摘要:

SummaryThe regulatory region spanning the divergently transcribednifF andnifLApromoters contains a NIFA‐specific upstream activator sequence (UAS) located around +59, and two NTRC binding sites centred at −142 and −163 with respect to thenifLAtranscription start site. We have constructed mutations in each of these binding sites and examined their role in transcriptional activation of the divergently transcribed promoters. Analysis of a mutation at +60 confirms that the UAS is required for efficient NIFA‐mediated activation ofnifFtranscription. This sequence is also required for maximal activation of thenifLApromoter. Mutations at −169 and −148, within the two NTRC binding sites, reduce activation of thenifLApromoter by NTRCin vivoand lower the affinity of the activator for these sitesin vitro.Phosphorylation of NTRC by NTRB is required for efficient binding of NTRC to

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1988.tb00049.x

出版商:Blackwell Publishing Ltd    

年代:1988

数据来源: WILEY

摘要:

SummaryA DNA fragment containing a gene for resistance to the antibiotic albicidin was isolated fromKlebsiella oxytocaand shown to be expressed inEscherichia coli, where it also protected bacteriophage T7 replication from inhibition by albicidin.In vivotranslation analysis demonstrated that the cloned 2.2kb DNA fragment coded for a 36 kiloDalton (kD) protein and a 25 kD protein.The DNA sequence was determined for a 654‐base‐pair open reading frame contained within a 1.2 kb subcloned DNA fragment encoding albicidin resistance. The predicted molecular weight of the polypeptide translated from the open reading frame was 25.8 kD. A putative Shine‐Dalgarno sequence precedes the open reading frame but a potential promoter sequence was not detected. A possible rho‐independent transcription termination signal was found directly following the stop codon. The functional protein for albicidin resistance was isolated and purified. Both the molecular weight and NHg‐terminal amino acid sequence of this protein correspond with that predicted from the DNA sequence of the open reading frame. The cloned albicidin resistance gene had no effect on thetsx (nupA)nucleoside uptake gene associated with spontaneous albicidin resistance in E.coli; also, it did not complement any of a range of E.coliDNAtsmutants at restrictive temperatures. The cloned resistance gene product remained intracellular in exponential cultures ofK. oxytocaandE. coli.Cell‐free extracts from E.colicontaining the resistance gene protected a sensitive strain ofE. colifrom inhibition by albicidin, as did the purified albicidin resistance protein. The mechanism of this albicidin resistance protein involved binding to albicidin to form a complex without antibiotic activity, but without catalysing further chemical modification of the

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1988.tb00050.x

出版商:Blackwell Publishing Ltd    

年代:1988

数据来源: WILEY

摘要:

SummaryTranscription and translation of theatpgenes encoding the subunits δ, α, γ, and ɛ of theEscherichiacoli H+‐ATPase were studied. The nature and quantities of the respective transcripts initiated from different promoters were compared with overall expression rates thus yielding accurate information about relative translational efficiency and its coupling to mRNA levels. Part of the highly efficient subunit c gene translational initiation region (TIR) was used as a tool in manipulating the TIRs of the other genes. Rate control ofatpcistron translation occurs at the initiation level and is determined locally by each gene's TIR. In this way, individual subunit synthesis rates are set to match the requirements for H+‐ATPase assembly. There is no (or very restricted) translational coupling between the cistrons. Translational initiation rates of the normally weakly expressedatpgenes could be increased by up to a factor of 27 by manipulating the sequences upstream of the start codons, despite biased codon usages. In the presence of an improved upstream sequence, the N‐terminal sequence of the subunit γ gene exerted a limiting effect. This could be relieved by altering the sequence of the first seven codons. The levels of subunit γ mRNA were more sensitive to changes in translational efficiency than the concentrations of the otheratpmRNAs. The relationships between initiation efficiency and primary and secondary structure in the natural and manipulatedatpTIRs are discusse

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1988.tb00051.x

出版商:Blackwell Publishing Ltd    

年代:1988

数据来源: WILEY

摘要:

SummaryThe expression of human transferrin and lactoferrin binding activity inHaemophilus influenzae, detected by a binding assay using human transferrin or lactoferrin conjugated to peroxidase, was regulated by the level of available iron in the medium. Transferrin binding activity was present in allH. influenzaeisolates tested but not detected in otherHaemophilusspecies or in species ofPasteurellaorActinobacillus.Lactoferrin binding activity was only detected in allH. influenzaeisolates tested. The transferrin and lactoferrin receptors were shown to be specific for the respective human proteins by means of a competition binding assay. Competition binding assays also showed that iron‐loaded transferrin was more effective at blocking the transferrin receptor than apotransferrin, but no differences in receptor blocking were observed between iron‐loaded lactoferrin and apolactofer

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1988.tb00052.x

出版商:Blackwell Publishing Ltd    

年代:1988

数据来源: WILEY

摘要:

SummaryThe structural gene for theEscherichia colienzyme amylomaltase,malQ, is the second gene in themalPQoperon. The nucleotide sequence ofmalQshows that the gene encodes an Mr78360 protein close to the experimentally determined Mrof purified amylomaltase (72000–74000). ThemalQinitiation codon was identified by sequence analysis of clustered deletions around the 5′ end of the gene. One of these deletions removed the first 5 bases from themalQcoding sequence. Strains carrying a plasmid with this truncatedmalQgene underlacZpromoter control and out‐of‐frame with the first four codons oflacZwere Mal−. The Mal+phenotype could be restored by inserting small, random fragments ofE. colichromosomal DNA into the unique EcoRI site. Nucleotide sequencing showed that the inserts either joined thelacZ and malQsequences in frame, or contained a new translation start signal and coding sequence in frame withmalQ.These results indicate that amylomaltase could be useful as a reporter protein in gene fusio

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1988.tb00053.x

出版商:Blackwell Publishing Ltd    

年代:1988

数据来源: WILEY

摘要:

SummaryThe El Tor haemolysin, product ofhlyA, is exported fromVibrio choleraeas a Mr80000 protein which can be subsequently cleaved to give two proteins of Mr65000 and 15000. Nucleotide sequence analysis has demonstrated thathlyAencodes a protein of Mr82250 with a potential 18‐amino‐acid signal sequence.The non‐haemolytic classical strain 569B has been shown to have a structural gene defect rather than a defect in secretion. By non‐reciprocal recombination it was possible to transfer this defect onto a plasmid and show that a truncatedhlyAproduct of Mr27000 is made inEscherichia coliK‐12 minicells. Nucleotide sequence analysis demonstrates an 11‐base‐pair deletion which would result in a Mr26940 protein probably loosely associated with

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1988.tb00054.x

出版商:Blackwell Publishing Ltd    

年代:1988

数据来源: WILEY

摘要:

SummaryThe expression withinPseudomonas aeruginosaPAO1 of three mutant pilin genes fromP. aeruginosaPAK was studied to determine their effects on pilin stability, translocation into the membrane, leader peptide removal, and methylation of the mature N‐terminal phenylalanine. The results revealed that a deletion of 4 or 8 amino acids within the immediate N‐terminus of pilin had deleterious effects upon leader peptide cleavage. In addition, while the 4‐amino‐acid deletion did not affect pilin partitioning into the membrane, the 8‐amino‐acid deletion decreased the amount of pilin found within the membrane fraction. Of considerable interest was the finding that the mutation within the mature pitin of the glutamate at position 5 to a lysine did not prevent leader peptide removal but did inhibit the methylation of the N‐terminal

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1988.tb00055.x

出版商:Blackwell Publishing Ltd    

年代:1988

数据来源: WILEY

摘要:

SummarySubgenomic mRNA from a virulent isolate of porcine transmissible gastroenteritis virus (TGEV) was used to produce cDNA clones. Part of a new clone and a previously reported clone were sequenced and used to construct the viral gene for integral membrane protein. A single open reading frame (ORF) encoding a polypeptide of 262 amino acids, relative molecular mass (Mr) 29459, was identified. The positive identification of the polypeptide as the integral membrane protein was demonstrated by the production in E. coli of a chimaeric protein comprising most of the ORF encoding the Mr29459 polypeptide and β‐galactosidase. The chimaeric protein reacted with a specific monoclonal antibody to viral integral membrane protein and antibodies raised against the chimaeric protein immune precipitated the viral protein. Comparison with the sequence of an avirulent isolate indicates amino acid residues that may be important in pathogenici

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1988.tb00056.x

出版商:Blackwell Publishing Ltd    

年代:1988

数据来源: WILEY

摘要:

SummaryThe nucleotide sequence of a 2510 base pair chromosomal fragment containing the aerolysin geneaerA, and its regulatory region aerC, from a clinical isolate ofAeromonas sobriawas determined. The aerolysin gene coded for a 54.5 kD polypeptide and had a G + C content of 59%, indicating that it is endogenous to the genusAeromonas.In contrast, theaerCregion was characterized by its high A + T content (61%) and the presence of a core motif, aATAAAa, repeated eight times within 300 base pairs. A 12 base pair repeat, 5′ AATAAAACCGGG3′, present within this region occurred as a direct repeat 544 base pairs away, within the coding region of aerolysin. RNA polymerase binding studies and S1 mapping allowed the detection of two divergent non‐overlapping promoters withinaerC.Despite having identical transcriptional start sites in bothA. sobriaandEscherichia coli, the amount of aerolysin transcript produced inE. coliis 30–40 times less than that found inA. sobria.The signal peptide of preproaerolysin was shown by deletion to be essential for export of the toxin to the external medium. The mature toxin is a hydrophilic protein with no hydrophobic stretches long enough to cross a membrane. A search for similarities to the primary sequence of aerolysin revealed that the toxin may share a functional similarity to haemolysin (hlyA) of

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1988.tb00057.x

出版商:Blackwell Publishing Ltd    

年代:1988

数据来源: WILEY

摘要:

SummaryReplacement of the amino‐terminal 40‐amino‐acid region of the 588‐ammo‐acid precursor of the membrane‐bound penicillin‐binding protein 3 (P6P3) by the decapeptide MKGKEFQAWI was carried out by altering the amino‐coding end of theftslgene. Insertion of the modified gene into a runaway‐replication plasmid under the control of a fusedIpppromoter andlacpromoter/operator, resulted in the overexpression byEscherichia coliof the modified PBP3 (designated PBP3**) in the cytoplasm. About 80% of the accumulated PBP3** underwent sequestration in the form of insoluble protein granules that were isolated by cell breakage or cell lysis. After selective removal of contaminants by an EDTA‐lysozyme/DNase (de‐oxyribonuclease)/Nonidet extraction, treatment of the granules with guanidinium chloride followed by dialysis against buffer containing 0.5 M NaCI yielded a refolded, water‐soluble PBP3**, which, upon chromatography on Superose 12, exhibited the expected 60000 molecular mass. The refolded PBP3** bound benzytpenicillin in a 1 to 1 molar ratio, was highly sensitive to aztreonam and showed the same degree of thermostability, in terms of penicillin‐binding capacity, as the parent, membrane‐bound PBP3, suggesting that protein refolding occurred with formation of the correct intramolecular interactions. Two to three mg of refolded PBP** can be obtained from 1 litre of culture

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1988.tb00058.x

出版商:Blackwell Publishing Ltd    

年代:1988

数据来源: WILEY