摘要:

SummaryActive transport of maltose inEscherichia colirequires the presence of both maltose‐binding protein (MBP) in the periplasm and a complex of MalF, MalG, and MalK proteins (FGK2) located in the cytoplasmic membrane. Earlier, mutants inmalForMalGwere isolated that are able to grow on maltose in the complete absence of MBP. When the wild‐typemalE+allele, coding for MBP, was introduced into these MBP‐independent mutants, they frequently lost their ability to grow on maltose. Furthermore, starting from these Mal‐strains, Mal+secondary mutants that contained suppressor mutations inmalEwere isolated. In this study, we examined the interaction of wild‐type and mutant MBPs with wild‐type and mutant FGK2 complexes by using right‐side‐out membrane vesicles. The vesicles from a MBP‐independent mutant (malG511) transported maltose in the absence of MBP, withKmandVmaxvalues similar to those found in intact cells. However, addition of wild‐type MBP to these mutant vesicles produced unexpected responses. Althoughmale+malG511cells could not utilize maltose, wild‐type MBP at low concentrations stimulated the maltose uptake bymalG511vesicles. At higher concentrations of the wild‐type MBP and maltose, however, maltose transport intomalG511vesicles became severely inhibited. This behaviour of the vesicles was also reflected in the phenotype ofmale+malG511cells, which were found to be capable of transporting maltose from a low external concentration (1μM), but apparently not from millimolar concentrations present in maltose minimal medium. We found that the mutant FGK2complex, containing MalG511, had a much higher apparent affinity towards the wild‐type MBP than did the wild‐type FGK2complex. We propose that the wild‐type FGK2complex exists in at least two conformations, active and inactive, and that the binding of the liganded MBP converts the latter into the former. The mutant complex presumably exists predominantly in the active form that has a higher affinity toward liganded MBP, and the Inhibition of the mutant complex by an excess of maltose and wild‐type MBP may be explained as a form o

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb01376.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummaryRhizobium melilotiFixL and FixJ are members of a symbiotically essential two‐component system that regulates nitrogen‐fixation genes in response to environmental oxygen concentrations. FixL is a membrane protein that is thought to relay information about oxygen availability to FixJ via a phosphotransfer mechanism. FixJ increases expression of thenifAandfixKgenes by activating transcription of thenifAandfixKpromoters (p‐nifAandp‐fixK, respectively). In this study, we examined the relationship between thein vivoactivity of FixJ as a transcriptional regulator and its ability to be phosphorylatedin vitroby the sensor FixL. FixJ mutants were isolated that showed decreased activity onp‐nifAinEscherichia coli.Most of the FixJ mutant proteins also showed decreased activity on thefixKpromoter. These mutants were analysed inR. melilotifor activity onp‐nifAduring vegetative growth, where similarities and differences were observed when compared with their phenotypes inE. coli.Three mutants showing significantly less activity inR. melilotiwere examined for symbiotic activityin plantaand were found to be ineffective. When these three mutant FixJ proteins were examinedin vitrofor their ability to be phosphorylated by FixL, two mutants were found to have a significantly decreased ability to accept phosphate from FixL. These findings are discussed in relation to signal transduction in the F

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb01377.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummaryThis paper describes the cloning of a gene (pep) coding for pyrrolidone carboxylyl peptidase (PYRase), an enzyme which selectively removesN‐terminal pyroglutamic acid residues from polypeptldes. This gene was isolated fromStreptococcus pyogenesby construction of a gene library with a bacteriophage λ‐derived cosmid‐Escherichia colihost system. Nucleotide sequence determination of a 1.3 kb restriction fragment revealed a 645 bp open reading frame encoding a 215‐amino‐acid product ofMr, 23 135 consistent with the 26 kDa polypeptide obtained fromin vivooverexpression inE. coll.Southern hybridization confirmed thatpepis a single‐copy gene on theS. pyogeneschromosome. 5’and 3’endpoint mapping of the 0.7 kb specific transcript observed by Northern analysis permitted the identification of transcriptional initiation and termination signals. Structural features of thepepgene product fromS. pyogenesare discussed and compared with that fromBacillus subtilis.The lack of sequence identity with any other known protein or nucleotide sequence suggests that this enzyme belongs to a new cl

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb01378.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummaryA cDNA (xynA), encoding xylanase A (XYLA), was isolated from a cDNA library, derived from mRNA extracted from the rumen anaerobic fungus,Neocallimastix patriciarum.Recombinant XYLA, purified fromEscherichia coliharbouringxynA, had aMr, of 53000 and hydrolysed oat‐spelt xylan to xylobiose and xylose. The enzyme did not hydrolyse any cellulosic substrates. The nucleotide sequence ofxynArevealed a single open reading frame of 1821 bp coding for a protein ofMr, 66192. The predicted primary structure of XYLA comprised anN‐terminal signal peptide followed by a 225‐amino‐acid repeated sequence, which was separated from a tandem 40‐residueC‐terminal repeat by a threonine/proline linker sequence. The largeN‐terminal reiterated regions consisted of distinct catalytic domains which displayed similar substrate specificities to the full‐length enzyme. The reiterated structure of XYLA suggests that the enzyme was derived from an ancestral gene which underwent two discrete duplications. Sequence comparison analysis revealed significant homology between XYLA and bacterial xylanases belonging to cellulase/xylanase family G. One of these homologous enzymes is derived from the rumen bacteriumRuminococcus flavefaciens.The homology observed between XYLA and a rumen prokaryote xylanase could be a consequence of the horizontal transfer of genes between rumen prokaryotes and lower eukaryotes, either when the organisms were resident in the rumen, or prior to their colonization of the ruminant. It should also be noted thatNeocallimastixXYLA is the first example of a xylanase which consists of reiterated sequences. It remains to be established whether this is a common phenomenon in other rumen fungal plant cell

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb01379.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummaryInEscherichia coli minBmutants, cell division can take place at the cell poles as well as non‐polarly in the cell. We have examined growth, division patterns, and nucleoid distribution in individual cells of aminCpoint mutant and aminBdeletion mutant, and compared them to the corresponding wild‐type strain and anintR1strain in which the chromosome is overreplicated. The main findings were as follows. In theminBmutants, polar and non‐polar divisions appeared to occur independently of each other. Furthermore, the timing of cell division in the cell cycle was found to be severely affected. In addition, nucleoid conformation and distribution were considerably disturbed. The results obtained call for a re‐evaluation of the role of the MinB system in theE colicell cycle, and of the concept that limiting quanta of cell division factors are regularly produced during the cel

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb01380.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummaryArginine auxotrophs of the dinitrogen‐fixing cyanobacteriumAnabaenaspecies strain PCC 7120 were isolated after ultraviolet light mutagenesis and penicillin enrichment. Two of these auxotrophs were complemented by a cosmid gene library of the wild‐type strain established inEscherichiacoli that was transferreden masseto the mutants by conjugation. The gene complementing one of those mutants was found to complement anE. coli argCmutant. Sequencing analysis of the gene showed that it encodes a 322‐residue polypeptide that is homologous to the ArgC protein ofE. coli, Bacillus subtilisandStreptomyces clavuligerusand to theC‐terminal moiety of theSac‐charomyces cerevisiae ARG5,6gene product,N‐acetylglutamate semialdehyde dehydrogenase. A cysteine residue present in a highly conserved domain in the five proteins is probably located in the active site of the enzyme. Conserved among the ArgC proteins, sequences resembling the primary structure of nucleotide‐binding domains are also found. Downstream of theAnabaena argCgene seven nearly perfect repeats of a heptanucleotide (consensus sequence: 5′‐CTAA

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb01381.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummaryA computer‐based analysis of hydropathy and surface probability of representative members of each class of the Cry family of proteins was performed. A highly conserved hydrophobic motif within the previously described block, D2, is present not only In lepidopteran toxin genes but also in toxins active against diptera and coleoptera. An interesting feature of this hydrophobic motif is the presence of an aspartic residue (highly hydrophilic) in its middle part. Comparison with the amino acid sequence from diphtheria toxin showed that it also contains a hydrophobic motif similar to the one present in theBacillus thuringiensistoxins. It also contains an aspartic residue in the middle part and some speculations are presented on the function of this specific region with regard to the toxic mechanism of actio

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb01382.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummaryA64kDa lipoprotein (LP 64) haemagglutinin (pI 4.9–5.0) was isolated from the membrane ofMycoplasma gallisepticum.Triton X‐114 phase partitioning has demonstrated that the hydrophobic nature of this haemagglutinin is due to a lipid portion of the molecule. Autoradiography of [3H]‐palmitate‐labelledM. gallisepticumrevealed the presence of several additional lipoproteins. Immunoelectron microscopy demonstrated the localization of LP 64 to the base of the terminal structure. Densitometric scans of stained polyacrylamide gels ofM. gallisepticumshowed that LP 64 constitutes 1.7% of the total protein. Scans of immunoblots ofM. gallisepticumindicate that LP 64 is highly immunogenic in chickens, accounting for 7.4% of the total serum IgG response at four weeks post‐infection. A quantitative value for the IgG response to LP 64, relative to the percentage of total protein (the Relative Immunogenicity Index) was 4.4. LP 64 is conserved among several strains ofM. gallisepticum, but its presence could not be detected inMycoplasma synoviae.Antiserum raised to electroeluted LP 64 reacted specifically with this lipoprotein when assessed on either one‐or two‐dimensional immunoblots ofM. gallisepticum.This antiserum, as well as Fab fragments, inhibited haemagglutination of chicken erythrocytes and inhibited the attachment of14ClabelledM. gallisepticumto chicken tracheal epitheliumi

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb01383.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummaryLength variations ofHaemophilus influenzaeouter membrane porin protein P2 were found at the DNA and protein levels, notably in non‐capsulate strains. Protein length, measured by SDS‐polyacrylamide gel electrophoresis, was found to correlate with the length of the gene, measured by polymerase chain reaction amplification, and ranged from 35–42 kDa and 970–1090 nucleotides, respectively. This represents a length variation of some 15%. The genetic location of these variations was studied by restriction enzyme mapping 10 of the non‐capsulate strains revealing further polymorphisms at the DNA level. All 10 strains were distinct and differed from a type b strain. The conservation and assortment of the different restriction sites in the alleles is discussed in relation to the very great diversity previously described for this protein and of the whole genome itself in non‐capsulate strains. The roles of selection, horizontal gene transfer, and transformation in generating this diversity ar

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb01384.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummaryvirRis the central regulatory locus required for coordinate temperature‐regulated virulence gene expression in the human enteric pathogens ofShigellaspecies. Detailed characterization of VirR+clones revealed thatvirRconsisted of a 411 bp open reading frame (ORF) that mapped to a chromosomally located 1.8kbEcoRI‐AcclDNA fragment fromShigella flexneri.Insertional inactivation of thevirRORF at a uniqueHpalrestriction site resulted in a loss of VirR+activity. ThevirR ORF nucleotide sequence was virtually identical to theEscherichia coli hnsgene, which encodes the histone‐like protein, H‐NS. Based on the predicted amino acid sequence ofE. coliH‐NS, only a single conservative base‐pair change was identified in thevirRgene. An additional clone, designated VirRP, which only partially complemented thevirRmutation, was also characterized and determined by Southern hybridization and nucleotide sequence analysis to be unique fromvirR.Subclone mapping of this clone indicated that the VirRPphenotype was a result of the multiple copy expression of theS. flexnerigene for tRNATyr.These data constitute the first direct genetic evidence thatvirRis an analogue of theE. coli hnsgene, and suggest a model for temperature regulation ofShigellaspecies virulence via the bacterial translationa

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb01385.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY