bluuebunny/crossref_metadata_2025 huggingface dataset:
Aerolysin — the ins and outs of a model channel‐forming toxin
Authors: Parker Michael W. , Van Der Goot F. Gisou , Buckley J. Thomas
Published: November 2003

Aerolysin is one of a large group of bacterial proteins that can kill target cells by forming discrete channels in their plasma membranes. The toxin has many properties in common with the porins of the Gram‐negative bacterial outer membrane, including an extensive amount of β‐structure, a high proportion of hydrophilic amino acid side‐chains and no hydrophobic stretches in the primary structure. It also oligomerizes to produce an insertion‐competent state. Aerolysin is secreted as a dimer by members of the Aeromonas family. It binds to a high‐affinity receptor on the target cell that has recently been shown to be a glycosylphosphatidylinositol‐anchored glycoprotein. Binding is followed by heptamerization to form a structure that we propose contains a β‐barrel which can insert into the membrane and produce a channel.
The local repressor AcrR plays a modulating role in the regulation of acrAB genes of Escherichia coli by global stress signals
Authors: Ma Dzwokai , Alberti Marie , Lynch Christy , Nikaido Hiroshi , Hearst John E.
Published: November 2003

Genes acrAB encode a multidrug efflux pump in Escherichia coli. We have previously reported that transcription of acrAB is increased under general stress conditions (i.e. 4% ethanol, 0.5 M NaCl, and the stationary phase in Luria‐Bertanl medium). In this study, lacZ transcriptional fusions and an in vitro gel mobility shift assay have been utilized to study the mechanisms governing the regulation of acrAB. We found that a closely linked gene, acrR, encoded a repressor of acrAB. Nevertheless, the general stress conditions increased transcription of acrAB in the absence of functional AcrR, and such conditions surprisingly increased the transcription of acrR even more strongly than that of acrAB. These results suggest that the general‐stress‐induced transcription of acrAB is primarily mediated by global regulatory pathway(s), and that one major role of AcrR is to function as a specific secondary modulator to fine tune the level of acrAB transcription and to prevent the unwanted overexpression of acrAB. To our knowledge, this represents a novel mechanism of regulating gene expression in E. coli. Evidence also suggests that the up‐regulation of acrAB expression under general stress conditions is not likely to be mediated by the known global regulators, such as MarA or SoxS, although elevated levels of these proteins were shown to increase the transcription of acrAB.
Post‐transcriptional regulation of CspA expression in Escherichia coli
Authors: Brandi Anna , Pietroni Paola , Gualerzi Claudio O. , Pon Cynthia L.
Published: November 2003

The Escherichia coli cspA gene, encoding the major cold‐shock protein CspA, was deprived of its natural promoter and placed in an expression vector under the control of the inducible λ PL promoter. After induction of transcription by thermal inactivation of the λ ts repressor, abundant expression of the product (CspA) was obtained if the cells were subsequently incubated at 10°C, but poor expression was obtained if the cells were incubated at 37°C or 30°C. The reason for this differential temperature‐dependent expression was investigated and it was found that: (i) the CspA content of the cells decreased more rapidly at 37°C compared to 10°C, regardless of whether transcription was turned off by addition of rifampicin; (ii) both the chemical and functional half‐lives of the cspA transcript were substantially longer at 10°C compared to 37°C; (iii) S30 extracts as well as 70S ribosomes prepared from cold‐shocked cells translated CspA mRNA (but not phage MS2 RNA) more efficiently than equivalent extracts or ribosomes obtained from control cells grown at 37°C; and (iv) purified CspA stimulated CspA mRNA translation. Overall, these results indicate that a selective modification of the cold‐shocked translational apparatus favouring translation of CspA mRNA, and an increased stability of this mRNA at low temperature, may play an important role in the induction of cspA expression during cold shock.
Differential mRNA stability of the cspA gene in the cold‐shock response of Escherichia coli
Authors: Goldenberg Daniel , Azar Idit , Oppenheim Amos B.
Published: November 2003

Exposure of bacterial cells to temperature changes induces the synthesis of a set proteins. We investigated the control of expression of the cspA gene, coding for the major cold‐shock protein of Escherichia coli. This protein was shown to be transiently induced upon shift to low temperature. We demonstrated that the cspA mRNA is extremely unstable at 37°C with a half‐life of approx. 10 s. Upon shift to 15°C cspA mRNA becomes highly stable. This mRNA stability is transient and is lost once the cells are adapted to the low temperature. Transcription fusions of lacZ containing part or most of the cspA gene do not show the rapid degradation at high temperature. Our results suggest that mRNA stability plays a major role in the control of the cspA gene. The expression of cspA is also regulated, to a smaller extent, by the relative increase in transcription after transfer to low temperature. A model by which cspA mRNA is regulated in response to temperature shift is discussed.
Molecular characterization of hybrid Tbp2 proteins from Neisseria meningitides
Authors: Legrain Michèle , Findeli Annie , Villeval Dominique , Quentin‐Millet Marie‐José , Jacobs Eric
Published: November 2003

Transferrin‐binding protein 2 (Tbp2) from Neisseria is an outer membrane‐associated extracellular lipoprotein that is involved in iron capture within the infected host. The analysis of tbp2 clones isolated from various bacterial strains revealed extensive divergences throughout the open reading frame (ORF), with predicted amino acid (aa) sequences displaying 47% to 83% identity. Such a variability is likely to have resulted from the selective pressure exerted by the host immune system, but raises questions regarding the existing constraints for conservation of protein function. Indeed, the neisserial Tbp2s include a large structured domain, extending throughout the N‐terminal half of the protein (∼270–290 aa), which is extremely stable and whose conformational integrity is required for efficient binding to human transferrin (hTf). In this work, a functional study of Tbp2s encoded by hybrid genes constructed by reassorting highly divergent tbp2 sequences in the region of the ORF encoding this structured domain was performed. The data demonstrate that the determinant intramolecular interactions allowing formation of a stable Tbp2 structure able to interact efficiently with hTf or/and that the Tbp2 residues involved in the interaction with hTf are not well conserved. However, a number of rearrangements appeared to generate genes encoding proteins which have retained structural stability and hTf‐binding capacity. This suggested that despite the extreme aa sequence divergence and the conformational constraints, horizontal genetic exchanges, which are known to occur in neisserial populations, may have contributed significantly to the generation of sequence variation within tbp2 ORFs. The analysis of two tbp2clones characterized in this work supports this hypothesis.
The unstable region of Streptomyces ambofaciens includes 210 kb terminal inverted repeats flanking the extremities of the linear chromosomal DNA
Authors: Leblond Pierre , Fischer Gilles , Francou François‐Xavier , Berger Franca , Guérineau Michel , Decaris Bernard
Published: November 2003

Physical maps of the chromosomes of three strains of Streptomyces ambofaciens were constructed by ordering AseI fragments generated from the genomic DNA as a single linear chromosome of about 8 Mb. The physical maps of the three strains were very similar. For strain DSM40697, a DraI map was obtained by positioning the DraI sites relative to the AseI map. Eighteen genetic markers as well as the deletable and amplifiable region were assigned to the AseI and DraI fragments in this strain. The resulting genetic map resembled that of Streptomyces coelicolor A3(2). The twoterminal AseI fragments exhibited retarded pulsed‐field gel electrophoresis mobility, demonstrating that proteins are covalently bound at this position. A restriction map of this region was made using four additional endonucleases. Repeated sequences present at both ends of the chromosome were mapped as long terminal inverted repeats stretching over 210 kb. This corresponds to the longest terminal inverted repeats so far characterized. The deletable region of S. ambofaciens was localized at the chromosomal extremities.
Ambidextrous transcriptional activation by SoxS: requirement for the C‐terminal domain of the RNA polymerase alpha subunit in a subset of Escherichia coli superoxide‐inducible genes
Authors: Jair Kam‐Wing , Fawcett William P. , Fujita Nobuyuki , Ishihama Akira , Wolf, Jr. Richard E.
Published: November 2003

Purified MalE–SoxS fusion protein specifically stimulated in vitro transcription of the Escherichia colizwffprfumCmicFnfo, and sodA genes, indicating that activation of the superoxide regulon requires only SoxS. As in vivo, a 21 bp sequence adjacent to the zwf promoter was able to activate transcription of an heterologous promoter in vitro. Activation of zwf and fpr transcription required the C‐terminal domain (CTD) of the RNA polymerase alpha subunit, while stimulation of fumCmicFnfo, and sodA transcription was independent of CTD truncation. Thus, like the catabolite gene activator protein (CAP), SoxS is an ‘ambidextrous’ activator, activation only requiring the α CTD in a subset of regulated promoters. Indeed, the −35 hexamers of the zwf and fpr promoters lie downstream of the respective MalE–SoxS binding sites, while the binding sites of fumCmicFnfo, and sodA overlap their −35 promoter hexamers.
Identification and characterization of genes required for post‐translational modification of Campylobacter coli VC167 flagellin
Authors: Guerry Patricia , Doig Peter , Alm Richard A. , Burr Donald H. , Kinsella Niamh , Trust Trevor J.
Published: November 2003

Two genes have been identified in Campylobacter coli VC167 which are required for the biosynthesis of post‐translational modifications on flagellin proteins. The ptmA gene encodes a protein of predicted Mr 28 486 which shows significant homology to a family of alcohol dehydrogenases from a variety of bacteria. The ptmB gene encodes a protein of predicted Mr 26 598 with significant homology to CMP‐N‐acetylneuraminic acid synthetase enyzmes involved in sialic acid capsular biosynthesis in Neisseria meninigitidis and Escherichia coli K1. Site‐specific mutation of either ptmA or ptmB caused loss of reactivity with antisera specific to the post‐translational modifications and a change in the isoelectric focusing fingerprints relative to the parent strains. Mutation of ptmB, but not of ptmA, caused a change in apparent Mr of the flagellin subunit in SDS–PAGE gels. The ptmA and ptmB genes are present in other strains of Campylobacter. In a rabbit model the ptmA mutant showed a reduced ability to elicit protection against subsequent challenge with heterologous strains of the same Lior serotype compared to the parental wild‐type strain. This suggests that the surface‐exposed post‐translational modifications may play a significant role in the protective immune response.
Characterization of a post‐translational modification of Campylobacter flagellin: identification of a sero‐specific glycosyl moiety
Authors: Doig Peter , Kinsella Niamh , Guerry Patricia , Trust Trevor J.
Published: November 2003

The flagellins of Campylobacter spp. differ antigenically. In variants of C. coli strain VC167, two antigenic flagellin types determined by sero‐specific antibodies have been described (termed T1 and T2). Post‐translational modification has been suggested to be responsible for T1 and T2 epitopes, and, using mild periodate treatment and biotin hydrazide labelling, flagellin from both VC167‐T1 and T2 were shown to be glycosylated. Glycosylation was also shown to be present on other Campylobacter flagellins. The ability to label all Campylobacter flagellins examined with the lectin LFA demonstrated the presence of a terminal sialic acid moiety. Furthermore, mild periodate treatment of the flagellins of VC167 eliminated reactivity with T1 and T2 specific antibodies LAH1 and LAH2, respectively, and LFA could also compete with LAH1 and LAH2 antibodies for binding to their respective flagellins. These data implicate terminal sialic acid as part of the LAH strain‐specific epitopes. However, using mutants in genes affecting LAH serorecognition of flagellin it was demonstrated that sialic acid alone is not the LAH epitope. Rather, the epitope(s) is complex, probably involving multiple glycosyl and/or amino acid residues.
Regulatory inputs for the synthesis of ComK, the competence transcription factor of Bacillus subtilis
Authors: Hahn J. , Luttinger A. , Dubnau D.
Published: November 2003

Competence in Bacillus subtilis is expressed post‐exponentially in response to signals which are interpreted by a complex network of regulatory proteins. This network culminates in the transcriptional activation of a set of late‐competence proteins that mediate DNA binding and uptake during transformation. ComK, a protein that binds to competence promoters and appears to activate their transcription, is itself synthesized in response to the signal‐transduction network. ComK is known to be required for the transcription of its own gene. We have placed comK under control of the xylose‐inducible PxylA promoter and used this construct to show that ComK synthesis is sufficient as well as necessary to induce competence. We have also confirmed that the Mec proteins act post‐transcriptionally to inactivate ComK, probably by protein–protein interaction. We have further demonstrated that ComS is required to generate an upstream signal that causes reversal of Mec‐induced inactivation of ComK. In addition to ComK itself, DegU, AbrB, and SinR are required for comK transcription; mutations in their genes are bypassed by PxylA–comK induction, and therefore their products appear not to act via the Mec proteins. Overproduction of ComK, in a loss‐of‐function mec mutant, is also known to bypass the need for DegU, SinR and AbrB. We propose that these proteins enhance the activity of ComK as a positive autoregulatory transcription factor, acting as co‐activator proteins when ComK is present at low concentrations. Finally, we demonstrate that when ComK is synthesized from the PxylA promoter and mecA is inactivated by mutation, no additional growth‐stage‐regulated control of competence can be detected.
Dramatic decay of phage transcripts in lactococcal cells carrying the abortive infection determinant AbiB
Authors: Parreira Ricardo , Ehrlich S. Dusko , Chopin Marie‐Christine
Published: November 2003

The abortive infection determinant AbiB prevents growth of the sensitive phage bIL170, but not of the resistant phage bIL41, on Lactococcus lactis strain IL1403. Here we show that AbiB promotes a dramatic degradation of sensitive phage transcripts, starting 10–15 min after infection. The decay of the transcripts is the probable cause of the arrest of the sensitive phage development. Mapping of the 5′ end of degradation products established that they result from endonucleolytic cleavage preferentially at U/U, A/U and U/A sites. We propose that an early product of the sensitive phage either induces the synthesis or stimulates the activity of an RNase in an AbiB+ cell.
The Escherichia coli enzoskeleton
Authors: Norris Vic , Turnock Geoff , Sigee Dave
Published: November 2003

The nature of the structure of the bacterial cell is becoming clearer. The envelope contains periseptal annuli, a discontinuous periplasm and adhesion sites, whilst the cytoplasmic membrane is probably organized into distinct proteolipid domains by the coupled transcription–translation–insertion (transertion) of membrane proteins. The structure of the nucleoid is determined by proteins which self‐associate and by attachment to membrane, which is achieved in part by transertion. Metabolic pathways form multi‐enzyme complexes which channel substrates and which connect membranes and nucleic acids to create the extensive, cross‐linked, intracellular structure we term the ‘enzoskeleton’. This enzoskeleton includes eukaryotic‐like cytoskeletal structures and elements such as the MukB and FtsZ proteins. We propose that the enzoskeleton is regulated by calcium and by protein phosphorylation during adaptation to different environments and during the cell cycle.
Translocation of surface antigen genes to a unique telomeric expression site in Pneumocystis carinii
Authors: Sunkin Susan M. , Stringer James R.
Published: November 2003

The surface of Pneumocystis carinii sp. f. carinii contains an antigen known as major surface glycoprotein (MSG), which is encoded by about 100 heterogeneous genes. Expression of MSG genes is not well understood. Previous work identified a sequence termed UCS, which is present at the beginning of nearly all MSG mRNAs, and which is likely to be involved in regulation of MSG gene transcription. Here we show that the UCS was present in one copy per haploid genome, but that different MSG genes were linked to the unique UCS locus in different members of a P. c. carinii population, predicting that individual organisms transcribe a limited number of MSG genes. This prediction was supported by indirect immunofluorescence observations. Comparison of three different populations of P. c. carinii showed that each contained a different set of MSG genes linked to the UCS, suggesting that UCS–MSG junctions are formed by recombination during population growth. Both the UCS and MSG genes were shown to be located at the ends of chromosomes, suggesting that the mechanism for UCS–MSG recombination is reciprocal exchange.
M‐related protein (Mrp) contributes to group A streptococcal resistance to phagocytosis by human granulocytes
Authors: Podbielski Andreas , Schnitzler Norbert , Beyhs Petra , Boyle Michael D. P.
Published: November 2003

The M protein has been postulated to be a major group A streptococcal (GAS) virulence factor because of its contribution to the bacterial resistance to opsono‐phagocytosis. Direct evidence of this was only provided for GAS strains which expressed a single M protein. The majority of GAS express additional, structurally similar M‐related proteins, Mrp and Enn, which have been described as IgG‐ and IgA‐binding proteins. To determine the involvement of Mrp and M protein in phagocytosis resistance, the mrp and emm genes from serotypes M2, M4, and M49 as well as from M‐untypeable strain 64/14 were insertionally inactivated. The mrp and emm mutants were subjected to direct bactericidal assays. As judged by numbers of surviving colony‐forming units, all mutant strains with the exception of the mrp4 mutant exhibited reduced multiplication factors as compared to the isogenic wild‐type strains. Subsequent analysis of phagocytosis by flow cytometry, measuring association of BCECF/AM‐labelled bacteria and granulocytes, paralleled the results from direct bactericidal assays regardless of whether isolated granulocytes or whole blood were utilized. Resistant wild‐type GAS strains bound to less than 24% of granulocytes, whereas phagocytosis‐sensitive controls attached to more than 90% of the white blood cells. 40 to 60% of the granulocytes associated with the mrp and emm mutants within 1 h of co‐incubation. Kinetic data suggested that attachment to granulocytes proceeds faster for emm mutants than for corresponding mrp mutants. By adding a dihydro‐rhodamine123 stain and measuring fluorescence induced by oxidative burst, the experimental data suggested that bacteria bound to granulocytes were also engulfed and integrated into phagolysosomes. Thus, these data indicated that, if present, both mrp and emm gene products contribute to phagocytosis resistance by decreasing bacterial binding to granulocytes.
Polynucleotide phosphorylase is necessary for competence development in Bacillus subtilis
Authors: Luttinger Amy , Hahn Jeanette , Dubnau David
Published: November 2003

comR (pnpA) is a newly identified gene in Bacillus subtilis that is necessary for the expression of late competence genes. Transformability of a comR (pnpA) mutant is 1–5% of that seen in comR + strains. Cloning and sequencing identified ComR as polynucleotide phosphorylase (PNPase). The PNPase amino acid sequence has 50% identity and 67% similarity with the Escherichia coli enzyme. Enzymatic assays show that this is the only PNPase activity in B. subtiliscomR (pnpA) is necessary for comG–lacZ and comK–lacZ expression, but this requirement is bypassed by a mecA disruption. In B. subtilis, the loss of PNPase has little effect on expression from a fusion of the srfA promoter directly to lacZ, butis necessary for normal expression from certain srfA–lacZ fusions that include portions of the normal srfA transcript. When a srfA–lacZ translational fusion is tested in isogenic pnpA + and pnpA derivatives of E. coli, lower expression is seen in the pnpA mutant. Since expression from lacZ fusions to comAsinR, and mecA appeared similar in the B. subtilispnpA and pnpA + strains, the loss of PNPase does not have a strong general effect on gene expression. These results suggest that PNPase may be necessary for modification of the srfA transcript in order to activate translation or stabilize the transcript, and that this may be necessary for competence development. This is the first evidence of post‐transcriptional effects on the development of competence in B. subtilis
Synthesis of the β and β′ subunits of Escherichia coli RNA polymerase is autogenously regulated in vivo by both transcriptional and translational mechanisms
Authors: Dykxhoorn Derek M. , St. Pierre Rebecca , Linn Thomas
Published: November 2003

Numerous experiments have indicated that the synthesis of RNA polymerase (ββ′α2σ70) in Escherichia coli is autogenously regulated. In the present study, we have examined expression of the rpoB and rpoC genes which encode the β and β′ subunits of RNA polymerase. These genes are the distal cistrons of the rplKAJLrpoBC ribosomal protein–RNA polymerase transcription unit. Both transcriptional (operon) and translational (gene) fusions of either rpoB or rpoC to the lacZ reporter were used to monitor their in vivo expression by inserting single copies of these fusions into the bacterial chromosome on integration‐proficient lambda vectors. In order to examine the expression of the rpoBC genes in response to varied intracellular concentrations of the RNA polymerase subunits, a series of expression plasmids carrying the rpoBrpoCrpoA (α) and rpoD (σ70) genes in different combinations were then introduced into these cells. Elevated concentrations of either β or β′ were found to repress the expression of both rpoB and rpoC at the translational level. However, the simultaneous increase in the concentration of all the subunits that comprise holoenzyme repressed the transcription of rpoBC. To determine the site of this repression, additional operon fusions were utilized which placed lacZ downstream of each of the transcriptional regulatory sites of this gene cluster, including two promoters, rplKp and rplJp, and a transcriptional attenuator in the rplL–rpoB intercistronic region. Expression from these fusions showed that transcriptional repression is achieved primarily by reducing initiation at both upstream promoters, coupled with a small increase in termination at the attenuator.
Cloning, characterization and disruption of a (p)ppGpp synthetase gene (relA) of Streptomyces coelicolor A3(2)
Authors: Chakraburtty Rekha , White Janet , Takano Eriko , Bibb Mervyn
Published: November 2003

An internal segment of the (p)ppGpp synthetase gene, relA, of Streptomyces coelicolor A3(2) was amplified from genomic DNA using the polymerase chain reaction and used as a hybridization probe to isolate the complete gene from a cosmid library. relA lies downstream of a gene (apt) that apparently encodes adenine phosphoribosyltransferase and is transcribed from two promoters, relAp1 and relAp2, and by transcriptional readthrough from apt. While the level of relAp2 transcripts remained relatively constant, relAp1 activity apparently peaked during transition phase, following a decline in readthrough transcription from apt. Disruption of relA using an att− derivative of the temperate phage φC31 abolished ppGpp synthesis on amino acid depletion. When grown on agar, the disruptants grew more slowly than a control lysogen made with an att+ phage vector and gave smaller colonies that sporulated normally. The relA mutation had no consistent or marked effect on actinorhodin production in either liquid‐ or agar‐grown cultures, indicating that elevated levels of (p)ppGpp are not essential for triggering the onset of antibiotic production.
Physical and genetic map of the Pseudomonas fluorescens SBW25 chromosome
Authors: Rainey Paul B. , Bailey Mark J.
Published: November 2003

Pseudomonas fluorescens is a saprophytic bacterium commonly isolated from soil, water, and the surfaces and tissues of plants and animals. The species has important applications in biotechnology because it can enhance plant growth and protect crops against disease. A complete physical map of the 6.63 Mbp P. fluorescens SBW25 chromosome was constructed using data obtained from combinations of one‐ and two‐dimensional electrophoresis of completely or partially digested chromosomal DNA with end labelling. In total, 139 restriction sites (15 PacI, 53 SpeI, 71 XbaI) were placed on the physical map and complete maps of the circular chromosome were obtained for both PacI and SpeI; only XbaI fragments linking SpeI fragments were positioned. The average resolution of restriction sites was 48 kbp. A genetic map was derived from the physical map by Southern hybridization and 31 genes were positioned including oriC, rDNA operons (rnnA–E), recAgacA and pyvD
Identification of an essential core element and stimulatory sequences in a Kluyveromyces lactis ARS element, KARS101
Authors: Fabiani Lucia , Frontali Laura , Newlon Carol S.
Published: November 2003

A Kluyveromyces lactis chromosomal sequence of 913 bp is sufficient for replication in Saccharomyces cerevisiae and K. lactis. This fragment contains a 12 bp sequence 5′‐ATTTATTGTTTT‐3′ that is related to the S. cerevisiae ACS (ARS consensus sequence). This dodecamer was removed by site‐directed mutagenesis and the effect on K. lactis and S. cerevisiae ARS (autonomous replicating sequence) activity was determined. The dodecamer is essential for S. cerevisiae ARS function but only contributes to K. lactis ARS activity; therefore, its role in K. lactis is unlikely to be the same as that of the essential S. cerevisiae ACS.

A 103 bp subclone was found to retain ARS activity in both yeasts, but the plasmid was very unstable in S. cerevisiae. Deletion and linker substitution mutagenesis of this fragment was undertaken to define the DNA sequence required for K. lactis ARS function and to test whether the sequence required for ARS activity in K. lactis and S. cerevisiae coincide. We found a 39 bp core region essential for K. lactis ARS function flanked by sequences that contribute to ARS efficiency. The instability of the plasmid in S. cerevisiae made a fine‐structure analysis of the S. cerevisiae ARS element impossible. However, the sequences that promote high‐frequency transformation in S. cerevisiae overlap the essential core of the K. lactis ARS element but have different end‐points.
Methylation segments are not required for chemotactic signalling by cytoplasmic fragments of Tsr, the methyl‐accepting serine chemoreceptor of Escherichia coli
Authors: Ames Peter , Yu Yong A. , Parkinson John S.
Published: November 2003

The serine chemoreceptor Tsr and other methyl‐accepting chemotaxis proteins (MCPs) control the swimming behaviour of Escherichia coli by generating signals that influence the direction of flagellar rotation. MCPs produce clockwise (CW) signals by stimulating the autophosphorylation activity of CheA, a cytoplasmic histidine kinase, and counter‐clockwise signals by inhibiting CheA. CheW couples CheA to chemoreceptor control by promoting formation of MCP/CheW/CheA ternary complexes. To identify MCP structural determinants essential for CheA stimulation, we inserted fragments of the tsr coding region into an inducible expression vector and used a swimming contest called ‘pseudotaxis’ to select for transformant cells carrying CW‐signalling plasmids. The shortest active fragment we found, Tsr (350–470), stimulated CheA in a CheW‐dependent manner, as full‐length Tsr molecules do. It spans a highly conserved ‘core’ (370–420) that probably specifies the CheA and CheW contact sites and other determinants needed for stimulatory control of CheA. Tsr (350–470) also carries portions of the left and right arms flanking the core, which probably play roles in regulating MCP signalling state. However, this Tsr fragment lacks all of the methylation sites characteristic of MCP molecules, indicating that methylation segments are not essential for generating receptor output signals.
Photobiology of microorganisms: how photosensors catch a photon to initialize signalling
Authors: Hellingwerf Klaas J. , Hoff Wouter D. , Crielaard Wim
Published: November 2003

Photobiological processes are relevant for microorganisms for energy generation, protection against excess and/or damaging radiation, and for signalling. In this review we give an overview of the knowledge on the functioning of photosensors in microorganisms, with special emphasis on the conformational changes that lead to signal generation and transduction. Light is absorbed by specific chromophores, which are tuned, by their proteinaceous environment, to function optimally. These chromophores belong to three classes: tetrapyrroles, polyenes and aromatics. The chemical structure of photosensing pigment/protein complexes has been resolved for many of the photobiological processes that have a characteristic sensitivity in the visible and infrared part of the spectrum of (solar) radiation. However, knowledge about the structure of photoreceptors responsible for several physiologically well‐characterized photoresponses to UV‐ and blue light is still lacking. For a limited number of phototransduction processes, the details of light‐induced signal transfer are beginning to be understood in atomic detail. This applies particularly to two photosensors involved in phototactic responses in bacteria: sensory rhodopsin I (SR‐I) from Halobacterium salinarium and photoactive yellow protein (PYP) from Ectothiorhodospira halophila. The SR‐I system is of special interest because the transducer accepting the signal from SR‐I was recently identified as Htr‐I, a homologue of the methyl‐accepting chemotaxis proteins which have been characterized in Escherichia coli. PYP, on the other hand, may be the first photosensor to actually reveal all relevant details of the kinetics, thermodynamics, and molecular motion of light‐induced signal generation, through an understanding of how the photo‐isomerization of the chromophore forces the sensor protein into the signalling state. Here we compare these photosensors and discuss common themes in the initiation of photosensory signal transduction in microorganisms in terms of the molecular properties of photosensors and their signalling state.
The bld mutants of Streptomyces coelicolor are defective in the regulation of carbon utilization, morphogenesis and cell–cell signalling
Authors: Pope Margaret K. , Green Brian D. , Westpheling Janet
Published: November 2003

Mutants of Streptomyces coelicolor blocked at the earliest visible stage of morphological differentiation are called bld mutants. These mutants fail to form aerial hyphae on rich medium and most are defective in antibiotic production. One striking feature of these mutants is that, with the exception of bldB, their morphological defect is carbon‐source dependent. In our investigation of catabolite control in Streptomyces, we identified mutants that were resistant to glucose repression and were also bld. The existence of these new bld mutants led us to examine the catabolite control phenotype of the previously described bld mutants which were not known to contain defects in carbon regulation. We report here that all of the characterized bld mutants of S.coelicolor are defective in the regulation of galP1, and that at least one of the bld mutants, bldB, is globally deregulated for carbon utilization. Complementation of the morphological defect of bldA and bldB mutants with a cloned copy of the wild‐type bld gene simultaneously restored normal regulation of galP1, indicating that both aspects of the mutant phenotype are caused by the same lesion. We suggest a new interpretation for the role of the bld genes in development in Streptomyces. We suggest that the primary defect in bld mutants is in the regulation of carbon utilization, not specifically in the activation of genes whose products regulate the development pathway as previously suggested. We speculate that the inability of bld mutants to initiate morphogenesis is a secondary consequence of their inability to sense and/or signal starvation.
Analysis of the role of the Pseudomonas syringae pv. syringae HrpZ harpin in elicitation of the hypersensitive response in tobacco using functionally non‐polar hrpZ deletion mutations, truncated HrpZ fragments, and hrmA mutations
Authors: Alfano James R. , Bauer David W. , Milos Timothy M. , Collmer Alan
Published: November 2003

Pseudomonas syringae pv. syringae, like many plant pathogenic bacteria, secretes a ‘harpin’ protein that can elicit the hypersensitive response (HR), a defensive cellular suicide, in non‐host plants. The harpin‐encoding hrpZ gene is located in an operon that also encodes Hrp secretion pathway components and is part of the functional cluster of hrp genes carried on cosmid pHIR11 that enables saprophytic bacteria like Escherichia coli and Pseudomonas fluorescens to elicit the HR in tobacco leaves. We have constructed functionally non‐polar hrpZ deletion mutations, revealing that HrpZ is necessary for saprophytic bacteria carrying pHIR11 to elicit a typical HR, whereas it only enhances the elicitation activity of P. s. syringae. Partial deletion mutations revealed that the N‐terminal 153 amino acids of HrpZ can enable E. coli MC4100‐(pHIR11) to elicit a strong HR. hrpZ subclone products comprising the N‐terminal 109 amino acids and C‐terminal 216 amino acids, respectively, of the 341 amino acid protein were isolated and found to elicit the HR. P. fluorescens (pHIR11 hrmA ::TnphoA) mutants do not elicit the HR, but cell fractionation and immunoblot analysis revealed that they produce and secrete wild‐type levels of HrpZ. Therefore, elicitor activity resides in multiple regions of HrpZ, P. syringae produces elicitor(s) in addition to HrpZ, and HrpZ is essential but not sufficient for HR elicitation by saprophytic bacteria carrying pHIR11.
A physical and genetic map of Neisseria meningitidis B1940
Authors: Gäher Martin , Einsiedler Kerstin , Crass Torsten , Bautsch Wilfried
Published: November 2003

A physical map of the chromosome of Neisseria meningitidis B1940 has been constructed by one‐ and two‐dimensional pulsed‐field gel electrophoresis techniques. Complete macrorestriction maps for the enzymes NheI (16 sites), SgfI (13 sites), SfiI (11 sites) and I‐CeuI (4 sites), as well as a partial restriction map for the restriction enzyme SpeI (15 of c. 28 sites) could be established. Altogether, 59 restriction sites were mapped on a single circular chromosome of 2.3 Mbp. By restriction endonuclease digestion and Southern hybridization of cloned genetic markers, 39 genetic loci were assigned to this map. Comparison with the metabolic maps of Neisseria gonorrhoeae MS11‐N198 and FA1090 revealed a high degree of conservation in the arrangement of gene loci among these two species, although four out of 24 genetic loci are located at different chromosomal positions, indicating several genomic rearrangements.
The Clp ATPases define a novel class of molecular chaperones
Authors: Wawrzynow Alicja , Banecki Bogdan , Zylicz Maciej
Published: November 2003

The Clp ATPases were originally identified as a regulatory component of the bacterial ATP‐dependent Clp serine proteases. Proteins homologous to the Escherichia coli Clp ATPases (ClpA, B, X or Y) have been identified in every organism examined so far. Recent data suggest that the Clp ATPases are not only specificity factors which help to ‘present’ various protein substrates to the ClpP or other catalytic proteases, but are also molecular chaperones which can function independently of ClpP. This review discusses the recent evidence that the Clp ATPases are indeed molecular chaperones capable of either repairing proteins damaged during stress conditions or activating the initiation proteins for Mu, λ or P1 DNA replication. A mechanism is suggested to explain how the Clp ATPases ‘decide’ whether to repair or destroy their protein substrates.
A Salmonella typhimurium htrA live vaccine expressing multiple copies of a peptide comprising amino acids 8–23 of herpes simplex virus glycoprotein D as a genetic fusion to tetanus toxin fragment C protects mice from herpes simplex virus infection
Authors: Chabalgoity J. A. , Khan C. M. A. , Nash A. A. , Hormaeche C. E.
Published: November 2003

Multiple tandem copies of an immunogenic epitope comprising amino acids 8–23 of glycoprotein D of herpes simplex virus (HSV) were expressed as C‐terminal fusions to tetanus toxin fragment C (TetC) in different Salmonella typhimurium live vaccine strains. Expression of the longer fusions was best in strains harbouring a lesion in htrA, a stress protein gene. SL3261, an aroA strain, did not effectively express the longer fusions. Mice immunised with an S. typhimurium C5 htrA mutant expressing fusions with two or four copies of the peptide made an antibody response to both the peptide and TetC, whereas constructs expressing one copy of the peptide only elicited antibody to TetC. A non‐immunogenic octameric fusion underwent rearrangements in vivo resulting in a predominantly monomeric fusion. In contrast, the S. typhimurium SL3261 aroA vaccine expressing the TetC‐tetrameric fusion did not elicit antibody to the peptide. Sera from mice immunised with a single dose of the dimer and tetramer fusions in the htrA strain neutralised HSV in vitro, and the mice were protected from HSV infection as measured by a reduction in virus load in the ear pinna. We have previously shown that mice vaccinated with salmonella expressing TetC are protected against tetanus toxin and virulent salmonella challenge. These results suggest that it may be possible to develop a multivalent vaccine against salmonellosis, tetanus and HSV.
Three novel plasmid R6K proteins act in concert to distort DNA within the α and β origins of DNA replication
Authors: Flashner Yehuda , Shlomai Joseph , Shafferman Avigdor
Published: November 2003

Three novel R6K genes which are responsible for expression of DNA distortion polypeptides (DDP) were identified. The DDPs act in vivo in concert to induce similar stepwise DNA helix distortions within two long inverted repeats (αLIR and βLIR), which are essential elements for the two distally located R6K α and β DNA replication origins. DDP1 and DDP2 are encoded by two tandem genes located at the 5′ end of αLIR, whereas a gene coding for DDP3 is located at the 3′ end of βLIR. DDP1 and DDP2 are required for primary DNA distortion within αLIR or βLIR, while DDP3 is essential for generation of secondary DNA distortion in these LIR sequences. Creation of DNA distortion within αLIR depends on its specific interaction with DDP1 and on the presence of the R6K primase DNA‐binding site. The possible relevance of these findings to R6K replication is discussed.
Overlapping functions for recF and priA in cell viability and UV‐inducible SOS expression are distinguished by dnaC809 in Escherichia coli K‐12
Authors: Sandler Steven J.
Published: November 2003

The recF and priA genes have roles in DNA repair and homologous recombination. Mutations in these genes also cause decreases in cell viability and alterations in UV‐inducible sulAp–lacZ (SOS) expression. To find out if the two genes are in the same or different pathways for viability and SOS expression, the phenotypes of the double mutant strains were studied. The recF priA double mutant showed a lower viability and SOS expression level than either of the single mutants. In the case of cell viability, recF missense mutations decreased viability of a priA2::kan strain two to fivefold whereas recF null priA2::kan double mutants were not viable at all. dnaC809, a mutation that suppresses the UV‐sensitive (UVS) and Rec− phenotypes of priA2::kan, restored cell viability, but not UV‐inducible SOS expression, to a priA recF strain. Since recF is epistatic with recO and recR (recOR) for UV resistance, recOR mutations were also tested with priA2::kan. No overlap was found between recOR and priA for viability and SOS expression. It is concluded that priA and recF have two different overlapping functions in viability and SOS expression that are distinguishable by the effects of dnaC809. The role of recF in a priA2::kan strain in cell viability is a new function for recF and unlike recF 's other roles in DNA repair and recombination, is independent of recOR. A new role for priA in UV‐inducible SOS expression in a recF mutant is also defined.
Expression of Shigella sonnei lipopolysaccharide in Vibrio cholerae
Authors: Viret Jean‐François , Cryz, Jr Stanley J. , Favre Didier
Published: November 2003

Making use of a newly designed mobilizable suicide vector, the genetic determinants encoding Shigella sonnei lipopolysaccharide (LPS) were stably integrated into the chromosome of the live attenuated Vibrio cholerae vaccine strain CVD103‐HgR. Expression studies showed that the production of complete S. sonnei O‐polysaccharide (O‐PS)‐bearing LPS was limited in bivalent recombinant strains that were also proficient in the synthesis of the host‐encoded Inaba O‐PS. Conversely, high amounts of LPS carrying S. sonnei O‐PS are produced in monovalent Inaba‐deficient derivatives, even in those strains which do not co‐express the compatible R1 LPS core. Thus, the non‐enterobacterial V. cholerae LPS core efficiently acts as a receptor for covalent binding of S. sonnei O‐PS provided that competition with the host O‐PS is avoided. Expression of the R1 core interferes with cell division in recombinant V. cholerae without affecting other physiological properties of vaccine strain CVD103‐HgR. Both monovalent and bivalent strains stimulated high serum‐antibody titres specific for their respective O‐serotype(s) when administered to rabbits. The potential of V. cholerae as an expression carrier for heterologous O‐serotypes is discussed.
The mmsA locus of Streptococcus pneumoniae encodes a RecG‐like protein involved in DNA repair and in three‐strand recombination
Authors: Martin Bernard , Sharples Gary J. , Humbert Odile , Lloyd Robert G. , Claverys Jean‐Pierre
Published: November 2003

We describe the characterization of a mutant strain of Streptococcus pneumoniae previously isolated on the basis of its sensitivity to Methyl Methane Sulphonate (MMS). The mutant strain also exhibited increased sensitivity to UV light and to X‐rays, together with a reduced capacity for recombination and Hex‐mediated generalized mismatch repair. We show that the original mutant contains two unlinked mutations in the mmsA and in the pms genes. The mmsA wild‐type region was cloned and the nucleotide sequence of the mmsA gene was determined. mmsA encodes a polypeptide of 671 amino acids related to a large family of DNA–RNA helicases, with the highest similarity to Escheri‐chia coli RecG, a protein involved in the branch migration of Holliday junctions. A plasmid carrying the intact mmsA coding region was shown to restore UV resistance to E. coli recG mutant strains. An mmsA‐null mutant constructed by insertion of a chloramphenicol‐resistance gene exhibited a 25‐fold reduction in recombination during transformation. We suggest that MmsA recognizes and branch migrates three‐strand transformation intermediates to extend donor–recipient heteroduplex regions. The mmsA‐null mutant exhibited the other phenotypes of the original mutant, apart from mismatch‐repair deficiency and, in addition, an alteration in colony‐forming ability was noticed. In the pms mutant background, all phenotypes caused by the mmsA mutation were attenuated. Therefore, the pms mutation, although it affected mismatch repair and, to some extent, DNA repair and recombination, acted as a suppressor of the mmsA mutation.
Suppression of transcription polarity in the Escherichia coli haemolysin operon by a short upstream element shared by polysaccharide and DNA transfer determinants
Authors: Nieto Jose M. , Bailey Marc J. A. , Hughes Colin , Koronakis Vassilis
Published: November 2003

Expression of the Escherichia coli hlyCABD operon encoding synthesis, maturation and export of haemolysin toxin was strongly dependent upon a 35 bp DNA sequence, spanning the element GGCGGTAG, located 2 kbp upstream. When the hly operon was placed under the control of the inducible tac promoter, expression remained dependent upon this element, when transcribed in its native orientation 3′ of the promoter. The increase in ptac‐directed transcription was strongest for the distal, export genes of the hly operon, and was particularly striking when ptac and the element were placed far upstream. The element did not influence transcript stability, and we suggest that it is a key component of a novel regulatory mechanism may suppresses transcription polarity within operons. The mechanism that be of widespread importance in bacterial gene expression because the 8 bp element is present in many Gram‐negative species as an upstream component of operons encoding the production of toxins and the surface assembly of polysaccharides and components required for the conjugal transfer of DNA. We name it the ops element for operon polarity suppressor.
A novel DnaA protein‐binding site at 94.7 min on the Escherichia coli chromosome
Authors: Kitagawa Risa , Mitsuki Hironobu , Okazaki Tuneko , Ogawa Tohru
Published: November 2003

There is a DNA sequence which has unusually high affinity for the DnaA protein of Escherichia coli between the glyV and amiB–mutL operons at 94.7 min on the genetic map. Affinity of DnaA protein for DNA was measured in vivo as the activity of β‐galactosidase produced from the lacZ gene on a plasmid fused to the 5′‐terminal portion of the mioC gene, which is under the control of the DnaA protein. The chromosomal DNA segment between the two operons, carried on a compatible plasmid, derepressed the β‐galactosidase activity by titrating DnaA protein. Derepression occurred on the chromosomal dnaA gene as well, since it is autoregulated. Hence, as measured by immunoassays, one plasmid molecule carrying the DnaA‐binding region titrated 370 DnaA molecules, which is a value eightfold higher than that for a plasmid containing the oriC region. We estimate that about 60% of the total cellular DnaA molecules are bound to this site. Four DnaA‐binding sequences (DnaA boxes) and a DnaA‐regulated promoter directing transcription of two small genes were present within a 250 bp stretch in this region but additional long DNA regions, including the fifth DnaA box located about 650 bp downstream, were required for maximum binding. A role for the DnaA‐binding site in controlling DnaA‐protein concentration in the cell cycle is discussed.