摘要:

SummaryRecent insight into the biochemical mechanism of protein translocation inEscherichia coliindicates that SecA ATPase is required both for the initial binding of preproteins to the inner membrane as well as subsequent translocation across this structure. SecA appears to promote these events by direct recognition of the preprotein or preprotein‐SecB complex, binding to inner‐membrane anionic phospholipids, insertion into the membrane biiayer and association with the preprotein translocator, SecY/SecE. ATP binding appears to control the affinity of SecA for the various components of the system and ATP hydrolysis promotes cycling between its different biochemical states. As a component likely to catalyse a rate‐determining step in protein secretion, SecA synthesis is co‐ordinated with the activity of the protein export pathway. This form of negative reguiation appears to rely on SecA protein binding to its mRNA and repressing translation if conditions of rapid protein secretion prevail within the cell. A precise biochemical scheme for SecA‐dependent catalysis of protein export and the details ofsecAregulation appear to be close at hand. The evolutionary conservation of SecA protein among eubacteria as well as the general requirement for translocation ATPases in other protein secretion systems argues for a mechanistic commonality of all prokaryotic protein export

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1993.tb01107.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

SummaryRhodobacter capsulatusis a member of the group α‐purple bacteria which are closely related to the ancestral endosymbiont that gave rise to mitochondria. It has therefore been hypothesized that the molecular mechanisms governing protein export in α‐purple bacteria have been conserved during the evolution of mitochondria. To enable analysis of protein export in α‐purple bacteria we describe here the development of a homologous cell‐free synthesis/export system consisting entirely of components ofR. capsulatus.Translocation of precytochromeC2into intracytoplasmic membrane vesicles of this organism was found to require the proton‐motive force and proceed at a significantly higher efficiency when membranes were present during protein synthesis. Furthermore, we show that, in this cell‐free system, translocation depends on a preparation of peripheral membrane proteins Which do not possess detectable SecA‐ and Sec

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1993.tb01108.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

SummaryIn eukaryotes, metallothioneins (MTs) are involved in cellular responses to elevated concentrations of certain metal ions. We report the isolation and analysis of a prokaryotic MT locus fromSynechococcusPCC 7942. The MT locus (smt) includessmtA, which encodes a class II MT, and a divergently transcribed gene,smtB.The sites of transcription initiation of both genes have been mapped and features within thesmtoperator‐promoter region identified. Elevated concentrations of the ionic species of Cd, Co, Cr, Cu, Hg, Ni, Pb and Zn elicited an increase in the abundance ofsmtAtranscripts. There was no detectable effect of elevated metal (Cd) on smtA transcript stability. Sequences upstream ofsmtA, fused to a promoterlesslacZgene, conferred metal‐dependent β‐galactosidase activity in Synechococcus PCC 7942 (strain R2‐PIM8). At maximum permissive concentrations, Zn was the most potent elicitorin vivo, followed by Cu and Cd with slight induction by Co and Ni. The deduced SmtB polypeptide has similarity to the ArsR and CadC proteins involved in resistance to arsenate/arsenite/antimonite and to Cd, contains a predicted helix‐turn‐helix DNA‐binding motif and is shown to be a repressor of transcription from thesmtAope

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1993.tb01109.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

SummaryGenomic rearrangements involving amplification of metallothionein (MT) genes have been reported in metal‐tolerant eukaryotes. Similarly, we have recently observed amplification and rearrangement of a prokaryotic MT locus,smt, in cells ofSynechococcusPCC 6301 selected for Cd tolerance. Following the characterization of this locus, the alteredsmtregion has now been isolated from a Cd‐tolerant cell line, C3.2, and its nucleotide sequence determined. This has identified a deletion withinsmtB, which encodes a trans‐acting repressor ofsmttranscription. Two identical palindromic octanucleotides (5′‐GCGATC‐GC‐3′) traverse both borders of the excised element. This palindromic sequence is highly represented in thesmtlocus (7 occurrences in 1326 nucleotides) and analysis of the GenBank/EMBL /DDB J DNA Nucleotide Sequence Data Libraries reveals that this is a highly iterated palindrome (HIP1) in other known sequences fromSynechococcusstrains (estimated to occur at an average frequency of once everyc.664 bp). HIP1 is also abundant in the genomes of other cyanobacteria. The functional significance ofsmtBdeletion and the possible role of HIP1 in genome plasticity and adaptation in cyanobacteri

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1993.tb01110.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

SummaryIn an attempt to unify the genetic and biological research onMycobacterium leprae, the aetiological agent of leprosy, a cosmid library was constructed and then ordered by a combination of fingerprinting and hybridization techniques. The genome of M. leprae is represented by four contigs of overlapping clones which, together, account for nearly 2.B Mb of DNA. Several arguments suggest that the gaps between the contigs are small in size and that virtually complete coverage of the chromosome has been obtained. All of the clonedM. lepraegenes have been positioned on the contig maps together with the 29 copies of the dispersed repetitive element, RLEP. These have been classified into four groups on the basis of differences in their organization. Several key housekeeping genes were identified and mapped by hybridization with heterologous probes, and the current genome map of this uncultivable pathogen comprises 72 loci.

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1993.tb01111.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

SummaryThe nucleotide sequence of cosmid B1790, carrying the Rif‐Str regions of theMycobacterium lepraechromosome, has been determined. Twelve open reading frames were identified in the 36716bp sequence, representing 40% of the coding capacity. Five ribosomal proteins, two elongation factors and the β and β'subunits of RNA polymerase have been characterized and two novel genes were found. One of these encodes a member of the so‐called ABC family of ATP‐binding proteins while the other appears to encode an enzyme involved in repairing genomic lesions caused by free radicals. This finding may well be significant asM. leprae, an intracellular pathogen, lives within macr

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1993.tb01112.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

SummaryAmet4mutant ofSaccharomyces cerevisiaewas unable to transcribe a number of genes encoding enzymes of the methionine biosynthetic pathway. The sequence of the cloned MET4 gene allowed the previously sequenced flankingLEU4andPOL1genes to be linked to MET4 into a 10 327 bp contiguous region of chromosome XIV. From the sequence and mapping of the transcriptional start points, MET4 is predicted to encode a protein of 634 amino acids (as opposed to 666 amino acids published by others) with a leucine zipper domain at the C‐terminus, preceded by both acidic and basic regions. Thus, MET4 belongs to the family of basic leucine zipper trans‐activator proteins. Disruption ofMET4resulted in methionine auxotrophy with no other phenotype. Transcriptional studies showed thatMET4was regulated by the general amino acid control and hence by another bZIP protein encoded by GCN4. GCN4 binding sequences are present between the divergently transcribedMET4andLEU4genes. Over‐expression ofMET4resulted in leaky expression from the otherwise tightly regulatedMET3promoter under its control. The presence of consensus sequences for other potential regulatory elements in theMET4promoter suggests a complex regulation of this

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1993.tb01113.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

SummaryAdherence of type‐1‐fimbriateSalmonella entericaandEscherichia colito immobilized proteins of the extracellular matrix and reconstituted basement membranes was studied. The type‐1‐fimbriate strain SH401 ofS. entericaserovar Enteritidis showed good adherence to laminin, whereas the adherence to fibronectin, type I, type III, type IV or type V collagens was poor. Only minimal adherence to the matrix proteins was seen with a non‐fimbriate strain of S.entericaserovar Typhimurium. A specific and mannoside‐inhibitable adhesion to laminin was exhibited by the recombinantE. colistrain HB101(plSF101) possessingfimgenes of Typhimurium. Adherence to laminin of strain SH401 was inhibited by Fab fragments against purified SH401 fimbriae, and a specific binding to laminin, of the purified fimbriae, was demonstrated using fimbriae‐coated fluorescent microparticles. Periodate treatment of laminin abolished the bacterial adhesion as well as the fimbrial binding. Specific adhesion to immobilized laminin was also shown by the type‐1 ‐fimbriate E.colistrain 2131 and the recombinant strainE. coliHB101(pPKL4) expressing the cloned type‐1‐fimbriae genes ofE. coli.Adhesion to laminin of strain HB101(pPKL4) was inhibited by mannoside, and no adherence was seen with thefimHmutantE. coliHB101(pPKL5/pPKL53) lacking the fimbrial lectin subunit. The type‐1 fimbriate strains also adhered to reconstituted basement membranes from mouse sarcoma cells and human placenta. Adhesion of strains HB101(plSF101) and HB101(pPKL4) to both basement membrane preparations was inhibited by mannoside. We conclude that type‐1 fimbriae of S. enterica and E. coli bind to oMgomannoside chains of the lamjnjn netw

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1993.tb01114.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

SummaryThe genetic determinants for the completeShigella sonneilipopolysaccharide (LPS) have been cloned, characterized by restriction mapping, and expressed in heterologous genetic backgrounds, includingSalmonella typhiandVibrio choleraelive attenuated vaccine strains. Therfb/rfclocus encoding the polymerized serotype‐specific O polysaccharide was mapped within 23 kb of DNA isolated from S. sonnei virulence plasmid pWR105. A highly similar chromosomal DNA sequence was identified by Southern hybridization analysis inPlesiomonas shigelloidesknown to have the same O serotype specificity asS. sonnei.Expression studies of therfb/rfclocus have shown thatS. sonnei.O polysaccharide is covalently bound to LPS cores of both the K‐12 and RI types, but neither toSalmonella(Ra‐type) nor toV. choleraeO1 cores. In order to express a compatible core structure in the latter organisms, chromosomal rfa loci encoding R1‐type LPS were isolated from both anEscherichia coliR1 strain (rfaR1) and fromS. sonnei(rfdsonnei). Restriction mapping and functional analysis of cloned DNA allowed us to localize the rfaR1 locus and to orient it with respect to the neighbouring cysE chromosomal marker. A high degree of sequence similarity was found at the DNA level between rfa loci of enterobacterial species characterized by Ri‐type LPS. Co‐expression studies involvingS. sonnei rfb/rfcand rfa loci propagated on compatible plasmids have shown that, at most, 13 to 14 kb of r/api DNA are required for the expression of complete phase‐l‐likeS. sonneiLPS inE. coliK‐12 andS. typhi, whereas an adjacent region of about 3.5 kb is needed in the more stringent host,V. cholerae, S. sonneiO antigen expressed in aV. eholeraerecombinant vaccine strain is present on the cell surface in a form suitable for the induction of a specific antibody response in v

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1993.tb01115.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

SummaryStrains ofEscherichia colihave been produced which express very high levels of the tRNAleu1isoacceptor. This was accomplished by transforming cells with plasmids containing theleuVoperon which encodes three copies of the tRNALeu1gene. Most transformants grew very slowly and exhibited a 15‐fold increase in cellular concentrations of tRNALeu1As a result, total cellular tRNA concentration was approximately doubled and 56% of the total was tRNALeu1. We examined a number of parameters which might be expected to be affected by imbalances in tRNA concentration: in vivo tRNA charging levels, misreading, ribosome step time, and tRNA modification. Surprisingly, no increase in intracellular ppGpp levels was detected even though only about 40% of total leucyl tRNA was found to be chargedin vivo.Gross ribosomal misreading was not detected, and it was shown that ribosomal step times were reduced between two‐ and threefold. Analyses of leucyl tRNA isolated from these slow‐growing strains showed that at least 90% of the detectable tRNALeu1was hypomodified as judged by altered mobility on RPC‐5 reverse‐phase columns, and by specific modification assays using tRNA(m1G)‐methyltransferase and pseudo‐uridylate synthetase. Analysis of fast‐growing revertants demonstrated that tRNA concentrationper semay not explain growth inhibition because selected revertants which grew at wild‐type growth rates displayed levels of tRNA comparable to that of control strains bearing theleuVoperon. A synthetic tRNALeu1operon under the control of the T7 promoter was prepared which, when induced, produced six‐ to sevenfold increases in tRNALeu1levels. This level of tRNALeu1titrated the modification system as judged by RPC‐5 column chromatography. Overall, our results suggest that hypomodified tRNA may explain, in part, the observed effects on growth, and that the protein‐synthesizing system can tolerate an enormous increase in the concent

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1993.tb01116.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY