摘要:

SummaryThe mechanism of iron utilization from transferrin has been most extensively characterized in the pathogenicNeisseriaspecies andHaemophilusspecies. Two transferrin‐binding proteins, Tbp1 and Tbp2, have been identified in these pathogens and are thought to be components of the transferrin receptor. Tbp1 appears to be an integral, TonB‐dependent outer membrane protein while Tbp2, a lipoprotein, may be peripherally associated with the outer membrane. The relative contribution of each of these proteins to transferrin binding and utilization is discussed and a model of iron uptake from transferrin is presented. Sequence comparisons of the genes encoding neisserial transferrin‐binding proteins suggest that they are probably under positive selection for variation and may have resulted from inter‐species genetic e

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb01320.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryTheBacillus thuringiensis cryIA(a)andcryIA(c)gene specificity regions were probed by creating and testing hybrid toxins bothin vivoandin vitroagainst cultured insect cells or dissociated midgut epithelial cells. Toxin threshold dose determinations revealed that CryIA(c) is highly active against culturedChoristoneure fumiterana cells(CF‐1) whereas CryIA(a) is nontoxic. In live insect bioassays, a reversed order of toxicity was observed. Hybrid analysis reversed that the CryIA(c) toxicity‐determining region is located between codons 258 and 510. Two smaller subsections of this region (residues 258–358 and 450–510) were able to confer toxicity, although at lower levels, and one region (358–450) was present where progressive substitutions of CryIA(a) with cryIA(c) sequences had no effect. Exchanging the non‐homologous N‐terminal regions of CryIA(c) with CryIE suggested that the W‐terminus does not play a role in specificity. One hybrid clone, MP80, displays a 99.3% homology to CryIA(b) but shows an 800‐fold increase in toxicity to CF–1 cells relative to that shown by CryIA(b). Direct comparison between liveBombyx moribioassays and a newly developedin vitrolawn assay using dissociated midgut epithelial cells from the same insect revealed striking differences in toxicity. The toxicity‐determining region for B.morilarvae was determined to be between codons 283 and 450, although the 450–620 codon region may exert an influence on toxicity. In general, native or hybrid toxins showing little or no insect intoxication were very active against the epithelial cells, suggesting that factors other than toxin amino acid sequence play an important role in determ

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb01321.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryEscherichia coliDnaK, DnaJ and GrpE are required for renaturation of heat‐inactivated λ CI857 repressor (Gaitanariset al., 1990). Here we demonstrate that in addition to the above three proteins, GroEL and GroES are necessary for the CI857 repressor to acquire full activity at the permissive temperature. Although full‐length soluble repressor is present at normal amounts, the protein has reduced specific activity and migrates abnormally on native gels. To determine where the different chaperones act in protein folding, we identified their cellular locations. DnaK and DnaJ are associated with nascent polypeptide chains in translating ribosomes. In contrast, GroEL, although it is transiently associated with newly synthesized proteins, is absent from the ribosomes. This suggests that DnaK and DnaJ ptay an early role in protein maturation, whereas GroEL acts at a later s

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb01322.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryThe 17 kbkpsgene cluster ofEscherichia coliK1, which encodes the information required for synthesis, assembly and translocation of the polysialic acid capsule ofE. coliK1, is divided into three functional regions. Region 3 contains two genes,kpsMandkpsT, essential for the transport of capsule polymer across the cytoplasmic membrane. The hydrophobicity profile of KpsM suggests that it is an integral membrane protein while KpsT contains a consensus ATP‐binding site. KpsM and KpsT belong to the ATP‐binding cassette (ABC) superfamily of membrane transporters. In this study, we investigate the topology of KpsM within the cytoplasmic membrane using β‐lactamase fusions and alkaline phosphatase sandwich fusions. Our analysis provides evidence for a model of KpsM having six membrane‐spanning regions, with theN‐ andC‐terminal domains facing the cytoplasm, and a short domain within the third periplasmic loop, which we refer to as the SV–SVI linker localizing in the membrane. Protease digestion studies are consistent with regions of KpsM exposed to the periplasmic space.In vivocross‐linking studies provide support for dimerization of KpsM within the cytoplasmic membrane. Linker‐insertion and site‐directed mutagenesis define theN‐terminus, the first cytoplasmic loop, and theSV‐SVIlinker as regions that are important for the function of KpsM

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb01323.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryA method of insertional mutagenesis for naturally transformable organisms has been adapted fromHaemophilus influenzaeand applied to the study of the pathogenesis ofCampylobacter jejuni.A series of kanamycin‐resistant Insertional mutants ofC. jejuni81–176 has been generated and screened for loss of ability to invade INT407 cells. Eight noninvasive mutants were identified which showed 18‐200‐fold reductions in the level of invasion compared with the parent. Three of these eight show defects in motility, and five are fully motile. The three mutants with motility defects were further characterized to evaluate the method. One mutant, K2–32, which is non‐adherent and non‐invasive, has an insertion of the kanamycin‐resistance cassette into theflaAflagellin gene and has greatly reduced motility and a truncated flagellar filament typical offlaAmutants. The adherent non‐invasive mutants K2–37 and K2–55 are phenotypically paralysed, i.e. they have a full‐length flagellar filament but are non‐motile. All three mutants show an aberration in flagellar structure at the point at which the filament attaches to the cell. Mutants K2–37 and K2–55 represent overlapping deletions affecting the same gene, termedpflA(paralysed flagella). This gene encodes a predicted protein of 788 amino acid residues and a molecular weight of 90 977 with no significant homology to known proteins. Site‐specific insertional mutants into this open reading frame result in the same paralysed flagellar phenotype and the same invasion

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb01324.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryThree peptides with pore‐forming activity were isolated from the cytoplasmic granules of pathogenicEntamoeba histolyticaby acidic extraction, gel filtration and reversed‐phase high‐performance liquid chromatography. Partial amino acid sequence analysis of the three active peptides revealed that the most abundant of them was amoebapore and the other two were isoforms thereof. Cloning and sequencing of genomic DNA resolved the amino acid sequence of the two newly recognized peptides. The three peptides designated amoebapores A, B and C were found to have the same molecular size but to differ markedly in their primary structure, although all six cysteine residues are conserved. Despite sequence divergence, structural implications predict for the three peptides a similar amphipathic α‐helical conformation stabilized by disulphide bonds. All three isoforms exhibit pore‐forming activity toward lipid vesicles, but they differ in their kinetics. They also are capable of perturbing the integrity of bacterial cytoplasmic membranes and thereby kill Gram‐positive bacteria. The amoebapores represent a distinct family of membrane‐active peptides that may function intracellularly as antimicrobial agents but may also confer cytolytic activity o

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb01325.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryThe chromosome ofYersinia enterocoliticaencodes an enterotoxin called Yst. We analysed transcription of chromosomalyst′–luxABand plasmid‐borneyst′–lacZoperon fusions and we observed that regulation ofystexpression occurs at transcriptional level. In a wild‐type strain,ystwas transcribed from at least two major promoters,ysttranscription reached a maximum at the entry to the stationary phase and significantly varied in differentY. enterocoliticastrains. In some strains, it gradually decreased during the course of our work, suggesting the existence of a mechanism switching the expression ofystto a silent state. Changes in the status of bacterial host factors rather than modifications in theystgene are responsible for this silencing. Negative regulator YmoA participates inystsilencing and temperature regulation ofystYmoA was also required for proper growth‐phase regulation ofyst, although it is not the only factor involved in this regulation. Physicochemical parameters of the environment play an important role inysttranscription. In usual culture media (e.g. tryptic soy broth), the enterotoxin gene was transcribed only at temperatures below 30°C, which argued against the role of Yst in a prolonged diarrhoea at body temperatures. However,ysttranscription could be induced at 37°C by increasing osmolarity and pH to the values normally present in the ileum lumen. This finding reconciles the observations concerningystexpression in a host environment and in bacterial cultures, thus supporting the idea that enterotoxin Yst is a virulence factor ofY.

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb01326.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryAspergillus fumigatussecretes a serine alkaline protease (ALP) and a metalloprotease (MEP) when the fungus is cultivated in the presence of collagen as sole nitrogen and carbon source. The gene encoding ALP was isolated and characterized previously. We report here the cloning and the sequencing of the gene encoding MEP. Genomic and cDNA clones were isolated fromA. fumigatuslibraries using synthetic oligonucleotides as probes. Stretches of the deduced amino acid sequence were found to be in agreement with theN‐terminal amino acid sequence of MEP and with internal peptide sequences. The amino acid sequence of the enzyme contains a putative active‐site sequence HEYTH homologous to the active site of other bacterial and eukaryotic zinc metalloproteases. Sequence analysis reveals that MEP has a pre‐proregion consisting of 245 amino acid residues preceding the 388 amino acid residues of the mature region (molecular mass of 42 kDa). Analp mepmutant, deficient in proteolytic activity at neutral pHin vitro, was constructed and tested for pathogenicity in a murine model. No difference in pathogenicity was observed between the wild‐type strain and thealp mepdouble mutant, suggesting that ALP and MEP are not essential for the invasion of the lung tissues byA. fu

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb01327.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryThe KdpD protein is a membrane‐located sensory kinase (or signal transducer) critically involved in the regulation of thekdpABCoperon that is responsible for a high‐affinity transport system inEscherichia coli.In this study, a set of KdpD mutants, each resulting in a single amino acid substitution around the membrane‐spanning regions of KdpD, was isolated. Amino acid substitutions in these KdpD mutants were located non‐randomly, particularly within theC‐terminal half of the membrane‐spanning regions. This set of KdpD mutants exhibited altered transmembrane‐signalling properties in response to external K+and other stimuli. In particular, these mutants were found to be insensitive, if not completely, to the K+signal. However, they were able to respond to other stimuli such as high‐salt stress, as in the wild type. Therefore, in contrast to the wild type, the cells carrying these mutations exhibited high levels of the steady‐state expression of kdp, regardless of external K+, provided that high concentrations of ionic solutes were supplemented to the cultures. More interestingly, the set of KdpD mutants could also respond to high concentrations of external non‐ionic solutes such as sucrose and D‐arabinose, thereby increasing substantially the steady‐state expression ofkdpin response to the medium osmolarity. Furthermore, it was found that certain chemicals, ethanol, chlorpromazine and procaine, could function as effectors for the KdpD mutants at relatively low concentrations in the media. Based on these findings, we have examined the primary signal(s) that regulates the function of KdpD. We propose here that KdpD can be considered to be an environmental sensor that exhibits sensing mechanisms in response to both the level of K+and the physico‐chemical state of

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb01328.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryAn alkali‐sensitive mutant, 38154, of the alkalophilicBacillussp. strain C‐125 could not grow at an alkaline pH. The nucleotide sequence of a 3.7 kb parental DNA fragment that recovers the growth of 38154 at alkaline pH has four open reading frames (ORF1–4). By sub‐cloning the fragment, we demonstrated that a 0.25 kb DNA region is responsible for the recovery. Direct sequencing of the mutant's corresponding region revealed a G to A substitution. The mutation resulted in an amino acid substitution from Gly‐393 to Arg of the putative 0RF1 product, which was deduced to be an 804‐amino‐acid polypeptide with a molecular weight of 89 070. The N‐terminal part of the putative ORF1 product showed amino acid similarity to those of the chain‐5 products of eukaryotic NADH quinine oxidoreductases. Membrane vesicles prepared from 38154 did not show membrane potential (δψ)‐driven Na+/H+antiporter activity. Antiporter activity was resumed by introducing a parental DNA fragment which recovered the mutant's alkalophily. These results indicate that the mutation in 38154 affects, either directly or indirectly, the electrogenic Na+/H+antiporter activity. This is the first report which shows that a gene responsible for the Na+/H+anti‐porter system is important in the alkalophily of alkal

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb01329.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY