摘要:

SummaryThe pathogenesis of clostridial myonecrosis, or gas gangrene, involves the growth of the anaerobic bacteriumClostridium perfringensin the infected tissues and the elaboration of numerous extracellular toxins and enzymes. The precise role of each of these toxins in tissue invasion and necrosis has not been determined. To enable genetic approaches to be used to studyC. perfringenspathogenesis we developed an allelic exchange method which involved the transformation ofC. perfringenscells with a suicide plasmid carrying a gene insertionally inactivated with an erythromycin‐resistance determinant. The frequency with which double reciprocal crossover events were observed was increased to a workable level by increasing the amount of homologous DNA located on either side of the inactivated gene. Allelic exchange was used to isolate mutations in the‘chromosomalpfoAgene, which encodes an oxygen‐labile haemolysin known as Θ‐toxin or perfringolysin O. and in the chromosomalpicgene, which encodes the α‐toxin or phospholipase C. The resultant mutants failed to produce detectable Θ‐toxin or α‐toxin activity, respectively, and could be complemented by recombinant plasmids that carried the respective wild‐type genes. The resultant strains were virulence tested in a mouse myonecrosis model. The results showed that thepicmutants had demonstrably reduced virulence and therefore provided definitive genetic evidence for the essential role of α‐toxin in gas gangrene or c

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.tb02234.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

SummaryTranscription of the ATP‐dependent deoxynuclease operon(addAB), as monitored by means of anaddAB–lacZtranscriptional fusion, has a low, constitutive level and is initiated from a σAtype promoter. Transcription ofaddABis independent of DNA‐damaging agents known to induce the SOS response inBacillus subtilis.However,addABtranscription increased significantly during competence development. This competence‐specific induction was dependent on the gene products ofsrfA., degUandcomK, but not on that ofrecA.Deletion analysis of theaddABpromoter region demonstrated that the competence‐specific transcription induction requires DNA sequences located upstream of theaddABpromoter 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

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.tb02235.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

SummaryDespite the lack of involvement of the competence‐specific, membrane‐associated deoxyribonuclease (DNase) in competence development, the expression of the gene encoding this protein,nucA, was shown to be dependent on the competence signal transduction pathway, and in particular on ComK, the competence transcription factor, which was shown to bind to the DNA region upstream ofnucA.The expression ofnucB, specifying an extracellular DNase, which was cloned on the basis of its homology tonucA, was shown to be sporulation‐specific and dependent on the gene products ofspo0AandspollG, the latter constituting an operon responsible for the synthesis of the mother‐cell‐specific sigma factor σE. The observed differential expression ofnucAandnucBdemarcates the appearance of DNase activities which are either associated with the cytoplasmic membrane or secreted into the medium during different post‐exponential growth‐p

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.tb02236.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

SummaryPeptide transport inSaccharomyces cerevisiaeis controlled by three genes:PTR1, PTR2, andPTR3. PTR1was cloned and sequenced and found to be identical toUBR1, a gene previously described as encoding the recognition component of theN‐end‐rule pathway of the ubiquitin‐dependent proteolytic system. Independently derivedubr1mutants, likeptr1mutants, were unable to transport small peptides into ceils. Concomitantly,ptr1mutants, likeubr1mutants, were unable to degrade an engineered substrate of theN‐end‐rule pathway. Further,ptr1mutants did not expressPTR2, a gene encoding the integral membrane component required for peptide transport inS. cerevisiae.These results establish a physiological role for a protein previously known to be required for the degradation ofN‐end‐rule substrates. Our findings show that peptide transport and the ubiquitin pathway—two dynamic phenomena universal to eukaryotic cells—share a common component

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.tb02237.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

SummaryThe toxicity of the powerful anti‐tuberculosis drug isoniazid (INH) is believed to be mediated by the haem‐containing enzyme catalase‐peroxidase, encoded by thekatGgene ofMycobacterium 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 inkatG.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 theN‐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 cytochromecperoxidase from yeast as a template. The second group comprises a frequently occurring amino acid substitution and a single mutation that are both located in theC‐terminal domain but do not noticeably alter either enzyme activity

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.tb02238.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

SummaryGrowth ofEscherichia coliK‐12 in low‐phosphate conditions results in the induction of the synthesis of many proteins, including the outer membrane porin PhoE, alkaline phosphatase, and the Pst system for the transport of phosphate (Pi). This response is controlled by a two‐component regulatory system of which PhoB and PhoR are the response‐regulator and the sensor/kinase, respectively. WhenShigella flexneriwas starved for Pi, neither PhoE nor alkaline phosphatase was produced. However, induction of the synthesis of the PstS protein was observed, indicating thatS. flexnericontains a functional PhoB/PhoR regulatory system. Consistent with this notion, the introduction of theB. coli phoAgene inS. flexneriresulted in the induction of alkaline phosphatase synthesis under phosphate limitation. However, introduction ofphoEon a plasmid did not lead to the expression of PhoE protein, indicating thatS. flexneriPhoB does not recognize thephoEpromoter region. ThephoBgene was cloned and sequenced and in the deduced amino acid sequence two deviations from that ofE. coliPhoB were detected. Site‐directed mutagenesis revealed that one of these deviations, i.e. Leu‐172, which is Arg inE. coliPhoB, is responsible for the lack of expression of the PhoE protein in

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.tb02239.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

SummaryOn amino acid starvation,Escherichia colicells 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 withE. coliRNA 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 anin vitrototal 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 o

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.tb02240.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

SummaryWe have investigated the function of thelsi‐1gene ofNeisseria gonorrhoeaepreviously implicated in lipopolysaccharide (LPS)‐inner‐core biosynthesis (Petricoinet al., 1991). Disruption of the gene in gonococcal strain MS11 resulted in the production of LPS that migrated faster than that from an isogenicgalEmutant, typical for a mutation that influences the inner‐core region. Complementation of a panel ofSalmonella typhimuriummutants with defined defects inrfaloci demonstrated conclusively that thelsi‐1gene of MS11 is functionally homologous to therfaFgene, which encodes heptosyltransferase II in bothE. coliandS. typhimurium.Comparison of deduced amino acid sequences of the gonococcal and theSalmonellaRfaF 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 andSalmonellaRd2‐Chemotypes were antigenically similar, further extending the genetic and functional homology. Infection experimentsin vitrodemonstrated that thelsi‐1mutant 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 gonoco

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.tb02241.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

SummaryHere we report the cloning and expression, inEscherichia coli, of PCR‐amplified DNA encoding the 63‐kDa stress‐inducible protein ofNeisseria gonorrhoeaestrains VP1 and PiD2,Neisseria meningitidis2996 and the commensalNeisseria flavescens.DNA sequence analysis revealed in all cases one open reading frame of 541‐544 amino acids corresponding to a protein of approximately 57 000 Da. The various neisserial proteins were>98% identical at the amino acid level and showed extensive homology with proteins belonging to the HspSO heat‐shock‐protein family. We constructed defined glutathione S‐transferase fusion polypeptides of the gonococcal Hsp60 homologue to locate antigenic domains on the recombinant protein. Variation in the immunoreactivity of two monoclonal antibodies recognizing a conserved and a neisseria‐unique antigenic Hsp60 determinant, respectively, could thus be deduced to result from single amino acid substitutions. Analysis of the antibody response in patients’sera demonstrated reactivity with the same fusion polypeptides in six out of nine sera, indicating that neisserial Hsp60 is expressed during the natural infection and that distinct domains on the protein are immu

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.tb02242.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

SummaryRegulation of DNA replication inBacillus subtilisinvolves 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 thehutmarker (‐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 oforiCSuch 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 elo

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.tb02243.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY