摘要:

SummaryA family of novel mobile DNA elements is described, examples of which are found at several independent locations and encode a variety of antibiotic resistance genes. The complete elements consist of two conserved segments separated by a segment of variable length and sequence which includes inserted antibiotic resistance genes. The conserved segment located 3′ to the inserted resistance genes was sequenced from Tn21 and R46, and the sequences are identical over a region of 2026 bases, which includes the sulphonamide resistance genesull, and two further open reading frames of unknown function. The complete sequences of both the 3′ and 5′ conserved regions of the DNA element have been determined. A 59‐base sequence element, found at the junctions of inserted DNA sequences and the conserved 3′ segment, is also present at this location in the R46 sequence. A copy of one half of this 59‐base element is found at the end of thesullgene, suggesting thatsull, though part of the conserved region, was also originally inserted into an ancestral element by site‐specific integration. Inverted or direct terminal repeats or short target site duplications, both of which are characteristics of class I and class II transposons, are not found at the outer boundaries of the elements described here. Furthermore, the conserved regions do not encode any proteins related to known transposition proteins, except the DNA integrase encoded by the 5′ conserved region which is implicated in the gene insertion process. Mobilization of this element has not been observed experimentally; mobility is implied from the identification of the element in at least four independent locations, inTn21, R46 (IncN), R388 (IncW) andTn1696.The definitive features of these novel elements are (i) that they include site‐specific integration functions (the Integrase and the insertion site); (ii) that they are able to acquire various gene units and act as an expression cassette by supplying the promoter for the inserted genes. As a consequence of acquiring different inserted genes, the element exists in a variety of forms which differ in the number and nature of the inserted genes. This family of elements appears formally distinct from other known mobile DNA elements and we propose the name DNA integration eleme

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1989.tb00153.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

SummaryThe complete nucleotide sequence of the 4746bp HindIII fragment encoding the genes for the biosynthesis and assembly of CS3 pili has been determined. By site‐directed mutagenesis in conjunction with analysis of the plasmid‐encoded proteins in minicells, the actual reading frames for the various products have been determined. This demonstrated that the genes for four of the proteins (63 kD, 48 kD, 33 kD, and 20kD in size) are encoded entirely within the same open reading frame as a fifth protein (104kD). However, for synthesis of this latter protein, suppression or readthrough of an internal amber codon is required. Termination at this codon is also necessary for synthesis of the former proteins. Two further proteins are also encoded within the HindIII fragment: a 27 kD precursor of a periplasmic protein and the 17.5kD precursor of the major CS3 fimbrial subu

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1989.tb00154.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

SummaryRegulation of the DNA damage‐inducibleRAD2gene was investigated in yeast cells transformed with centromeric plasmids containingRAD2‐lacZfusion constructs. Deletion analysis defined several regions in the 350 bp region upstream of the translational start codon which are required for induction of β‐galactosidase activity. No deletions resulted in constitutivety enhanced expression. We therefore conclude that induction ofRAD2by DNA‐damaging agents is positively regulated. Two domains required for induction have a similar sequence and are located ∼70 and ∼140bp upstream of the major transcriptional start site. Four other sequence domains required for induction contain uninterrupted poly(dA) poly(dT) stretches 9‐13 bp long. Deletion of some of these AT‐rich domains also affects constitutive expression ofRAD2.Expression ofRAD2is not cell‐cycle‐regulated in mitotic cells. However, meiosis is accompanied by increased steady‐state levels ofRAD2mRNA in the absence of DNA damage. This enhanced transcription is not dependent on the presence of upstream sequences required for regulation of induction by DNA damage. Increased steady‐state levels ofRAD2mRNA are induced by cycloheximide in asynchronously dividing populations of cells, but not in non‐replicating cells arrested in G1 phase of the cell cycle. Following exposure to u.v. irradiation induction is also dramatically reduce

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1989.tb00155.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

SummaryHydrogenase activity and other hydrogenase‐related functions can be restored tohydCmutants by the specific addition of nickel salts to the growth medium. These mutants are defective in all three hydrogenase isoenzymes and the restoration is dependent upon protein synthesis. The cellular nickel content of the mutant when grown in LB medium is less than 1% of that of the parental strain. Partial suppression of the hydrogenase phenotype ofhydCmutants occurs when growth takes place in a different medium. This correlates with an increased cellular nickel content. The phenotype of the mutant is also fully suppressed by growth in media of very low magnesium content. Such media facilitate nickel uptake via the magnesium transport system, which leads to the acquisition of a normal cellular nickel content. Mutations in thefnrgene, which encodes a transcriptional regulator for several anaerobically expressed enzymes, abolisheshydCexpression and gives rise to a defective hydrogenase phenotype. The hydrogenase phenotype offnris closely similar to that ofhydCin all respects examined. The hydrogenase activity offnrstrains can be restored by the presence of a functionalhydCgene on a multicopy plasmid. The hydrogenase phenotype offnrstrains therefore arises indirectly via suppression ofhydC, which leads to a low cellular nickel content. Nickel has no influence on fumarate reductase or nitrate reductase activities infnrstrains. The hydrogen‐metabolism phenotype offnrstrains is, therefore, dependent upon their ability to acquire nickel from growth media. It is likely thathydCencodes a specific transport system for nic

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1989.tb00156.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

SummaryBordetella bronchisepticais a common respiratory tract pathogen of many mammalian species. Nucleotide sequences from the iocus involved in coordinate regulation of B. pertussis virulence factors,vir, were shown to have a high degree of homology to chromosomal DNA from virulent (Vir+) and avirulent (Vir−) strains ofB. bronchiseptica.Small deletions, 50 bp to 500 bp, within thevirlocus were found in some of the Vir−phase variants. Thevirlocus and the adjacent 5′ portion of thefhaBstructural gene were cloned from the parental Vir+B. bronchisepticastrain on a 23.5kbBamHlfragment. Restriction enzyme mapping of the clonedB. bronchiseptica virlocus revealed similarities with and differences from the previously clonedB. pertussis virlocus. The clonedB. bronchiseptica virlocus complemented spontaneous Vir−variants ofBordetella pertussisandB. bronchisepticaas well aswr::Tn5mutants ofB. pertussis.Comparison of various functions of thevirloci ofB. bronchisepticaandB. pertussisrevealed some interesting differences in the coordinate regulation of virulence

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1989.tb00157.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

SummaryEnterotoxigenicEscherichia coli(ETEC) andShigellaaccount for a substantial proportion of acute diarrhoeal illnesses among Third‐World children. Rapid detection of these infectious agents in faeces followed by the prompt implementation of public health measures could help reduce their spread during the early phase of epidemics. Towards this end, three pairs of synthetic oligonucleotide primers were prepared and shown to hybridize specifically to the genes encoding the heat‐stable (ST) and the heat‐labile (LT) enterotoxins of ETEC and to invasion‐associated loci (ial) of the largeShigellavirulence plasmid. When the three primer pairs were used together in the polymerase chain reaction (PCR), the three corresponding genetic loci could be simultaneously amplified using DNA extracted directly from stool; the amplified products were readily detected by ST‐, LT‐ and ial‐specific, alkaline phosphatase‐labelled oligonucleotide probes (AP probes). The performance of this system was evaluated in a Mayan community in southeastern Mexico, where diarrhoeal illnesses are a common cause of childhood morbidity and mortality. Using only simple and inexpensive laboratory equipment, multigene amplification with these primers and probes led to the identification of ETEC and/orShigellain the stools of 20 out of 71 children with diarrhoea; the procedure could be completed in seven hours and was more sensitive than conventional diagnostic tests or DNA probes used withou

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1989.tb00158.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

SummaryThe S fimbrial adhesin (Sfa) enablesEscherichia colito attach to sialic acid‐containing receptor molecules of eukaryotic cells. As previously reported, the genetic determinant coding for the Sfa of anE. coliO6 strain was cloned, the gene coding for the major fimbrial subunit was identified and sequenced and the S specific adhesin was detected. Here we present evidence that in addition to the major subunit protein SfaA three other minor subunit proteins, SfaG (17kD), SfaS (14 kD) and SfaH (31 kD) can be isolated from the S‐specific fimbrial adhesin complex. The genes coding for these minor subunits were identified, mutagenized separately and sequenced. Using haemagglutination tests, electron‐microscopy and quantitative ELISA assays with monoclonal anti‐SfaA and anti‐SfaS antibodies the functions of the minor subunits were determined. It was determined that SfaS is identical to the S‐specific adhesin, which also plays a role in determination of the degree of fimbriation of the cell. The minor subunit SfaH also had some influence on the level of fimbriation of the cell, while SfaG is necessary for full expression of S‐specific binding. It was further shown that the amino‐terminal protein sequence of the isolated SfaS protein was identical to the protein sequence calculated from the DNA sequence of th

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1989.tb00159.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

SummaryThe average copy number, the level of ampicillin resistance conferred by one plasmid, and the degree of plasmid multimerization were determined for several ColE1‐like and pBR322‐like plasmids. From the results obtained, the variance of the units of partition corresponding to each plasmid studied was calculated. Experimentally determined plasmid stability was compared with that calculated using the variance of the units of partition and the ratio between the generation times of plasmid‐free and of plasmid‐carrying cells, assuming that the units of partition are distributed randomly between daughter cells.Stability of the pBR322‐like plasmids present mainly as monomers in the bacterial host was consistent with random partitioning, whereas pBR322‐like plasmids, present mainly as dimers, and the ColE1‐like plasmid showed greater stability than that predicted with random partitioning at

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1989.tb00160.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

SummaryNucleotide sequence analysis of a 2.5kb region downstream of thenifAgene fromRhizobium leguminosarumbiovartrifoliihas resulted in linkage, at the DNA sequence level, of thenifEN, nifHDK, fixABCX, nifAgene cluster with thenodEF, nodD, nodABCIJgenes. Four genes have been identified within this intervening region. Immediately 3’to thenifAgene is thenifBgene and thenifB‐linked ferredoxin‐encodingfdxNgene. Downstream offdxNinR. leguminosarumbv.trifoliiand inRhizobium meliloti, we have identified an open reading frame which has not been described previously and which we propose to designatefixU.Downstream offixUin R.leguminosarumby.trifoliiis anodgene,nodT, which is contiguous withnodJ(B. Surinet al., manuscript in preparation). As a result of this study, the linkage relationships of 22 symbiotic genes spanning a 24 kb region of the symbiotic plasmid from R.leguminosarumbv.trifoiiiare now

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1989.tb00161.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

SummaryTwo open reading frames (ORFs), designated ORF95 and ORF162, downstream of theKlebsiella pneumoniaeσ54structural gene(rpoN)have been sequenced and shown to encode polypeptides of 12kD and 16kD, respectively. ORFs homologous to ORF95 are present downstream of four out of fiverpoNgenes sequenced to date from a range of Gram‐negative bacteria, and ORF162 is also conserved, at least inPseudomonas putida.Chromosomal mutations have been created in each gene using akancassette and both have the same phenotype, i.e. they cause an increase in the level of expression from σ54‐dependent promoters. We propose that the products of both genes function to modulate the activity of Eσ54, although a physiological role for these proteins has not yet been iden

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1989.tb00162.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY