ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1990.tb00583.x

出版商:Blackwell Publishing Ltd    

年代:1990

数据来源: WILEY

摘要:

SummaryBacteria swim by rotating their flagella, the rotation being due to a motor located at the base of each flagellum. In this paper the correlation between motor function and mode of swimming is reviewed, with special emphasis on recent data that indicate that the motor is a three‐state device. Novel findings with regard to the motor function and bioenergetics are surveyed, and mechanisms are proposed to account for these finding

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1990.tb00584.x

出版商:Blackwell Publishing Ltd    

年代:1990

数据来源: WILEY

摘要:

SummaryThis paper describes software (written in Pascal and running on Macintosh computers) allowing localization of unknown DNA fragments from theEscherichia colichromosome on the restriction map established by Koharaet al.(1987). The program identifies the segment's map position using a restriction pattern analysis obtained with all, or some, of the eight enzymes used by Koharaet al.(1987). Therefore, the sequenced genes available in the EMBL library may be localized on theE. colichromosome restriction map. This allowed correction of the map (mainly by introducing missing sites in the published maps) at the corresponding positions. Analysis of the data indicates that there is only a very low level of polymorphism, at the nucleotide level, between theE. coliK12 strains used by the various laboratories involved in DNA sequencing. The program is versatile enough to be used with other genomes.

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1990.tb00585.x

出版商:Blackwell Publishing Ltd    

年代:1990

数据来源: WILEY

摘要:

SummaryIncreased levels of alginate biosynthesis cause mucoidy inPseudomonas aeruginosa, a virulence factor of particular importance in cystic fibrosis. ThealgRgene product, which controls transcription of a key alginate biosynthetic gene,algD, is homologous to the activator members of the two‐component, environmentally responsive systems (NtrC, OmpR, PhoB, ArcA, etc). In this report, we show that mutations in themucloci, (muc‐2,muc‐22, andmuc‐23, in the standard geneticP. aeruginosastrain PAO, as well as a mappedmucallele in an isolate from a cystic fibrosis patient) affect transcription ofalgDandalgR.This influence was strongly dependent on environmental factors. Regulation by nitrogen was observed in all strains examined, but the absolute transcriptional levels, determining the mucoid or non‐mucoid status, were strain (mucallele)‐dependent. Increased concentrations of NaCl in the medium, an osmolyte which is elevated in cystic fibrosis lung secretions, resulted in an increasedalgDtranscription and mucoid phenotype in amuc‐2 strain; the same conditions, however, produced a nonmucoid phenotype in themuc‐23 background and abolishedalgDtranscription. Mutations in themucloci may cause mucoidy by deregulating the normal response of the alginate system to enviro

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1990.tb00586.x

出版商:Blackwell Publishing Ltd    

年代:1990

数据来源: WILEY

摘要:

SummaryChitin synthase activity was studied in yeast and hyphal forms ofCandida albicans.pH‐activity profiles showed that yeast and hyphae contain a protease‐dependent activity that has an optimum at pH 6.8. In addition, there is an activity that is not activated by proteolysisin vitroand which shows a peak at pH 8.0. This suggests there are two distinct chitin syntheses inC albicans.A gene for chitin synthase fromC. albicans (CHS1)was cloned by heterologous expression in aSaccharomyces cerevisiae chs1 mutant. Proof that the cloned chitin synthase is aC. albicansmembrane‐bound zymogen capable of chitin biosynthesisin vitrowas based on several criteria, (i) theCHS1 gene complemented theS. cerevisiae chs1 mutation and encoded enzymatic activity which was stimulated by partial proteolysis; (ii) the enzyme catalyses incorporation of [14C]‐GlcNAc from the substrate, UDP[U‐14C]‐GlcNAc, into alkali‐insoluble chitin; (iii) Southern analysis showed hybridization of aC. albicans CHS1probe only withC. albicansDNA and not withS. cerevisiaeDNA; (iv) pH profiles of the cloned enzyme showed an optimum at pH 6.8. This overlaps with the pH‐activity profiles for chitin synthase measured in yeast and hyphal forms ofC. albicans.Thus,CHS1 encodes only part of the chitin synthase activity inC. albicans.A gene for a second chitin synthase inC. albicanswith a pH optimum at 6.0 is proposed.DNA sequencing revealed an open reading frame of 2328 nucleotides which predicts a polypeptide ofMr88281 with 776 amino acids. The alignment of derived amino acid sequences revealed that theCHS1 gene fromC. albicans (canCHS1) is homologous (37% amino acid identity) to theCHS1 gene fromS. cerev

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1990.tb00587.x

出版商:Blackwell Publishing Ltd    

年代:1990

数据来源: WILEY

摘要:

SummaryThe putative structural genemepAof the penicillin‐insensitive murein endopeptidase fromEscherichia coliwas cloned and sequenced.N‐terminal sequence determination with the isolated endopeptidase protein showed that this enzyme is coded by themepAgene and that it is synthesized initially with an N‐terminal signal peptide. No significant sequence homoiogy with the other (penicillin‐sensitive) murein endopeptidase(dacB)or any other protein was found. The precise chromosomal mapping position ofmepArelative to two other genes,aroCandfabB, was shown to be 50.4 min.E. colistrains carrying multicopy plasmids with themepAgene produced 5‐6‐fold more endopeptidase and secreted it into the periplasm, where it appeared to function normallyin vivosince the release of ceil wall peptides into the medium increased in parallel. The transformed cells were, however, not unusually sensitive to penicillin and their murein had a normal degree of cr

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1990.tb00588.x

出版商:Blackwell Publishing Ltd    

年代:1990

数据来源: WILEY

摘要:

SummaryAHaemophilus influenzaeDNA library was prepared in the vector lambda EMBL3, and recombinant phage were screened for the pilin gene (pil) using a synthetic oligonucleotide. Southern blot analysis of the positive clones revealed a 2.5kbPstl/Pvulfragment that hybridized with the oligonucleotide probe. This fragment was subcloned into pBR322 and sequenced. The nucleotide sequence disclosed an open reading frame of 653 bases. The deduced amino acid sequence corresponded with the known amino acid sequence of the purified pilin protein. Primer extension analysis using total RNA from piliatedH. influenzaecells delineated a start site for the gene, ‐10 and ‐35 promoter regions, and a ribosome‐binding site. No transcripts were seen with the RNA derived from a non‐piliated strain. Southern blots of DNA from a number ofH. influenzaestrains revealed homology with thepilstructural gene. DNA from a non‐piliated strain ofH. influenzaealso hybridized with thepilprobe. Transcriptional and translational studies were performed inEscherichia coliwith plasmids containing: (i) thepilgene on the 2.5kbPstl/Pvulfragment, (ii) thepilgene fused to thephoAgene, and (iii) thepilgene present on a 12.2 kb insert containing extensiveH. influenzaeDNA flanking thepilgene. The results suggest that theH. influenzae pilgene is expressed inEscherichia coli, but from a promoter other than the one used inH. i

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1990.tb00589.x

出版商:Blackwell Publishing Ltd    

年代:1990

数据来源: WILEY

摘要:

SummaryAn 8kb segment of DNA from the 58/59 min region of theE. colichromosome, which complements the defect of a mutant devoid of hydrogenase 3 activity, has been sequenced. Eight open reading frames were identified which are arranged in a transcriptional unit; all open reading frames were transcribed and translatedin vivoin a T7 promoter/polymerase system. Analysis of the amino acid sequences derived from the nucleic acid sequences revealed that one of them, open reading frame 5 (0RF5), exhibits significant sequence similarity to conserved regions of the large subunit from Ni/Fe hydrogenases. Two of the open reading frames (orf2,orf6) code for proteins apparently carrying iron‐sulphur clusters of the 4Fe/4S ferredoxin type. The product of one of the open reading frames,orf7, displays extensive sequence similarity with protein G from the chloroplast electron transport chain. ORF3 and ORF4, on the other hand, are extremely hydrophobic proteins with nine and six putative transmembrane helices, respectively. Over a limited hydrophilic sequence stretch, bordered by putative transmembrane areas, ORF3 and ORF4 exhibit homology with subunits 4 and 1 of mitochondrial and plastid NADH‐ubiquinol oxidoreductases, respectively. The operon described, therefore, appears to comprise genes for redox carriers linking formate oxidation to proton reduction and for a hydrogenase of hitherto unique composit

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1990.tb00590.x

出版商:Blackwell Publishing Ltd    

年代:1990

数据来源: WILEY

摘要:

SummaryDNA sequencing of thenodlJregion fromRhizobium leguminosarumbiovartrifoliirevealed thenodTgene immediately downstream ofnodJ.DNA hybridizations using a nodT‐specific probe showed thatnodTis present in severalR. leguminosarumstrains. Interestingly, a flavonoid‐induciblenodTgene homologue inR. leguminosarumbv.viciaeis not in thenodABCIJoperon but is located downstream ofnodMN.The sequence of thenodTgene from bv.viciaewas determined and a comparison of the predicted aminoacid sequences of the twonodTgenes shows them to be conserved; the predicted protein sequences appear to have a potential transit sequence typical of outer‐membrane proteins. Mutations affectingnodTin either biovar had no observed effect on nodulation of the legumes t

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1990.tb00591.x

出版商:Blackwell Publishing Ltd    

年代:1990

数据来源: WILEY

摘要:

SummaryAs an initial approach in the study of the mechanism of secretion of the extracellular heat‐stable enterotoxin ofEscherichia coli(STA), and in order to use this polypeptide as an extracellular carrier we previously constructed a fusion between the complete STAtoxin (pre‐pro‐STA) and the mature B subunit of the periplasmic heat‐labile enterotoxin (LTB); the resulting STA‐LTBhybrid was not secreted to the extracellular environment, and cells expressing the hybrid lysed at temperatures above 35°C. In this work we have established that the hybrid is initially detected as pre‐pro‐STA‐LTBand converted to pro‐STA‐LTB, which lacks the 19amino acids that share the properties of a signal peptide; the sequenced 17 amino‐terminal residues of pro‐STA‐LTBdefined the processing site of pre‐pro‐STA‐LTBat pro_3phe_2ala_1 ↓ gln+1. This process was sensitive to an energy uncoupler (CCCP) and was correlated with translocation of pro‐STA‐LTBacross the inner membrane. Additionally, we are able to show that although pre‐pro‐STA‐LTBis processed at 37°C and 29°C, it is more efficiently processed at the latter temperature. At 37°C, pro‐STA‐LTBwas poorly released into the periplasm, resulting in accumulation of this protein, pre‐pro‐STA‐LTB, and pre‐β‐lactamase in the inner membrane, and in cell lysis. In contrast, at 29°C pro‐STA‐LTBwas localized in the periplasm and in the inner membrane, and pre‐pro‐STA‐LTBand pre‐β‐lactamase did not accumulate; however, translocation of periplasmic pro‐STA‐LTBacross the outer membrane still did not occur, and a second processing step that would eliminate the pro segment from pro‐STA‐LTBwas never observed. Thus, the fusion of pre‐pro‐STAand LTBresulted in a polypeptide that, while incompatible with secretion to the extracellular medium, is exported to the periplasm in a temperature‐conditional fashion. This latter observation is consistent with an STAsecretion pathway whereby pre‐

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1990.tb00592.x

出版商:Blackwell Publishing Ltd    

年代:1990

数据来源: WILEY