bluuebunny/crossref_metadata_2025 huggingface dataset:
Candida albicans ALS1: domains related to a Saccharomyces cerevisiae sexual agglutinin separated by a repeating motif
Authors: Hoyer L. L. , Scherer S. , Shatzman A. R. , Livi G. P.
Published: October 2006

Transfer of budding Candida albicans yeast cells from the rich, complex medium YEPD to the defined tissue culture medium RPMl 1640 (RPMI) at 37°C and 5% CO2 causes rapid onset of hyphal induction. Among the genes induced under these conditions are hyphal‐specific genes as well as genes expressed in response to changes in temperature, CO2 and specific media components. A cDNA library constructed from cells incubated for 20 min in RPMI was differentially screened with yeast (YEPD)‐ and hyphal (RPMI)‐specific probes resuming in identification of a gene expressed in response to culture conditions but not regulated by the yeast‐hyphal transition. The deduced gene product displays significant identity to Saccharomyces cerevisiaeα‐agglutinin, encoded by AGα1, an adhesion glycoprotein that mediates mating of haploid cells. The presence of this gene in C albicans is curious since the organism has not been observed to undergo meiosis. We designate the C. albicans gene ALS1 (for agglutinin‐like sequence). While the N‐ and C‐termini of the predicted 1260‐amino‐acid ALS1 protein resemble those of the 650‐amino‐acid AGα1, ALS1 contains a central domain of tandem repeats consisting of a highly conserved 36‐amino‐acid sequence not present in AGb1. These repeats are also present on the nucleotide level as a highly conserved 108bp motif. Southern and Northern blot analyses indicate a family of C. albicans genes that contain the tandem repeat motif; at least one gene in addition to ALS1 is expressed under conditions similar to those for ALS1 expression. Genomic Southern blots from several C. albicans isolates indicate that the number of copies of the tandem repeat element in ALS1 differs across strains and. In some cases, between ALS1 alleles in the same strain, suggesting a strain‐dependent variability in ALS1 protein size. Potential roles for the ALS1 protein are discussed.
Molecular characterization of an Aeromonas salmonicida mutant with altered surface morphology and increased systemic virulence
Authors: Noonan Brian , Trust Trevor J.
Published: October 2006

The asoA gene of Aeromonas salmonicida is located approximately 7 kb downstream of the A‐layer structural gene, vapA. A 6 kb Bam HI fragment containing aso A was cloned and marker‐exchange mutagenesis using a kanamycin‐resistance cassette was performed to generate an aso A mutation in the low‐virulence strain A449L. When analysed by electron microscopy, the mutant A449L‐MB exhibited an altered surface morphology. Strands and blebs of membranous material were observed protruding from the disorganized cell surface. This material was shown to contain lipopolysaccharide and A‐layer subunit protein. The disorganization of the surface of A449L‐IV1B had no apparent effect on virulence when the bacteria were administered to rainbow trout (Oncorhynchus mykiss) by bath Immersion. However, when administered by intraperitoneal injection, the mutant A449L‐MB was found to exhibit significantly increased virulence. The predicted amino acid sequence of AsoA shows homology to a number of polytopic membrane proteins involved in translocation across the cytoplasmic membrane.
Reversibility of DnaA protein activity in the‘irreversible’dnaA204 mutant of Escherichia coli
Authors: Hansen Flemming G. , Atlung Tove
Published: October 2006

The dnaA204 mutant, one of the so‐called irreversible dnaA mutants which cannot reinitiate chromosome replication upon a shift from non‐permissive to permissive growth temperature in the absence of protein synthesis, was reinvestigated using flow cytometry and marker frequency analysis. In a temperature downshift experiment and in the presence of protein synthesis the dnaA204 mutant reinitiates chromosome replication very fast. Using a lac promoter‐controlled wild type or a dnaA204 mutant gene carried on a plasmid, we have observed instantaneous initiation of replication when synthesis of DnaA protein is induced in the dnaA204 mutant at 42δC. The data indicate that the dnaA204 mutant after a shift to 42δC still contains functional DnaA protein, but that the activity level is below the initiation threshold. Thus, after synthesis of very small amounts of additional DnaA protein, initiation occurs very fast both after a shift to 30δC, and after induction of DnaA protein synthesis at 42 C. A model describing the processing of DnaA protein in mutants and in the wild type Is presented.
The hrpRS locus of Pseudomonas syringae pv. phaseolicola constitutes a complex regulatory unit
Authors: Grimm C. , Aufsatz W. , Panopoulos N. J.
Published: October 2006

The right part of the hrp cluster of Pseudomonas syringae pv. phaseolicola contains two regulatory genes, the previously described hrpS gene and an adjacent locus, hrpR. In this study we determined the sequence of hrpR and analysed the functional organization of the two genes. HrpR and HrpS show high sequence similarities to each other and to other response regulators of the two‐component regulatory system. This has recently also been described for the hrpRS system of the closely related pathogen Pseudomonas syringae pv. syringae. The results of our genetic analyses strongly indicate that hrpS expression is regulated by the hrpR gene product. DNA‐protein binding studies and site‐directed mutagenesis of the hrpR sequence provided further evidence that HrpR activates hrpS transcription by binding to an activator site. This HrpR binding site has mapped in a fragment which is located 378–609 nucleotides upstream of the hrpS transcription start site. The hrpS transcription start site maps 179 nucleotides upstream of the initiation codon ATG, as determined by primer extension analysis, and is preceded by a typical ‐12/‐24 promoter motif.
Expression of the ATP‐dependent deoxyribonuclease of Bacillus subtilis is under competence‐mediated control
Authors: Haijema Bert Jan , Hamoen Leendert Willem , Kooistra Jan , Venema Gerard , van Sinderen Douwe
Published: October 2006

Transcription of the ATP‐dependent deoxynuclease operon (addAB), as monitored by means of an addAB–lacZ transcriptional fusion, has a low, constitutive level and is initiated from a σA type promoter. Transcription of addAB is independent of DNA‐damaging agents known to induce the SOS response in Bacillus subtilis. However, addAB transcription increased significantly during competence development. This competence‐specific induction was dependent on the gene products of srfA., degU and comK, but not on that of recA. Deletion analysis of the addAB promoter region demonstrated that the competence‐specific transcription induction requires DNA sequences located upstream of the addAB promoter that associated with ComK, the competence transcription factor. The latter finding indicates that a direct regulatory link exists between the establishment of the competent state and the synthesis of AddAB, required for recombination of internalized donor DNA.
Missense mutations in the catalase‐peroxidase gene, katG, are associated with isoniazid resistance in Mycobacterium tuberculosis
Authors: Heym Beate , Alzari Pedro M. , Honore Nadine , Cole Stewart T.
Published: October 2006

The toxicity of the powerful anti‐tuberculosis drug isoniazid (INH) is believed to be mediated by the haem‐containing enzyme catalase‐peroxidase, encoded by the katG gene of Mycobacterium tuberculosis. Compelling evidence for this was obtained by studying a panel of INH‐resistant clinical isolates using a novel strategy based on the polymerase chain reaction and single‐strand‐conformation polymorphism analysis (PCR‐SSCP) to detect mutations in katG. In most cases INH resistance was associated with missense mutations while in a small number of strains the gene had been completely, or partially, deleted. The missense mutations fell into two groups, the larger of which contained several independent mutations that affected the N‐terminal peroxidase domain of the protein, resulting in the production of a catalase peroxidase with strongly reduced enzyme activity and increased heat lability. The effects of these substitutions could be interpreted by means of molecular modelling using the crystal structure of the related enzyme cytochrome c peroxidase from yeast as a template. The second group comprises a frequently occurring amino acid substitution and a single mutation that are both located in the C‐terminal domain but do not noticeably alter either enzyme activity or heat stability.
Evidence for a ppGpp‐binding site on Escherichia coli RNA polymerase: proximity relationship with the rifampicin‐binding domain
Authors: Reddy Padmalatha S. , Raghavan Arvind , Chatterji Dipankar
Published: October 2006

On amino acid starvation, Escherichia coli cells exhibit an adaptive facility termed the stringent response. This is characterized by the production of high levels of a regulatory nucleotide, ppGpp, and concomitant curtailment in rRNA synthesis. Various studies reported earlier indicated that RNA polymerase is the site of action of ppGpp although a direct demonstration of the interaction of ppGpp with E. coli RNA polymerase is still lacking. Here we report the labelling of PpGpp with a fluorescent probe, 1‐aminonapthalene‐5‐sulphonate (AmNS), at the terminal phosphates. AmNS‐ppGpp responded much like a ppGpp molecule in an in vitro total transcription assay at selective promoters. Fluorescence titration of the tryptophan emission of RNA polymerase by AmNS‐ppGpp indicated a unique binding site in the absence of template DNA. Competition experiments showed that unlabelled ppGpp binds to the enzyme at the same site. Sigma factor seems to have no effect on this binding. The titration profile is also characterized by a single slope in the Scatchard analysis. The presence of GTP or GDP does not influence the binding of AmNS‐ppGpp with RNA polymerase. Forster's distance measurement was carried out which placed AmNS‐ppGpp 27Å away from the rifampicin‐binding domain of RNA polymerase.
Gonococcal rfaF mutants express Rd2 chemotype LPS and do not enter epithelial host cells
Authors: Schwan E. Thomas , Robertson Brian D. , Brade Helmut , van Putten Jos P. M.
Published: October 2006

We have investigated the function of the lsi‐1 gene of Neisseria gonorrhoeae previously implicated in lipopolysaccharide (LPS)‐inner‐core biosynthesis (Petricoin et al., 1991). Disruption of the gene in gonococcal strain MS11 resulted in the production of LPS that migrated faster than that from an isogenic galE mutant, typical for a mutation that influences the inner‐core region. Complementation of a panel of Salmonella typhimurium mutants with defined defects in rfa loci demonstrated conclusively that the lsi‐1 gene of MS11 is functionally homologous to the rfaF gene, which encodes heptosyltransferase II in both E. coli and S. typhimurium. Comparison of deduced amino acid sequences of the gonococcal and the Salmonella RfaF demonstrated 70% similarity, including 47% identical amino acid residues. Immunochemical analysis of the LPS using monoclonal antibodies directed against chemically defined inner‐core glycoconjugates revealed that the gonococcal and Salmonella Rd2‐Chemotypes were antigenically similar, further extending the genetic and functional homology. Infection experiments in vitro demonstrated that the lsi‐1 mutant could not invade human Chang epithelial cells despite expression of a genetically defined invasion‐promoting gonococcal opacity protein. These data imply that the LPS phenotype is a critical factor for gonococcal invasiveness.
A checkpoint involving RTP, the replication terminator protein, arrests replication downstream of the origin during the Stringent Response in Bacillus subtilis
Authors: Levine Alain , Autret Sabine , Séeror Simone J.
Published: October 2006

Regulation of DNA replication in Bacillus subtilis involves a post‐initiation mechanism which is subject to control by the Stringent System, an essential regulatory network, mediated by the alarmone, ppGpp. In detailed studies using DNA‐DNA hybridization procedures, we have now shown that, following the induction of the Stringent Response, replication is blocked downstream of the origin, on the left, close to the hut marker (‐175 kb) and on the right, beyond the soft10 marker (+199 kb). In addition, we provide evidence that inhibition of replication under these conditions requires the replication terminator protein (RTP). In a mutant lacking RTP, a protein normally involved in termination of chromosomal replication through recognition of specific terminator sequences, replication continues past the sites normally blocked by the Stringent Response. These data strengthen the argument that this second level of control of DNA replication occurs at specific sites, the Stringent Terminus (STer) sites, either side of oriC Such sites are presumably related to the sequence involved in RTP recognition at the terminus, terC. We propose that the binding of RTP must be modulated, perhaps through the action of ppGpp, to recognize post‐initiation control sequences during the Stringent Response, in order to block replisome movement. This, therefore, acts as a checkpoint in chromosome elongation.
Iron‐responsive gene expression in Pseudomonas fluorescens M114; cloning and characterization of a transcription‐activating factor, PbrA
Authors: Sexton Ray , Gill Paul R. , Callanan Michael J. , O'Sullivan Daniel J. , Dowling David N. , O'Gara Fergal
Published: October 2006

In response to iron limitation, Pseudomonas fluorescens M114 induces a number of genes including an iron‐scavenging siderophore termed pseudobactin M114, its cognate receptor, PbuA, and a casein protease. A Tn5lacZ‐induced mutant (M114FA1) was isolated that exhibits a pleiotropic phenotype and lacks the ability to express these iron‐regulated genes. A cosmid clone was identified which complements this mutation. This clone is capable of activating a number of iron‐regulated promoter fusion constructs from P. fluorescens M114 and Pseudomonas putida WCS358 and can also promote expression of these fusions in Escherichia coli. A series of insertion mutants was constructed by homologous recombination which were unable to transcribe the promoter fusions. DNA sequence analysis of the complementing region identified one open reading frame (ORF) termed pbrA (pseuciobactin regulation activation) and the deduced amino acid sequence shows domains with significant homology to a number of ECF (extracytoplasmic function) transcriptional regulators of the σ70 sigma factor family, including fecl required for expression of the ferric dicitrate outer‐membrane receptor protein of E. coli. Sequences upstream of the pbrA gene suggest that transcription of pbrA may also be iron regulated.
Cold‐shock induction of a family of TIP1‐related proteins associated with the membrane in Saccharomyces cerevisiae
Authors: Kowalski Leslie R. Z. , Kondo Keiji , Inouye Masayori
Published: October 2006

TIP1 is the first known cold‐shock‐and heat‐shock‐induced gene in Saccharomyces cerevisiae. Here it is demonstrated that a TIP1 homologue, TIR1, which had been previously cloned as SRP1 (serine‐rich protein), is strongly induced by a downshift in growth temperature from 30 to 10°C. We further cloned TIR2, which is transcribed at a low basal level but is increased strongly by cold shock and, to a lesser extent, by heat shock. The predicted protein sequence of TIR2 demonstrates remarkable homology to T1R1 (72.2%) and is also homologous with TIP1 (49%). TIP1, TIR1 and TIR2 are rich in both serine and alanine residues and each contains serine‐rich tandem repeats. The proteins contain putative N‐terminal signal peptides as well as hydro‐phobic C‐terminal sequences, indicating that the proteins may be membrane bound. The predicted protein sequences are also consistent with extensive O‐mannosylation as well as glycosyl‐phosphatidyl inositol (GPI) membrane anchoring. Cell fractionation analysis as well as studies using a yeast strain that is conditionally deficient in glycosylation demonstrate that TIP1 is a heavily modified membrane‐associated protein. Single, double combinations and triple mutants were created and none demonstrated any obvious phenotype, indicating that this family of genes is not essential for normal growth.
The recA gene of Streptococcus pneumoniae is part of a competence‐induced operon and controls lysogenic induction
Authors: Martin Bernard , Garcia Pedro , Castanié Marie‐Pierre , Claverys Jean‐Pierre
Published: October 2006

The recently identified recA gene of the naturally transformable bacterium Streptococcus pneumoniae has been further characterized by constructing a recA null mutation and by investigating its regulation. The recA mutation has been shown to confer both DNA repair (as judged from sensitivity to u.v. and methyl methane sulphonate) and recombination deficiencies. Plasmid transformation into the recA mutant was also drastically reduced. Western blotting established that recA gene expression is increased several fold at the onset of competence for genetic transformation, increased expression was associated with the appearance of a recA‐specific transcript, approximately 5.7 kb long. This transcript indicated that recA is part of a competence‐inducible (cin) operon. The major (about 4.3 kb) transcript detected from non‐competent cells did not include cinA, the first gene in the operon, suggesting that this gene could be specifically required at some stage in the transformation process. Detection of small amounts of the 5.7 kb polycistronic mRNA in cells treated with mitomycin C suggested that the operon could also be damage inducible. In addition, mitomycin C treatment of a recA lysogenic strain did not lead to prophage induction and cell lysis. This is unlike the situation of a recA+ lysogen. Together these results demonstrate that RecA controls lysogenic induction and suggest the existence of a SOS repair system in S. pneumoniae.
Phage‐exclusion enzymes: a bonanza of biochemical and cell biology reagents?
Authors: Snyder Larry
Published: October 2006

Many parasitic DNA elements including prophages and plasmids synthesize proteins that kill the cell after infection by other phages, thereby blocking the multiplication of the infecting phages and their spread to other nearby cells. The only known function of these proteins is to exclude the infecting phage, and therefore to protect their hosts, and thereby the DNA elements themselves, against phage contagion. Many of these exclusions have been studied extensively and some have long been used in molecular genetics, but their molecular basis was unknown. The most famous of the phage exclusions are those caused by the Rex proteins of λ prophage. The Rex exclusions are still not completely understood, but recent evidence has begun to lead to more specific models for their action. One of the Rex proteins, RexA, may be activated by a DNA‐protein complex, perhaps a recombination or replication intermediate, produced after phage infection. In the activated state, RexA may activate RexB, which has been proposed to be a membrane ion channel that allows the passage of monovalent cations, destroying the cellular membrane potential, and killing the cell. We now understand two other phage exclusions at the molecular level which use strategies that are remarkably similar to each other. The parasitic DNA elements responsible for the exclusions both constitutively synthesize enzymes that are inactive as synthesized by the DNA element but are activated after phage infection by a short peptide determinant encoded by the infecting phage. In the activated state, the enzymes cleave evolutionarily conserved components of the translation apparatus, in one case EF‐Tu, and in the other case tRNALys. Translation is blocked and development of the phage is arrested. A myriad of different phage‐exclusion systems are known to exist and many of these may also be specific for highly conserved cellular components, furnishing generally useful enzymes for biochemical and biomedical research.
Isolation and analysis of mutants of the dnaK operon of Bacillus subtilis
Authors: Schulz Anja , Tzschaschel Barbara , Schumann Wolfgang
Published: October 2006

Bacillus subtilis contains at least three classes of heat‐shock genes regulated by different mechanisms. We are studying class I heat‐shock genes encoded by the operons dnaK and groE. These two operons are both expressed from a vegetative promoter, and their regulation involves a novel heat‐shock element designated CIRCE. Here we show that induction of both operons results from enhanced synthesis of mRNA and is independent of de novo protein synthesis. To answer the question of whether dnaK is involved in the deregulation of the heat‐shock response as reported for Escherichia coli, two different insertion mutations were isolated within the tetracistronic dnaK operon (orf39–grpE–dnaK–dnaJ). In one mutant a cat cassette was inserted at the beginning of orf39. Transcriptional analysis revealed that this mutation abolished expression of the whole operon. In contrast, the basal level of groE mRNA was significantly increased at 37°C, followed by a prolonged delay in the shut off after temperature upshift. These data point to a crucial role for the orf39 gene in the regulation of class I heat‐shock genes. In the other mutant an internal 0.8 kb Bgl II fragment of dnaK was replaced by the cat cassette. In contrast to E. coli dnaK null mutants, the two B. subtilis dnaK operon mutants could grow within a temperature range from 16–52°C. At temperatures above 52°C, they failed to form colonies on agar plates, started to filament, and lost motility. Furthermore, the induction profile of the groE and dnaK operons was not impaired in the dnaK::cat mutant. The expression of the flagellin gene is influenced on both the transcriptional and post‐transcriptional level. By Northern‐blot analysis we previously showed that expression of the dnaK operon resulted in two mRNA species of 4.9 and 2.6 kb. Our mutational analysis suggests the formation of the 2.6 kb transcript as a processing product of the 4.9 kb species.
comK encodes the competence transcription factor, the key regulatory protein for competence development in Bacillus subtilis
Authors: van Sinderen Douwe , Luttinger Amy , Kong Liyun , Dubnau David , Venema Gerard , Hamoen Leendert
Published: October 2006

comK is a positive autoregulatory gene occupying a central position in the competence‐signal‐transduction network. All regulatory routes identified in this network converge at the level of comK expression. The ComK protein is required for the transcriptional induction of comK and the late competence genes, which specify morphogenetic and structural proteins necessary for construction of the DNA‐binding and uptake apparatus. In this report we demonstrate that ComK specifically binds to DNA fragments containing promoter and upstream sequences of the genes it affects (comC, comE, comF, comG and comK) Using portions of the region upstream of comC we show that the ComK‐binding sequences are essential for the expression of competence. Moreover, we demonstrate that the presence of ComK stimulates the expression of comF–lacZ and comG–lacZ translational fusions in vivo in Escherichia coli These results indicate that the gene product of comK is identical to the previously inferred competence transcription factor (CTF).
Regulation of succinate dehydrogenase (sdhCDAB) operon expression in Escherichia coli in response to carbon supply and anaerobiosis: role of ArcA and Fnr
Authors: Park Soon‐Jung , Tseng Ching‐Ping , Gunsalus Robert P.
Published: October 2006

Succinate dehydrogenase (SDH) of Escherichia coli, the sole membrane‐bound enzyme of the tricarboxylic acid cycle, participates in the aerobic electron‐transport pathway to generate energy via oxidative phosphorylation reactions. Previous studies have established that succinate dehydrogenase (SDiH) synthesis is elevated by aerobiosis and supressed during growth with glucose. To examine how the sdhCDAB genes that encode SDH are regulated by changes in the environment, sdh–lacZ fusions were constructed and analysed in vivo following cell growth under a variety of alternative culture conditions. Expression of sdh–lacZ was highest under aerobic conditions and was decreased 10‐foid in the absence of oxygen. The fnr and arcA gene products are required for this oxygen control and each acts to repress sdhC–lacZ expression. Expression of sdh–lacZ also varied 10‐ to 14‐foid depending on the type of carbon substrate used or the medium richness. This control was shown to be independent of the crp and fruR gene products, and indicates that some other regulatory element exists in the ceil to adjust SDH enzyme levels accordingly. Iron and haem availability affected sdhC–lacZ expression by two‐ to threefold. Lastly, fold. Lastly, sdhC–lacZ expression was shown to vary with the cell growth rate during aerobic and anaerobic conditions.
Comparative characterization of the iga gene encoding IgA1 protease in Neisseria meningitidis, Neisseria gonorrhoeae and Haemophilus influenzae
Authors: Lomholt Hans , Poulsen Knud , Kilian Mogens
Published: October 2006

Cloning and sequencing of the IgA1 protease gene (iga) from Neisseria meningitidis strain HF13 showed an overall structure equivalent to iga genes from Neisseria gonorrhoeae and Haemophilus influenzae, although no region corresponding to the gonococcal α‐peptide was evident. An additional 18 N. meningitidis and 3 H. influenzae iga genes were amplified by the polymerase chain reaction technique and sequenced corresponding approximately to the N‐terminal half of the mature enzyme. Comparative analyses of a total of 29 iga genes showed that pathogenic Neisseria have iga genes with a significantly lower degree of heterogeneity than H. influenzae iga genes. Recombinational events indicated by mosaic‐like structures corresponding to those found among N. gonorrhoeae protease genes were detected among N. meningitidis iga genes. One region showed characteristic differences in sequence and length which correlated with each of the different cleavage specificities. Meningococci were extremely conserved in this region with no evidence of recombination between isolates of different cleavage specificities. Sequences further downstream showed no obvious relationship with enzyme cleavage type. This region consisted of conserved areas interspersed with highly variable areas. Amino acid sequence homologies in the variable regions of meningococci reflected the antigenic types defined by using polyclonal neutralizing antibodies.
Overexpression of the Shigella flexneri genes coding for DNA topoisomerase IV compensates for loss of DNA topoisomerase I: effect on virulence gene expression
Authors: McNairn Elizabeth , Bhriain Niamh Ni , Dorman Charles J.
Published: October 2006

Introducing the Escherichia coli topA20::Tn10 allele to Shigella flexneri results in osmotic sensitivity, a reduced growth rate, an increase in reporter plasmid supercoiling (all common to the E. coli mutants), an inability to grow on MacConkey agar and a loss of virulence gene expression. E. coli mutants harbouring this topA allele often compensate for the loss of DNA topoisomerase l by amplifying the genes coding for topoismerase IV. Unlike the E. coli topA mutants, derivatives of S. flexneri harbouring this topA allele did not appear to acquire any compensatory mutations. We investigated the possibility that this was due in part to an inability of the S. flexneri topoisomerase IV genes to compensate for loss of DNA topoisomerase I when overexpressed. The S. flexneri genes encoding the alpha‐ and beta subunits of topoisomerase IV were detected and cloned in separate multicopy plasmids. These plasmids complemented well‐characterized Salmonella typhimurium temperature‐sensitive topoisomerase IV mutations, showing that the S. flexneri and S. typhimurium proteins are capable of combining to form active complexes. When the S. flexneri topoisomerase IV genes were cloned in the same multicopy plasmid and introduced into a S. flexneri topA mutant, the plasmid restored osmotic tolerance, improved the growth rate, allowed growth on MacConkey indicator plates and resulted in a relaxation of reporter plasmid supercoiling. The same plasmid also partially restored S. flexneri virulence gene transcription. These data show that overexpression of the S. flexneri topoisomerase IV genes can compensate for the loss of topoisomerase I in terms of general viability of the cell, DNA supercoiling, and (partially) virulence gene expression. The fact that S. flexneri does not exploit topoisomerase IV gene amplification as E. coli does points to a significant difference in the abilities of these species to adapt to the loss of topoisomerase I.
Purification, characterization and mode of action of PdhR, the transcriptional repressor of the pdhR–aceEF–Ipd operon of Escherichia coli
Authors: Quail M. A. , Guest J. R.
Published: October 2006

The repressor of the pdhR–aceEF–lpd operon of Escherichia coli, PdhR, was amplified to 23% of total cell protein and purified to homogeneity by heparin–agarose and catlon‐exchange chromatography. The purified protein is a monomer (Mr 29300) which binds specifically to DNA fragments containing the pdh promoter (Ppdh) in the absence of pyruvate. The pdh operator was identified by DNase I footprinting as a region of hyphenated dyad symmetry, +11AATTGGTaagACCAATT+27, situated just downstream of the transcript start site. In vitro transcription from Ppdh was repressed > 1000‐fold by PdhR and this repression was antagonized in a concentration‐dependent manner by its co‐effector, pyruvate. Studies on RNA polymerase binding at Ppdh showed that RNA polymerase protects the ‐44 to +21 region in the absence of PdhR, but no RNA polymerase binding or protection upstream of +9 could be detected in the presence of PdhR. It is concluded that PdhR represses transcription by binding to an operator site centred at +19 such that effective binding of RNA polymerase is prevented.
Identification of an mRNA element promoting rate‐limiting cleavage of the polycistronic puf mRNA in Rhodobacter capsulatus by an enzyme to RNase E
Authors: Fritsch Jürgen , Rothfuchs Rüdiger , Rauhut Reinhard , Klug Gabrrele
Published: October 2006

We have identified an mRNA element that is involved in the initial cleavage of the pufBALMX mRNA species in Rhodobacter capsulatus. This endoribonuclease recognition site, the first to be identified in a bacterial species other than Escherichia coli, shows strong similarities to mRNA sequences cleaved by the endoribonuclease E in E. coli. The presence of an RNase E‐like enzyme in R capsulatus is further supported by in vitro cleavage of E. coli transcripts by R. capsulatus extracts at sites attributed to RNase E and by the cross‐reaction of a polypeptide from R capsulatus with antisera against E. coli RNase E. Our data provide evidence that mRNAs are degraded in different bacterial species by enzymes with similar recognition sequences and activities. We present a model that attributes the segmental differences in stability of the polycistronic puf transcript to a specific distribution of mRNA decay‐promoting and mRNA decay‐impeding elements.
F plasmid CcdB killer protein ccdB gene mutants coding for non‐cytotoxic proteins which retain their regulatory functions
Authors: Bahassi EI Mustapha , Salmon Michel A. , van Melderen Laurence , Bernard Philippe , Couturier Martine
Published: October 2006

The ccd locus of the F plasmid codes for two gene products, CcdA and CcdB, which contribute to the plasmid's high stability by post‐segregational killing of plasmid‐free bacteria. Like the quinolones, the CcdB protein is a poison of the DNA‐topoisomerase II complexes, while CcdA acts as an antidote against CcdB. in addition to these poison‐antipoison properties, the CcdA and CcdB proteins act together at transcription level to repress their own synthesis. In this work, we have isolated, in vivo., and characterized several non‐killer CcdB mutants. All missense mutations which inactivate CcdB killer activity are located in the region coding for the last three C‐terminal residues. However, the resulting mutant CcdB proteins retain their auto‐regulatory properties. We conclude that the last three C‐terminal residues of CcdB play a key role in poisoning but are not involved in repressor formation.
Iron regulation of siderophore biosynthesis and transport in Pseudomonas putida WCS358: involvement of a transcriptional activator and of the Fur protein
Authors: Venturi Vittorio , Ottevanger Clemens , Bracke Madelon , Weisbeek Peter
Published: October 2006

Pseudobactin 358 is the yellow‐green fluorescent siderophore produced by Pseudomonas putida WCS358 in conditions of iron limitation. The genes encoding for siderophore biosynthesis are iron‐regulated at the transcriptional level. Previous work has shown that a positive regulator, PfrA, is absolutely required for the activation under iron‐limiting conditions of pseudobactin 358 biosynthesis. In this study we identified a set of Tn5 insertion mutants of strain WCS358 which lost the ability to activate an iron‐regulated siderophore promoter. These mutants no longer produced pseudobactin 358. Molecular analysis revealed that they carried a Tn5 insertion in a gene, designated pfrl (Pseudomonas ferric regulator), which codes for a protein (Pfrl) of 19.5kDa. Pfrl contains a putative helix‐turn‐helix motif typical of DNA‐binding proteins and has homology to two DNA‐binding transcriptional activators, Fecl from Escherichia coli and Pupl from P. putida. The proposed role of Pfrl in strain WCS358 is an activator protein regulating pseudobactin 358 biosynthesis under iron limitation. The pfrl promoter region contains a sequence which displays high identity to the Fur‐box consensus. This 19bp consensus sequence is recognized by Fur, an iron‐binding repressor protein found in many different bacteria. The E. coli Fur protein can bind to the pfrl promoter region, indicating that this activator gene is likely to be iron‐regulated by Fur. We also report the identification and characterization of the P. putida WCS358 fur gene. The Fur protein of strain WCS358 is structurally and functionally similar to all other cloned Fur proteins from other bacterial species.
Branched‐chain fatty acids: the case for a novel form of cell‐cell signalling during Myxococcus xanthus development
Authors: Downward John , Toal Douglas
Published: October 2006

The esg locus is required for the formation of muiti‐cellular fruiting bodies and spores by the developmental bacterium Myxococcus xanthus Studies have suggested that esg mutants are defective in the production of an essential signal (E‐signal) used in cell‐cell communication and that E‐signalling is required for the expression of many developmental genes. Recently we have determined that the esg locus encodes components of a branched‐chain keto acid dehydrogenase. a multienzyme complex involved in branched‐chain amino acid metabolism in many bacteria and higher organisms. During vegetative growth in M. xanthus. this enzyme complex appears to participate in the production of the branched‐chain fatty acids found in this organism. M. xanthus fatty acids (including the branched‐chain fatty acids) have been observed to have a variety of effects on developing cells. These effects include; (i) the lysis of M. xanthus cells (autocide activity), (ii) acceleration of the rate of sporulation and (iii) rescue of sporulation by certain development‐defective mutants. These and other results suggest a model in which the branched‐chain fatty acids. Synthesized during growth, are released from cellular phospholipid by a developmentally regulated phospholipase during fruiting‐body formation. This model proposes that one or more of the branched‐chain fatty acids that are released constitutes the E‐signal which must be transmitted between cells to complete M. xanthus development.
The esg locus of Myxococcus xanthus encodes the E1α and E1β subunits of a branched‐chain keto acid dehydrogenase
Authors: Toal Douglas R. , Clifton Sandra W. , Roe Bruce A. , Downard John
Published: October 2006

The esg locus of Myxococcus xanthus appears to control the production of a signal that must be transmitted between cells for the completion of multicellular development DNA sequence analysis suggested that the esg locus encodes the E1 decarboxylase (composed of E1α and E1β subunits) of a branched‐chain keto acid dehydrogenase (BCKAD) that is involved in branched‐chain amino acid (BCAA) metabolism. The properties of an esg ::Tn5 insertion mutant supported this conclusion. These properties include: (i) the growth yield of the mutant was reduced with increasing concentrations of the BCAAs in the medium while the growth yield of wild‐type cells increased, (ii) mutant extracts were deficient in BCKAD activity, and (iii) growth of the mutant in media with short branched‐chain fatty acids related to the expected products of the BCKAD helped to correct the mutant defects in growth, pigmentation and development. The esg BCKAD appears to be involved in the synthesis of long branched‐chain fatty acids since the mutant contained reduced levels of this class of compounds. Our results are consistent with a model in which the esg‐encoded enzyme is involved in the synthesis of branched‐chain fatty acids during vegetative growth, and these compounds are used later in cell‐cell signalling during development.
A family of bacteriocin ABC transporters carry out proteolytic processing of their substrates concomitant with export
Authors: Havarstein Leiv Sigve , Diep Dzung Bao , Nes Ingolf F.
Published: October 2006

Lantibiotic and non‐lantibiotic bacteriocins are synthesized as precursor peptides containing N‐terminal extensions (leader peptides) which are cleaved off during maturation. Most non‐lantibiotics and also some lantibiotics have leader peptides of the so‐ called double‐glycine type. These leader peptides share consensus sequences and also a common processing site with two conserved glycine residues In positions ‐1 and 2. The double‐glycine‐type leader peptides are unrelated to the N‐terminal signal sequences which direct proteins across the cytoplasmic membrane via the sec pathway. Their processing sites are also different from typical signal peptidase cleavage sites, suggesting that a different processing enzyme is involved. Peptide bacteriocins are exported across the cytoplasmic membrane by a dedicated ATP‐binding cassette (ABC) transporter. Here we show that the ABC transporter is the maturation protease and that its proteolytic domain resides in the N‐terminal part of the protein. This result demonstrates that the ABC transporter has a dual function: (i) removal of the leader peptide from its substrate, and (ii) translocation of its substrate across the cytoplasmic membrane. This represents a novel strategy for secretion of bacterial proteins.
Entry of Listeria monocytogenes into hepatocytes requires expression of InIB, a surface protein of the internalin multigene family
Authors: Dramsi Shaynoor , Biswas Indranil , Maguin Emmanuelle , Braun Laurence , Mastroeni Pietro , Cossart Pascale
Published: October 2006

The intracellular bacterium Listeria monocytogenes can invade several types of normally non‐phagocytic cells. Entry into cultured epithelial cells requires the expression of inIA, the first gene of an operon, comprising two genes: inIA, which encodes internalin, an 800‐amino‐acid protein, and inIB, which encodes a 630‐amino‐acid protein. Several genes homologous to inIA are detected in the genome of L. monocytogenes; InIB is one of them. We have assessed the role of inIB In invasiveness of L. monocytogenes by constructing isogenic chromosomal deletion mutants in the inIAB locus. Our findings indicate that: i) inIB is required for entry of L. monocytogenes into hepatocytes, but not into intestinal epithelial cells; ii) inIB encodes a surface protein; iii) internalin plays a role for entry into some hepatocyte cell lines. These results provide the first insight into the cell tropism displayed by L. monocytogenes.
Activation of a temporally regulated Caulobacter promoter by upstream and downstream sequence elements
Authors: Marques Marills V. , Gober James W.
Published: October 2006

The flagellar genes of Caulobacter crescentus are expressed under cell‐cycle control. Expression is regulated by both flagellar assembly cues and cell‐cycle events. In this paper we define the sequences required for the expression of the fIgF operon, a new class of σ54 flagellar promoter. This promoter type is expressed in the middle portion of the cell cycle and regulates the expression of basal‐body genes. DNase I footprinting and mutagenesis demonstrates that an integration host factor (IHF)‐binding site is required for maximal levels of transcription of the fIgF promoter. In addition to containing a conventional upstream enhancer element (RE‐1), this promoter is unusual in that it also requires sequences (element RE‐2) immediately downstream of the transcriptional start site for maximal levels of gene expression. Cell‐cycle experiments indicate that RE‐1 and RE‐2 contribute equally to the regulation of temporal transcription. The presence of two intact elements in the promoter results in a fourfold increase in promoter activity compared with a promoter containing only one intact element, suggesting that these two elements may function synergistically to activate transcription.
A second ABC transporter is involved in oleandomycin resistance and its secretion by Streptomyces antibioticus
Authors: Olano Carlos , Rodriguez Ana Maria , Méndez Carmen , Salas José A.
Published: October 2006

A 3.2 kb Sstl‐Sphl DNA fragment of Streptomyces antibioticus, an oleandomycin producer, conferring resistance to oleandomycin was sequenced and found to contain an open reading frame of 1710 bp (oleB). Its deduced gene product (OleB) showed a high degree of similarity with other proteins belonging to the ABC‐transporter superfamily including the gene product of another oleandomycin‐resistance gene (OleC). The OleB protein contains two ATP‐binding domains, each of approximately 200 amino acids in length, and no hydrophobic transmembrane regions. Functional analysis of the oleB gene was carried out by deleting specific regions of the gene and assaying for oleandomycin resistance. These experiments showed that either the first or the second half of the gene containing only one ATP‐binding domain was sufficient to confer resistance to oleandomycin. The gene oleB was expressed in Escherichia coli fused to a maltose‐binding protein (MBP) using the pMal‐c2 vector. The MBP‐OleB hybrid protein was purified by affinity chromatography on an amylose resin and polyclonal antibodies were raised against the fusion protein. These were used to monitor the biosynthesis and physical location of OleB during growth. By Western analysis, the OleB protein was detected both in the soluble and in the membrane fraction and its synthesis paralleled oleandomycin biosynthesis. It was also shown that a Streptomyces albus strain, containing both a glycosyltransferase (OleD) able to inactivate oleandomycin and the OleB protein, was capable of glycosylating oleandomycin and secreting the inactive glycosylated molecule. It is proposed that OleB constitutes the secretion system by which oleandomycin or its inactive glycosylated form could be secreted by S. antibioticus.
Effects of upstream deletions on light‐ and oxygen‐regulated bacterio‐opsin gene expression in Halobacterium halobium
Authors: Gropp Felix , Gropp Roswitha , Betlach Mary C.
Published: October 2006

The bacterio‐opsin gene (bop) of Halobacterium halobium is located within a cluster with three other genes. Growth conditions of high light intensity and low oxygen tension induce bop gene cluster expression. To identify putative regulatory factor binding sites upstream of the bop gene, we have compared sequences upstream of the bop gene with the corresponding sequences from two other genes in the bop gene cluster. Conserved sequence motifs were observed which may mediate the effect of high light intensity and/or low oxygen tension on bop gene expression. Based on these motifs, a set of mutants was constructed which contained deletions upstream of the bop gene. These constructs were tested in a host strain where bop gene expression is independent of oxygen regulation and in another strain where it is regulated by oxygen and light. The minimal upstream sequence required for both light‐ and oxygen‐regulated bop gene expression was determined to be 54 bp.
Open complex formation by Escherichia coli RNA polymerase: the mechanism of polymerase‐induced strand separation of double helical DNA
Authors: DeHaseth Pieter L. , Helmann John D.
Published: October 2006

Escherichia coli RNA polymerase is able to site‐specifically melt 12 bp of promoter DNA at temperatures far below those normally associated with DNA melting. Here we consider several models to explain how RNA polymerase destabilizes duplex DNA. One popular model proposes that upon binding to the promoter, RNA polymerase untwists the spacer DNA between the –10 and –35 regions, which results in a destabilization of the –10 region at a TA base step where melting initiates. Promoter untwisting may result, in part, from extensive wrapping of the DNA around RNA polymerase. Formation of the strand‐separated open complex appears to be facilitated by specific protein‐DNA interactions which occur predominantly on the non‐template strand. Recent evidence suggests that these include important contacts with Sigma factor region 2.3, which we propose binds the displaced single strand of DNA.
The PTR family: a new group of peptide transporters
Authors: Steiner Henry‐York , Naider Fred , Becker Jeffrey M.
Published: October 2006

The transport of peptides into cells is a well‐documented biological phenomenon which is accomplished by specific, energy‐dependent transporters found in a number of organisms as diverse as bacteria and humans. Until recently, the majority of peptide transporters cloned and characterized were found to be proteins of the ATP‐binding cassette (ABC) family. We report the identification of a new family of peptide transporters, which we call the PTR family. This group of proteins, distinct from the ABC‐type peptide transporters, was uncovered by sequence analyses of a number of recently discovered peptide transport proteins. Alignment of these proteins demonstrated a high number of identical and similar residues and identified conserved glycosylation and phosphorylation sites, as well as a structural motif unique to this group of proteins. Cluster analysis among the proteins indicated these sequences were indeed related and could be further divided into two subfamilies. A phylogenetic analysis of these new peptide transport sequences, compared to over 50 other peptide and membrane‐bound transporters, showed that these proteins comprise a distinct, separate group of proteins.
Molecular mechanisms and implications for infection of lipopolysaccharide variation in Neisseria
Authors: van Putten Jos p. M. , Robertson Brian D.
Published: October 2006

The lipopolysaccharides of the pathogenic Neisseria species are subject to structural variation owing to a combination of intrinsic changes in lipopolysaccharide (LPS) biosynthesis and external modification of the LPS molecule with sialic acid. This variation appears to control bacterial behaviour by altering their ability to interact with human cells and to evade host Immune defences. This interconversion of LPS phenotypes, which is also observed during the natural infection, is probably due to environmental regulation of LPS biosynthesis superimposed on spontaneous changes in the DNA of distinct LPS loci. LPS variation may be a common strategy of mucosal pathogens to colonize and persist within the human host.