a heterologous protein fused to its amino‐ or carboxy‐terminus
Authors: Milne Jill C. , Blanket Steven R. , Hanna Philip C. , Collier R. John
Published: October 2006

The edema factor (EF) and lethal factor (LF) components of anthrax toxin require anthrax protective antigen (PA) for binding and entry into mammalian cells. After internalization by receptor‐mediated endocytosis, PA facilitates the translocation of EF and LF across the membrane of an acidic intracellular compartment. To characterize the translocation process, we generated chimeric proteins composed of the PA recognition domain of LF (LFN; residues 1–255) fused to either the amino‐terminus or the carboxy‐terminus of the catalytic chain of diphtheria toxin (DTA). The purified fusion proteins retained ADP‐ribosyltransferase activity and reacted with anti‐sera against LF and diphtheria toxin. Both fusion proteins strongly inhibited protein synthesis in CHO‐K1 cells in the presence of PA, but not in its absence, and they showed similar levels of activity. This activity could be inhibited by adding LF or the LFN fragment (which blocked the interaction of the fusion proteins with PA), by adding inhibitors of endo‐some acidification known to block entry of EF and LF into cells, or by introducing mutations that attenuated the ADP‐ribosylation activity of the DTA moiety. The results demonstrate that LFN fused to either the amino‐terminus or the carboxy‐terminus of a heterologous protein retains its ability to complement PA in mediating translocation of the protein to the cytoplasm. Besides its importance in understanding translocation, this finding provides the basis for constructing a translocation vector that mediates entry of a variety of heterologous proteins, which may require a free amino‐ or carboxy‐terminus for biological activity, into the cytoplasm of mammalian cells.
Phosphorylation and functional analysis of PilA, a protein involved in the transcriptional regulation of the pilin gene in Neisseria gonorrhoeae
Authors: Taha Muhamed‐Kheir , Giorgini Dario
Published: October 2006

The transcriptional regulation of the pilE gene, coding for the pilin in Neisseria gonorrhoeae, by PilA/PilB proteins is quite complex. Sequence analysis of PilA suggested that it has multiple domains. PilA appears to have in its N‐terminal half a DNA‐binding site followed by a region showing sequence similarity with other bacterial transcriptional regulators. In its C‐terminal half, PilA has extensive homology with the 54kDa protein of the eukaryotic signal‐recognition particle which is involved in protein secretion. A transcriptional fusion between the promoter of pilE and the lacZ gene was constructed and integrated into the gonococcal chromosome. We show that transcription of the pilE‐lacZ fusion is affected in pHA mutants in the absence of any possible interference with pilin secretion. Moreover, pilE transcription depends on a 24/‐12‐type promoter which could be a member of a family of promoters recognized by the alternative Sigma subunit, RpoN, of the RNA polymerase. We also show that PilA binds specifically to the promoter region of pilE and that it is phosphorylated in a manner dependent on acidic residues Glu‐59, Asp‐149 and Asp‐186. The functional organization of PilA suggests that it may be an unusual transcriptional regulator different from other RpoN‐dependent activators.
Localization of the start sites of lagging‐strand replication of rolling‐circle plasmids from Gram‐positive bacteria
Authors: Dempsey Laurie A. , Zhao Adam C. , Khan Saleem A.
Published: October 2006

A number of small, multicopy plasmids from Gram‐positive bacteria replicate by an asymmetric rolling‐circle mechanism. Previous studies with several of these plasmids have identified a palindromic sequence, SSOA, that acts as the single‐strand origin (SSO) for the replication of the lagging‐strand DNA. Although not all the SSOA sequences share ONA sequence homology, they are structurally very similar. We have used an in vitro system to study the lagging‐strand replication of several plasmids from Gram‐positive bacteria using the SSOA sequences of pT181, pE194 and pSN2 as representative of three different groups of Staphylococcus aureus plasmids. In addition, we have investigated the lagging‐strand replication of the pUB110 plasmid that contains an alternative single‐strand origin, ssou. Our results confirm that RNA polymerase is involved in lagging‐strand synthesis from both SSOA and ssou‐type lagging‐strand origins. Interestingly, while initiation of lagging‐strand DNA synthesis of pUB110 occurred predominantly at a single position within ssou, replication of pT181, pSN2 and pE194 plasmids initiated at multiple positions from SSOA.
A gene required for nutritional repression of the Bacillus subtilis dipeptide permease operon
Authors: Slack Frank J. , Serror Pascale , Joyce Elizabeth , Sonenshein Abraham L.
Published: October 2006

An insertion mutation was isolated that resulted in derepressed expression of the Bacillus subtilis dipeptide transport operon (dpp) during the exponential growth phase in rich medium. DNA flanking the site of insertion was found to encode an operon (codVWXY) of four potential open reading frames (ORFs). The deduced product of the codV ORF is similar to members of the λ Int family; CodW and CodX are homologous to HsIV and HsIU, two putative heat‐shock proteins from Escherichia coli, and to LapC and LapA, two gene products of unknown function from Pasteurella haemolytica. CodX also shares homology with a family of ATPases, including CIpX, a regulatory subunit of the E. coli ClpP protease. CodY does not have any homologues in the databases. The insertion mutation and all previously isolated spontaneous cod mutations were found to map In codY. In‐frame deletion mutations in each of the other cod genes revealed that only codY is required for repression of dpp in nutrient‐rich medium. The cody mutations partially relieved amino acid repression of the histidine utilization (hut) operon but had no effect on regulation of certain other early stationary phase‐induced genes, such as spoVG and gsiA.
The Pseudomonas aeruginosa pilK gene encodes a chemotactic methyltransferase (CheR) homologue that is translationally regulated
Authors: Darzins Aldis
Published: October 2006

A new locus, designated pilK, located immediately adjacent to the previously described Pseudomonas aeruginosa pilG–J gene cluster, has been identified. Sequence analysis of a 1.3 kb region revealed the presence of a single open reading frame of 291 amino acid residues (Mr, 33 338) that contained significant homology to the chemotactic methyltransferase proteins of Escherichia coli. Bacillus subtilis and the gliding bacterium Myxococcus xanthus. The 60 bp pilJ–pilK intergenic region was devoid of promoter consensus sequences, suggesting that pilj and pilK are contained within the same transcriptional unit. The intergenic region did contain, however, a large, highly GC‐rich, inverted repeat that prevented PilK production in expression studies. To investigate the regulatory role of these sequences, pilK‐lacZ gene fusions, as well as derivatives containing sequence alterations in the potential stem‐loop region, were constructed and analysed in E. coli and P. aeruginosa. Modification of the inverted repeat region in pilK‐lacZ protein fusion constructs resulted in as much as a 24'fold increase in β‐galactosidase activity, whereas similar modifications in pilK‐lacZ transcriptional fusions had only a marginal effect on β‐galactosidase levels. These results indicated that PilK production may be largely regulated at the level of translation. In stark contrast to pilG‐J mutants, which are dramatically impaired in pilus production and/or function, a PAO1 pilK deletion mutant was indistinguishable from the wild type. In addition, complementation studies suggested that the PilK and E. coli CheR proteins are not functionally interchangeable.
NoIR controls expression of the Rhizobium meliloti nodulation genes involved in the core Nod factor synthesis
Authors: Cren Michèle , Kondorosi Adam , Kondorosi Eva
Published: October 2006

The synthesis of Rhizobium meliloti Nod signal molecules, encoded by the nod gene products, is finely regulated. A negative control of plasmid‐borne nod gene expression is provided by the NoIR repressor encoded by the chromosomal noIR gene. NoIR was previously shown to downregulate the expression of the activator nodD1 gene and the common nodABC operon by binding to an overlapping region of the two promoters adjacent to the n1 nod‐box (Kondorosi et al., 1989). We demonstrate here that NoIR also controls the expression of two additional genes, nodD2 and nodM, but does not directly regulate the expression of the host‐specific nod genes located downstream of the n2, n3 and n5 nod‐boxes. Thus, the nod genes are differentially regulated by NoIR and only those providing common nodulation functions, by determining the synthesis of the core Nod factor structure, are subjected to this negative regulation. Furthermore, NoIR has a strong negative effect on the production of Nod metabolites, the level of which may serve as a fine‐tuning mechanism for optimal nodulation, specific to host‐plant genotypes. In addition, it elicits preferential synthesis of Nod factors carrying unsaturated C16 fatty acids. Expression of noIR was high both in the free‐living bacterium and in the bacteroid and it was downregulated by its own product and by the nod gene inducer luteolin.
A 40 kb chromosomal fragment encoding Salmonella typhimurium invasion genes is absent from the corresponding region of the Escherichia coli K‐12 chromosome
Authors: Mills Debra M. , Bajaj Vivek , Lee Catherine A.
Published: October 2006

Many Salmonella typhimurium genes are required for bacterial entry into host cells. P22 transduction analysis has localized several invasion loci near minute 59 on the S. typhimurium chromosome. To further characterize the 59–60 min chromosomal region, we determined the physical and genetic map of 85 kb of S. typhimurium DNA between srl and cysC. It was previously shown that some of the invasion genes from this region are not present in Escherichia coli K‐12. We examined whether other S. typhimurium genes on the 85 kb of DNA were similarly absent from E. coli We found that a contiguous 40 kb fragment of the S. typhimurium chromosome which encodes invasion genes is absent from the corresponding region of the E. coli K‐12 chromosome and may represent a pathogenicity island. We speculate that acquisition of the 40 kb region must have significantly advanced the evolution of Salmonella as a pathogen.
Topological analyses of the l‐fucose‐H+ symport protein, FucP, from Escherichia coli
Authors: Gunn Frank J. , Tate Christopher G. , Sansom Clare E. , Henderson Peter J. F.
Published: October 2006

The transport of l‐fucose into Escherichia coli is mediated by the l‐fucose‐H+ symport protein (FucP). The fucP gene has been sequenced and encodes a hydrophobic protein that contains 438 amino acid residues, with a predicted Mr of 47773. The hydropathic profile of FucP indicates 10 to 12 hydrophobic regions that could span the membrane as α‐helices. A 12‐helix model with the N‐ and C‐termini located in the cytoplasm was derived from the hydropathic profile and from application of the ‘positive inside’ rule. This model was tested using β‐lactamase fusion technology. Analyses of 62 different FucP‐β‐lactamase fusions suggested that the FucP protein crosses the cytoplasmic membrane of E. coli 12 times, with the N‐ and C‐termini in the cytoplasm. From measurements of [14C]‐l‐fucose uptake, it was deduced that the last putative transmembrane region must be complete for transport activity to be retained and that the four C‐terminal residues were unnecessary for transport activity. Fourier transform analyses show that all the predicted helices contain a periodicity that enables hydrophobic/hydrophilic faces to be identified; these were particularly evident in putative helices 1, 3, 4, 5, 6, 10 and 11.
The regulation of transcription initiation by integration host factor
Authors: Goosen Nora , van de Putte Pieter
Published: October 2006

Integration host factor (IHF) of Escherichia coli is an asymmetric histone‐like protein that binds and bends the DNA at specific sequences. IHF functions as an accessory factor in a wide variety of processes including replication, site‐specific recombination and transcription. In many of these processes IHF was shown to act as an architectural element which helps the formation of nucleo‐protein complexes by bending of the DNA at specific sites. This MicroReview shows how such a structural role of IHF can influence the initiation of transcription. In addition, it summarizes the evidence indicating that IHF can stimulate transcription via a direct interaction with RNA polymerase and explores the possibility that the asymmetry of the IHF protein might reflect such an interaction
Mutational analysis of the ganglioside‐binding activity of the type II Escherichia coli heat‐labile enterotoxin LT‐IIb
Authors: Connell Terry D. , Holmes Randall K.
Published: October 2006

The Escherichia coli type II heat‐labile enterotoxin LT‐IIb consists of a single A polypeptide and five B polypeptides. The A polypeptide is responsible for the toxic activity, and the B polypeptides function to bind the toxin to gangliosides on the surface of the plasma membrane. Previous studies on the related type II enterotoxin LT‐IIa demonstrated the importance of threonine (Thr) residues at positions 13, 14, and 34 in the mature B polypeptide for ganglioside GD1b‐binding activity. In this study, we used site‐specific mutagenesis to investigate ganglioside GD1a‐binding activity of the B polypeptide of LT‐IIb. We determined that Thr‐13 and Thr‐14 were involved in binding of ganglioside GD1 a by the B polypeptides of LT‐IIb but that Thr‐34 was not essential. Substitution of serine, but not other amino acids, at position 13 or 14 in the B polypeptide of LT‐IIb resulted in retention of ganglioside‐binding activity equivalent to that of the wild‐type enterotoxin, providing strong evidence that the hydroxyl groups of threonine or serine at positions 13 and 14 are important for the ganglioside‐binding activity of LT‐IIb. Chimeric genes that expressed hybrids of the B polypeptides of LT‐IIb and LT‐IIa were also constructed, and analysis of the hybrids showed that the specificity of their ganglioside‐binding activity was determined by the N‐terminal half of the molecule.
Genes acrA and acrB encode a stress‐induced efflux system of Escherichia coli
Authors: Ma Dzwokai , Cook David N. , Alberti Marie , Pon Ning G. , Nikaido Hiroshi , Hearst John E.
Published: October 2006

Defined mutations of acrA or acrB (formerly acrE) genes increased the susceptibility of Escherichia coli to a range of small inhibitor molecules. Deletion of acrAB increased susceptibility to cephalothin and cephaloridine, but the permeability of these β‐lactams across the outer membrane was not increased. This finding is inconsistent with the earlier hypothesis that acrAB mutations increase drug susceptibility by increasing the permeability of the outer membrane, and supports our model that acrAB codes for a multi‐drug efflux pump. The natural environment of an enteric bacterium such as E. coli is enriched in bile salts and fatty acids. An acrAB deletion mutant was found to be hypersusceptible to bile salts and to decanoate. In addition, acrAB expression was elevated by growth in 5mM decanoate. These results suggest that one major physiological function of AcrAB is to protect E. coli against these and other hydrophobic inhibitors. Transcription of acrAB is increased by other stress conditions including 4% ethanol, 0.5 M NaCl, and stationary phase in Luria‐Bertani medium. Finally, acrAB expression was shown to be increased in mar (multiple‐antibiotic‐resistant) mutants.
Characterization and distribution of two insertion sequences, IS1191 and iso‐IS981, in Streptococcus thermophilus: does intergeneric transfer of insertion sequences occur in lactic acid bacteria co‐cultures?
Authors: Guédon G. , Bourgoin F. , Pébay M. , Roussel Y. , Colmin C. , Simonet J. M. , Decaris B.
Published: October 2006

A chromosomal repeated sequence from Streptococcus thermophilus was identified as a new insertion sequence (IS), IS1191. This is the first IS element characterized in this species. This 1313 bp element has 28 bp imperfect terminal inverted repeats and is flanked by short direct repeats of 8bp. The single large open reading frame of IS1191 encodes a 391‐amino‐acid protein which displays homologies with transposases encoded by IS1201 from Lactobacillus helveticus (44.5% amino‐acid sequence identity) and by the other ISs of the IS256 family. One of the copies of IS 1191 is inserted into a truncated iso‐IS981 element. The nucleotide sequences of two truncated iso‐IS981 s from S. thermophilus and the sequence of IS981 element from Lactococcus lactis share more than 99% identity. The distribution of these insertion sequences in L. lactis and S. thermophilus strains suggests that intergeneric transfers occur during co‐cultures used in the manufacture of cheese.
Overexpression of wild‐type and mutant NDP kinase in Dictyostelium discoideum
Authors: Sellam Olivier , Véron Michel , Hildebrandt Martin
Published: October 2006

Nucleoside diphosphate (NDP) kinase has a central role in the synthesis of (deoxy‐)trinucleotides. In addition, mutations in the gene encoding NDP kinase have been shown to have important consequences for Drosophila development and mammalian tumorogenesis. We have overexpressed, in Dictyostelium discoideum, a genomic clone encoding the enzyme NDP kinase. The concomitant increase in the levels of RNA and enzyme activity identifies a 5′ non‐coding genomic region of 0.9 kb as being the complete promoter region. Overexpression of wild‐type NDP kinase has no effect on development. This is also true for an inactive mutant H122C that does not have a dominant inhibitor effect. Overexpression of the P105G mutant NDP kinase, which is known to be affected in its stability in vitro, only leads to a small increase in total NDP‐kinase activity. Thermal and chemical denaturation experiments demonstrate the formation of hexameric hybrids between wild‐type and mutant monomers.
Protein exporter function and in vitro ATPase activity are correlated in ABC‐domain mutants of HlyB
Authors: Koronakis Eva , Hughes Colin , Milisav Irina , Koronakis Vassilis
Published: October 2006

The Escherichia coli toxin exporter HlyB comprises an integral membrane domain fused to a cytoplasmic domain of the ATP‐binding casette (ABC) super‐family, and it directs translocation of the 110kDa haemolysin protein out of the bacterial cell without using an N‐terminal secretion signal peptide. We have exploited the ability to purify the soluble HlyB ABC domain as a fusion with glutathione S‐transferase to obtain a direct correlation of the in vivo export of protein by HlyB with the degree of ATP binding and hydrolysis measured in vitro. Mutations in residues that are invariant or highly conserved in the ATP‐binding fold and glycine‐rich linker peptide of prokaryotic and eukaryotic ABC transporters caused a complete less of both HlyB exporter function and ATPase activity in proteins still able to bind ATP effectively and undergo ATP‐induced conformational change. Mutation of less‐conserved residues caused reduced export and ATP hydrolysis, but not ATP binding, whereas substitutions of poorly conserved residues did not impair activity either in vivo or in vitro. The data show that protein export by HlyB has an absolute requirement for the hydrolysis of ATP bound by its cytoplasmic domain and indicate that comparable mutations that disable other prokaryotic and eukaryotic ABC transporters also cause a specific loss of enzymatic activity.
Differential expression of mal genes under cAMP and endogenous inducer control in nutrient‐stressed Escherichia coli
Authors: Notley Lucinda , Ferenci Thomas
Published: October 2006

LamB glycoporin has an important general role in carbohydrate uptake during growth at low extracellular sugar concentrations. lamB and mal regulon induction during glucose starvation and glucose‐limited continuous culture was investigated using lacZ fusions. A low‐level burst of lamB induction occurred upon entry into glucose starvation‐induced stationary phase but returned to basal levels during continued nutrient deprivation. Glucose‐limited continuous culture elicited much higher expression of transporter genes in the mal regulon, as well as [14C]‐maltose‐transport activity; malEFG and malKlamB operons in glucose‐limited chemostats were expressed to close to half of the level of maltose‐induced batch cultures. Limitation‐induced expression was dependent on both Crp‐cAMP and MalT activation but was independent of RpoS function. As expected for a gene with a Crp‐controlled promoter, malT expression was maximal under conditions which elicited the highest cAMP levels, but lamB induction did not behave in a corresponding fashion. Rather, maximal lamB induction occurred at rapid but suboptimal growth rates with micromolar or submicromolar medium glucose. Maximal transport and lamB induction coincided with increased endogenous maltotriose (inducer) concentrations during growth on glucose. Hence regulation of glycoporin and the maltose‐transport system is not a starvation‐ or stationary‐phase response but facilitates the adaptation of Escherichia coli to low‐nutrient environments through endoinduction.
Control of lytic development in the Streptomyces temperate phage φC31
Authors: Wilson Stuart E. , Ingham Colin J. , Hunter Iain S. , Smith Margaret C. M.
Published: October 2006

The repressor gene. c, is required for maintenance of lysogeny in the Streptomyces phage φC31. The c gene expresses three in‐frame N‐terminally different protein isoforms at least one of which is thought to bind to a 17bp highly conserved inverted repeat (CIR) sequence found at 18 (or more) loci throughout the φC31 genome. Here we present evidence that one of these loci, CIR6, and its interaction with the products of the repressor gene are critical in the control of the lytic pathway in φC31. To the right of CIR6, according to the standard map of φC31, an ‘immediate‐early’ promoter. ap1, was discovered after insertion of a fragment containing CIR6 upstream of a promoterless kanamycin‐resistance gene. aphll, to form pCIA2. pCIA2 conferred kanamycin resistance upon Streptomyces coelicolor A3(2) but not upon a φC31 lysogen of S. coelicolor. Operator‐constitutive (Oc) mutants of pCIA2 were isolated and the mutations lay in CIR6, i.e. CIR6:G14T and CIR6:C2A. Primer extension analysis of RNA prepared from an induced, temperature‐sensitive lysogen of S. coelicolor localized a mRNA 5′ endpoint 21 bp to the right of CIR6. The importance of the ap1/CIR6 region in the regulation of lytic growth was demonstrated by the analysis of a virulent mutant, φC31 vir1, capable of forming plaques on an S. coelicolorφC31 lysogen, φC31 vir1 contained a DNA inversion with the breakpoints lying within the integrase gene (which lies approximately 7kbp to the right of CIR6) and in the essential early region between CIR6 and the ‐10 sequence for ap1. The separation of ap1 from its operator was thought to be the basis for the virulent phenotype in φC31 vir1. Band‐shift assays and DNase I footprinting experiments using purified 42kDa repressor isoform confirmed that CIRs 5 and 6 were indeed the targets for binding of this protein. The 42 kDa repressor bound to CIR6 with higher affinity than to CIR5 in spite of their identical core sequences. Repressor bound at CIR6 facilitated binding at CIR5. The high‐affinity binding to CIR6 was abolished with the Oc mutant, CIR6:G14T. Hydroxyl radical footprinting and dimethyl sulphate methylation protection of the 42 kDa repressor–CIR6 interaction suggested that the protein bound in the major groove and to one face of the DNA.
Identification of novel HXT genes in Saccharomyces cerevisiae reveals the impact of individual hexose transporters on qlycolytic flux
Authors: Reifenberger E. , Freidel K. , Ciriacy M.
Published: October 2006

In Saccharomyces cerevisiae, hexose uptake is mediated by HXT proteins which belong to a superfamily of monosaccharide facilitators. We have identified three more genes that encode hexose transporters (HXT5, 6, 7). Genes HXT6 and HXT7 are almost identical and located in tandem 3′ adjacent to HXT3 on chromosome IV. We have constructed a set of congenic strains expressing none or any one of the seven known HXT genes and followed growth and flux rates for glucose utilization. The hxt null strain does not grow on glucose, fructose or mannose, and both glucose uptake and flux rate were below the detection level. Expression of either HXT1, 2, 3, 4, 6 or 7 is basically sufficient for aerobic growth on these sugars. In most of the constructs, glucose was the preferred substrate compared to fructose or mannose. There is a considerable variation in flux and growth rates with 1% glucose, dependent on the expression of the individual HXT genes. Expression of either HXT2, 6 or 7 in the null background is sufficient for growth on 0.1% glucose, while growth of strains with only HXT1, 3 or 4 requires higher (≥1%) glucose concentrations. These results demonstrate that individual HXT proteins can function independently as hexose transporters, and that most of the metabolically relevant HXT transporters from S. cerevisiae have been identified.
Collapse and repair of replication forks in Escherichia coli
Authors: Kuzminov Andrei
Published: October 2006

Single‐strand interruptions in a template DNA are likely to cause collapse of replication forks. We propose a model for the repair of collapsed replication forks in Escherichia coli by the RecBCD recombinational pathway. The model gives reasons for the preferential orientation of Chi sites in the E. coli chromosome and accounts for the hyper‐rec phenotype of the strains with increased numbers of single‐strand interruptions in their DNA. On the basis of the model we offer schemes for various repeat‐mediated recombinational events and discuss a mechanism for quasi‐conservative DNA replication explaining the recombinational repair‐associated mutagenesis.
The role of the YAP1 and YAP2 genes in the regulation of the adaptive oxidative stress responses of Saccharomyces cerevisiae
Authors: Stephen Duncan W. S. , Rivers Stuart L. , Jamieson Derek J.
Published: October 2006

The YAP1 and YAP2 genes encode yeast transcription factors of the c‐jun family. We show that yeast mutants deleted for either the YAP1 or the YAP2 genes are hypersensitive to oxidants, particularly H2O2, and that these genes play a role in regulating the induction of the H2O2 adaptive stress response in Saccharomyces cerevisiae. They do not significantly affect the regulation of the superoxide adaptive stress response. The intrinsic resistance of stationary‐phase and respiring yeast cells towards superoxide anions is unaffected by deletion of the YAP1 and YAP2 genes. However, resistance towards H2O2 under these conditions is significantly reduced. We show that expression of the yeast GSH1 gene (encoding γ‐glutamylcysteine synthetase) and the SSA1 gene (encoding an HSP70 isoform) are induced by oxidants. Unlike the SSA1 and thioredoxin (TRX2) genes, expression of the GSH1 gene is more strongly induced by superoxide anions than by H2O2. In the absence of added oxidants, transcription of the GSH1 gene is reduced in strains carrying the yap1 deletion. However, we show that Yap1 is not required for the superoxide anion‐mediated induction of GSH1 gene expression. Furthermore, while the H2O2‐mediated induction of SSA1 expression is shown to be YAP1 dependent, the heat‐shock‐mediated induction of the SSA1 gene does not require YAP1. We also present evidence to show that the YAP2 gene does not regulate the expression of the TRX2, SSA1 or GSH1 genes.
Molecular cloning and functional expression of bacteriophage PK1E‐encoded endoneuraminidase Endo NE
Authors: Gerardy‐Schahn Rita , Bethe Andrea , Brennecke Thomas , Mühlenhoff Martina , Eckhardt Matthias , Ziesing Stefan , Lottspeich Friedrich , Frosch Matthias
Published: October 2006

Homopolymeric α‐2,8‐linked sialic acid (PSA) has been found as a capsular component of sepsis‐ and meningitis‐causing bacterial pathogens, and on eukaryotic cells as a post‐translational modification of the neural cell adhesion molecule (NCAM). The polysaccharide is specifically recognized and degraded by a phage‐encoded enzyme, the endo‐N‐acetylneuraminidase E (Endo NE). Endo NE therefore has become a valuable tool in the study of bacterial pathogenesis and eukaryotic morphogenesis. In this report we describe the molecular cloning of Endo NE and the expression of a functionally active recombinant enzyme. The cloned DNA sequence (2436 bp) encodes a polypeptide of 811 amino acids, which at the 5′ end contains a totally conserved neuraminidase motif. Expressed in Escherichia coli, the enzyme migrates as a single band of approximately 74 kDa in SDS‐PAGE. A central domain of 669 amino acid residues is about 90% homologous to the recently cloned Endo NF. Both phage‐induced lysis of bacteria and the catalysis of PSA degradation by the recombinant enzyme are efficiently inhibited by a polyclonal antiserum raised against the intact phage particle. The C‐terminal region seems to be essential to enzymatic functions, as truncation of 32 amino acids outside the homology domain completely abolishes Endo NE activity. Our data also indicate that the 38 kDa protein, previously assumed to be a subunit of the Endo NE holoenzyme, is the product of a separate gene locus and is not necessary for in vitro depolymerase activity.
Identification and characterization of pilG, a highly conserved pilus‐assembly gene in pathogenic Neisseria
Authors: Tønjum Tone , Freitag Nancy E. , Namork Ellen , Koomey Michael
Published: October 2006

Expression of type IV pili appears to be a requisite determinant of infectivity for the strict human pathogens Neisseria gonorrhoeae and Neisseria meningitidis. The assembly of these colonization factors is a complex process. This report describes a new pilus‐assembly gene, pilG, that immediately precedes the gonococcal (Gc) pilD gene encoding the pre‐pilin leader peptidase. The nucleotide sequence of this region revealed a single complete open reading frame whose derived polypeptide displayed significant identities to the pilus‐assembty protein PilC of Pseudomonas aeruginosa and other polytopic integral cytoplasmic membrane constituents involved in protein export and competence. A unique polypeptide of Mr 38kDa corresponding to the gene product was identified. A highly related gene and flanking sequences were cloned from a group E polysaccharide‐producing strain of N. meningitidis (Mc). The results indicate that the pilG genes and genetic organization at these loci in Gc and Me are extremely conserved. Hybridization studies strongly suggest that pilG‐related genes exist in commensal Neisseria species and other species known to express type IV pili. Defined genetic lesions were created by using insertional and transposon mutagenesis and moved into the Gc and Me chromosomes by allelic replacement. Chromosomal pilG insertion mutants were devoid of pili and displayed dramatically reduced competence for transformation. These findings could not be ascribed to pilin‐gene alterations or to polarity exerted on pilD expression. The results indicated that PilG exerts its own independent role in neisserial pilus biogenesis.
The phoP locus influences processing and presentation of Salmonella typhimurium antigens by activated macrophages
Authors: Jo Wick Mary , Harding Clifford V. , Twesten Nicholas J. , Normark Staffan J. , Pfeifer John D.
Published: October 2006

The destruction and processing of bacteria by activated macrophages facilitates the presentation of antigens to T cells and thereby promotes the induction of specific immunity. The PhoP‐PhoQ regulatory system that controls the synthesis of many Salmonella proteins required for virulence and survival within macrophages is one mechanism that this particular intracellular pathogen has evolved to resist destruction. To address whether the phoP locus also influences antigen processing during the interaction of Salmonella typhimurium with macrophages, we tested the effect of phoP mutations on the processing and presentation of model antigens expressed by the bacteria. Activated macrophages processed phoP− bacteria with greater efficiency than wild‐type bacteria, as measured by the response of antigen‐specific T‐hybridoma cells; Salmonella constitutively expressing PhoP were processed even less efficiently than wild‐type Salmonella. After heat‐inactivation, however, both wild‐type and phoP− bacteria were efficiently processed. The altered processing and presentation efficiency was not due to differences in the level of antigen expressed by the bacteria or differences in the level of bacterial uptake by the macrophages. In addition, phoP‐regulated gene expression was shown to influence processing of antigen phagocytosed independently of the bacteria. Thus, phoP‐regulated gene products decrease the processing and presentation of S. typhimurium antigens, demonstrating a rote 1or this virulence locus in the inhibition of the induction of specific immunity.
Identification of a gene, pilV, required for type 4 fimbrial biogenesis in Pseudomonas aeruginosa, whose product possesses a pre‐pilin‐like leader sequence
Authors: Alm Richard A. , Mattick John S.
Published: October 2006

Type 4 fimbriae are important colonization factors in Pseudomonas aeruginosa and other pathogens that mediate attachment to epithelial cells of the host. They are also responsible for a form of translocation termed ‘twitching motility’ and are implicated in the susceptibility to fimbrial‐specific bacteriophage. Analysis of a transposon mutant which lacks functional fimbriae has identified a new gene which is required for fimbrial biogenesis. This gene, termed pilV, is located on chromosomal Spel fragment E, 2 kb downstream of the previously characterized pilSR genes involved in transcriptional activation of the fimbrial subunit gene. The pilV gene encodes a 20kDa membrane‐located protein with considerable amino‐terminal homology to the type 4 consensus pre‐pilin leader sequence, suggesting that it is processed by a leader peptidase. Site‐directed mutagenesis has shown that PilV requires such cleavage to be functional. PilV also exhibits close similarity to a group of proteins involved in extracellular protein secretion from a number of Gram‐negative bacteria, suggesting that the biogenesis of type 4 fimbriae may have a similar basis.
Characterization of a five‐cluster required for the biogenesis of type 4 fimbriae in Pseudomonas aeruginosa
Authors: Martin Paul R. , Watson Alison A. , McCaul Thomas F. , Mattick John S.
Published: October 2006

The opportunistic pathogen Pseudomonas aeruginosa produces type 4 fimbriae which promote adhesion to epithelial cells and are associated with a form of surface translocation called twitching motility. Transposon mutagenesis was used to identify loci required for fimbrial assembly or function by screening for mutants that lack the spreading colony morphology characteristic of twitching motility. Six mutants were isolated that contain transposon insertions upstream of the previously characterized gene pilQ. This region contains four genes: pilM‐P, which encode proteins with predicted sizes of 37.9, 22.2, 22.8 and 19.0 kDa, respectively, pilM‐P appear to form an operon and to be expressed from a promoter in the intergenic region between pilM and the divergently transcribed upstream gene ponA. PilM‐P were found to be required for fimbrial biogenesis by complementation studies using twitching motility and sensitivity to fimbrial‐specific phage as indicators of the presence of functional fimbriae. This was confirmed by electron microscopy. PilO and PilP did not have homologues in the sequence databases, but the predicted PilN amino acid sequence displayed similarity to XpsL from Xanthamonas campestris, a protein required for protein secretion. PilP contained a hydro‐phobic leader sequence characteristic of lipoproteins, while PilN and PilO have long internal hydrophobic domains which may serve to localize them to the cytoplasmic membrane. PilM has shared sequence motifs with the cell division protein FtsA from Bacillus subtilis and Escherichia coli, as well as the rod‐shapedetermining protein MreB from E. coli. These motifs are also conserved in eukaryotic actin, in which they are involved in forming an ATPase domain. Deletion mutants of pilM and pilQ displayed a dominant negative phenotype when transformed into wild‐type cells, suggesting that these genes encode proteins involved in multimeric structures.
Molecular population genetic analysis of the streptokinase gene of Streptococcus pyogenes: mosaic alleles generated by recombination
Authors: Kapur Vivek , Kanjilal Sagarika , Hamrick Marcie R. , Li Ling‐Ling , Whittam Thomas S. , Sawyer Stanley A. , Musser James M.
Published: October 2006

To understand the mechanisms governing molecular evolution of the streptokinase gene (skn), a 384 bp DNA fragment encoding two variable regions of the molecule was characterized in 47 isolates of Streptococcus pyogenes. The results reveal that alleles of the streptokinase gene have a mosaic structure, and provide strong evidence for intragenic recombination. Moreover, organisms that are well differentiated in overall chromosomal character have identical skn alleles, which suggests that horizontal gene transfer and recombination have participated in the evolution of this locus. No simple relationship between skn allele and serum opacity factor production or specific disease was identified. The predicted amino acid sequences of highly divergent skn alleles are strikingly similar in hydrophobicity and hydrophobicity profiles, distribution of amphipathic and flexible regions, surface probability plots, and antigenic indices, indicating that despite extensive nucleotide polymorphism in the two skn variable regions, selective pressure has constrained overall structural divergence. These results add to an important emerging theme that intragenic recombination plays a critical role in diversifying genes coding for streptococcal virulence factors.
Transcriptional regulation of the proton translocating NADH dehydrogenase (nuoA‐N) of Escherichia coli by electron acceptors, electron donors and gene regulators
Authors: Bongaerts Johannes , Zoske Sascha , Weidner Uwe , Linden Gottfried
Published: October 2006

The promoter region and transcriptional regulation of the nuoA‐N gene locus encoding the proton‐translocating NADH:quinone oxidoreductase was analysed. A 560 bp intergenic region upstream of the nuo locus was followed by a gene (designated irhA for LysRhomo‐logue A) coding for a gene regulator similar to those of the LysR family. Disruption of irhA did not affect growth (respiratory or non‐respiratory) or expression of nuo significantly. Transcriptional regulation of nuo by electron acceptors, electron donors and the transcriptional regulators ArcA, FNR, NarL and NarP, and by IHF (integration host factor) was studied with protein and operon fusions containing the promoter region up to base pair ‐277 (‘nuo277’) or up to base pair ‐899 (‘nuo899’). The expression of the nuo277‐lacZ fusions was subject to ArcA‐mediated anaerobic repression and NarL(+nitrate)‐mediated anaerobic activation. FNR and IHF acted as weak repressors under anaerobic conditions. Expression of nuo899‐lacZ was stimulated during anaerobic fumarate respiration and aerobically by C4 dicarboxylates. Therefore, expression of nuo is regulated by O2 and nitrate via ArcA, NarL, FNR and IHF at sites within the ‐277 region, and by other factors including C4 dicarboxylates at a site between ‐277 and ‐899. A physiological role for the transcriptional stimulation by O2 and nitrate is suggested.
Molecular genetics of the chloramphenicol‐resistance transposon Tn4451 from Clostridium perfringens: the TnpX site‐specific recombinase excises a circular transposon molecule
Authors: Bannam Trudi L , Crellin Paul K. , Rood Julian I.
Published: October 2006

The chloramphenicol‐resistance transposon Tn4451 undergoes precise conjugative deletion from its parent plasmid piP401 in Clostridium perfringens and precise spontaneous excision from multicopy plasmids in Escherichia coli. The complete nucleotide sequence of the 6338 bp transposon was determined and it was found to encode six genes. Genetic analysis demonstrated that the largest Tn4451‐encoded gene, tnpX, was required for the spontaneous excision of the transposon in both E. coli and C. perfringens, since a Tn4451 derivative that lacked a functional tnpX gene was completely stable in both organisms. Because the ability of this derivative to excise was restored by providing the tnpX gene on a compatible plasmid, it was concluded that this gene encoded a trans‐acting site‐specific recombinase. Allelic exchange was used to introduce the tnpXΔ allele onto plP401 and it was shown that TnpX was also required for the conjugative excision of Tn4451 in C. perfringens. It was also shown by hybridization and polymerase chain reaction (PCR) studies that TnpX‐mediated transposon excision resulted in the formation of a circular form of the transposon. The TnpX recombinase was unique because it potentially contained the motifs of two independent site‐specific recombinase families, namely the resolvase/invertase and integrase families. Sequence analysis indicated that the resolvase/invertase domain of TnpX was likely to be involved in the excision process by catalysing the formation of a 2bp staggered nick on either side of the GA dinucleotide located at the ends of the transposon and at the junction of the circular form. The other Tn4451‐encoded genes include tnpZ, which appears to encode a second potential site‐specific recombinase. This protein has similarity to plasmid‐encoded Mob/Pre proteins, which are involved in plasmid mobilization and multimer formation. Located upstream of the tnpZ gene was a region with similarity to the site of interaction of these mobilization proteins.