摘要:

SummaryAn integron is a genetic unit that includes the determinants of the components of a site‐specific recombination system capable of capturing and mobilizing genes that are contained in mobile elements called gene cassettes. An integron also provides a promoter for expression of the cassette genes, and integrons thus act both as natural cloning systems and as expression vectors. The essential components of an integron are anintgene encoding a site‐specific recombinase belonging to the integrase family, an adjacent site,attl, that is recognized by the integrase and is the receptor site for the cassettes, and a promoter suitably oriented for expression of the cassette‐encoded genes. The cassettes are mobile elements that include a gene (most commonly an antibiotic‐resistance gene) and an integrase‐specific recombination site that is a member of a family of sites known as 59‐base elements. Cassettes can exist either free in a circularized form or integrated at theattlsite, and only when integrated is a cassette formally part of an integron. A single site‐specific recombination event involving the integron‐associatedattlsite and a cassette‐associated 59‐base element leads to insertion of a free circular cassette into a recipient integron. Multiple cassette insertions can occur, and integrons containing several cassettes have been found in the wild. The integrase also catalyses excisive recombination events that can lead to loss of cassettes from an integron and generate free circular cassettes. Due to their ability to acquire new genes, integrons have a clear role in the evolution of the genomes of the plasmids and transposons that contain them. However, a more general role in evolution is also likely. Events involving recombination between a specific 59‐base‐element site and a nonspecific secondary site have recently been shown to occur. Such events should lead either to the insertion of cassettes at non‐specific sites or to the formation of stable cointegrates between different plasmid molecules, and a cassette situated outside the integron context has

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.tb02368.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

SummaryDNA topoisomerases are essential to the cell for the regulation of DNA supercoiling levels and for chromosome decatenation. The proposed mechanisms for these reactions are essentially the same, except that a change in supercoiling is due to an intramolecular event, while decatenation requires an intermolecular event. The characterized bacterial topoisomerases appear capable of both types of reactionin vitro.Four DNA topoisomerases have been identified in Escherichia coli. Topoisomerase I, gyrase, and topoisomerase IV normally appear to have distinct essential functions within the cell, Gyrase and topoisomerase I are responsible for the regulation of DNA supercoiling. Both gyrase and topoisomerase IV are necessary for chromosomal decatenation. Multiple topoisomerases with distinct functions may give the cell more precise control over DNA topology by allowing tighter regulation of the principal enzymatic activities of these different proteins.

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.tb02369.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

SummaryFew bacterial pathogens are as widespread in nature or as capable of eliciting such a diversity of disease syndromes as are the chlamydiae. As obligate intra‐cellular organisms, they pose a special research challenge in defining the molecular components and mechanisms for productive growth within host cells and the overall progress of infection throughout host tissue. Although a comprehensive view of chlamydial envelope composition and respective functions in pathogenesis is far from complete, ongoing investigations continue to expose new and intriguing avenues for exploratio

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.tb02370.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

SummaryChlamydiae are obligate intracellular bacteria which occupy a non‐acidified vacuole (the inclusion) throughout their developmental cycle. Little is known about events leading to the establishment and maintenance of the chlamydial inclusion membrane. To identify chlamydial proteins which are unique to the intracellular phase of the life cycle, an expression library ofChlamydia psittaciDNA was screened with convalescent antisera from infected animals and hyperimmune antisera generated against formalin‐killed purified chlamydiae. Overlapping genomic clones were identified which expressed a 39 kDa protein only recognized by the convalescent sera. Sequence analysis of the clones identified two open reading frames (ORFs), one of which (ORF1) coded for a predicted 39 kDa gene product. The ORF1 sequence was amplified and fused to themalEgene ofEscherichia coliand antisera were raised against the resulting fusion protein. Immunoblotting with these antisera demonstrated that the 39 kDa protein was present in lysates of infected cells and in reticulate bodies (RBs), but was at the limit of detection in lysates of purifiedC. psittacielementary bodies. Fluorescence microscopy experiments demonstrated that this protein was localized in the inclusion membrane of infected HeLa cells, but was not detected on the developmental forms within the inclusion. Because the protein produced by ORF1 is deposited on the inclusion membrane of infected cells, this gene has been designatedincA, (inclusion membrane proteinA) and its gene product, IncA. In addition to the inclusion membrane, these antisera labelled structures that extended from the inclusion over the nucleus or into the cytoplasm of infected cells. Immunoblotting also demonstrated that IncA, in lysates of infected cells, had a migration pattern that seemed indicative of post‐translational modification. This pattern was not observed in immunoblots of RBs or in theE. coliexpressing IncA. Collectively, these data identify a chlamydial gene which codes for a protein that is released from RB and is localized in the inclusion membrane of infected

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.tb02371.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

SummaryRhizobium lotiis a fast‐growingRhizobiumspecies that has been described as a microsymbiont of plants of the genusLotus.Nodulation studies show thatLotusplants are nodulated byR loti, but not by most otherRhizobiumstrains, indicating thatR. lotiproduces specific lipo‐chitin oligosaccharides (LCOs) which are necessary for the nodulation of Lotus plants. The LCOs produced by five differentRhizobium iotistrains have been purified and were shown to beN‐acetylglucosamine pentasaccharides of which the non‐reducing residue isN‐methylated andN‐acylated with c/s‐vaccenic acid (C18:1) or stearic acid (C18:O) and carries a carbamoyl group. In oneR. lotistrain, NZP2037, an additional carbamoyl group is present on the non‐reducing terminal residue. The major class of LCO molecules is substituted on the reducing terminal residue with 4‐O‐acetylfucose. Addition of LCOs to the roots ofLotusplants results in abundant distortion, swelling and branching of the root hairs, whereas spot inoculation leads to the formation

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.tb02372.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

SummaryThe location of thecpegene, encoding the enterotoxin responsible for food poisoning in humans, has been studied in a series of enterotoxigenicCiostridium perfringensstrains by means of pulsed field gel electrophoresis of genomic DNA. The cpe gene was found at the same chromosomal locus in strains associated with food poisoning in humans and was shown to be linked to a repetitive sequence, theHindlll repeat, and an open reading frame, ORF3, that may be part of an insertion sequence. In contrast, when the strains originated from domesticated livestockcpewas located on a large episome where it was often close to a copy of the transposable element IS1151.In these cases, theHindlll repeat was not linked to thecpegene although this was generally preceded by ORF3.

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.tb02373.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

SummaryBacteriophage T4 middle promoters, which are transcribed using phage‐modified host RNA polymerase and the T4 transcriptional activator, MotA, match the host σ70consensus sequence at – 10, but they have a different consensus ((t/a)(t/a)TGCTT(t/c)A) (a MotA box) at – 30. While the T4 middle promoter Puvsxhas these – 10 and –30 motifs, it also has matches to the MotA box at –35, –51, –70, and –87. We show that MotA binds to PuvsxDNA, footprinting a region that includes the MotA boxes at –30, –35, and –51. Very high levels of MotA are required for footprinting and gel‐shift experiments, and protein‐DNA complexes formed in the presence of both phage‐modified polymerase and MotA are more resistant to Hindlll cleavage than those formed with either protein alone. These results suggest that MotA‐DNA interactions may be stabilized by phage‐modified polymerase. Sequences between –18 and –38 are absolutely required for MotA activation of transcription, but sequences upstream of –38 are stimulatory, particularly when chloride instead of glutamate is the major anion. Our results dissect Puvsxinto a core promoter, downstream of ‐38, which is required for MotA activation, and an upstream region that enhances transcription especially under conditions le

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.tb02374.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

SummaryThe edema factor (EF) and lethal factor (LF) components of anthrax toxin require anthrax protective antigen (PA) for binding and entry into mammalian cells. After internalization by receptor‐mediated endocytosis, PA facilitates the translocation of EF and LF across the membrane of an acidic intracellular compartment. To characterize the translocation process, we generated chimeric proteins composed of the PA recognition domain of LF (LFN; residues 1–255) fused to either the amino‐terminus or the carboxy‐terminus of the catalytic chain of diphtheria toxin (DTA). The purified fusion proteins retained ADP‐ribosyltransferase activity and reacted with anti‐sera against LF and diphtheria toxin. Both fusion proteins strongly inhibited protein synthesis in CHO‐K1 cells in the presence of PA, but not in its absence, and they showed similar levels of activity. This activity could be inhibited by adding LF or the LFNfragment (which blocked the interaction of the fusion proteins with PA), by adding inhibitors of endo‐some acidification known to block entry of EF and LF into cells, or by introducing mutations that attenuated the ADP‐ribosylation activity of the DTA moiety. The results demonstrate that LFNfused to either the amino‐terminus or the carboxy‐terminus of a heterologous protein retains its ability to complement PA in mediating translocation of the protein to the cytoplasm. Besides its importance in understanding translocation, this finding provides the basis for constructing a translocation vector that mediates entry of a variety of heterologous proteins, which may require a free amino‐ or carboxy‐terminus for biological activity, into the cyt

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.tb02375.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

SummaryThe transcriptional regulation of thepilE gene, coding for the pilin inNeisseria gonorrhoeae, by PilA/PilB proteins is quite complex. Sequence analysis of PilA suggested that it has multiple domains. PilA appears to have in its N‐terminal half a DNA‐binding site followed by a region showing sequence similarity with other bacterial transcriptional regulators. In its C‐terminal half, PilA has extensive homology with the 54kDa protein of the eukaryotic signal‐recognition particle which is involved in protein secretion. A transcriptional fusion between the promoter ofpilEand thelacZgene was constructed and integrated into the gonococcal chromosome. We show that transcription of thepilE‐lacZfusion is affected inpHAmutants in the absence of any possible interference with pilin secretion. Moreover,pilEtranscription depends on a 24/‐12‐type promoter which could be a member of a family of promoters recognized by the alternative Sigma subunit, RpoN, of the RNA polymerase. We also show that PilA binds specifically to the promoter region ofpilEand that it is phosphorylated in a manner dependent on acidic residues Glu‐59, Asp‐149 and Asp‐186. The functional organization of PilA suggests that it may be an unusual transcriptional regulator different from other RpoN

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.tb02376.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

SummaryA number of small, multicopy plasmids from Gram‐positive bacteria replicate by an asymmetric rolling‐circle mechanism. Previous studies with several of these plasmids have identified a palindromic sequence,SSOA, that acts as the single‐strand origin (SSO) for the replication of the lagging‐strand DNA. Although not all theSSOAsequences share ONA sequence homology, they are structurally very similar. We have used anin vitrosystem to study the lagging‐strand replication of several plasmids from Gram‐positive bacteria using theSSOAsequences of pT181, pE194 and pSN2 as representative of three different groups ofStaphylococcus aureusplasmids. In addition, we have investigated the lagging‐strand replication of the pUB110 plasmid that contains an alternative single‐strand origin,ssou. Our results confirm that RNA polymerase is involved in lagging‐strand synthesis from bothSSOAand ssou‐type lagging‐strand origins. Interestingly, while initiation of lagging‐strand DNA synthesis of pUB110 occurred predominantly at a single position withinssou, replication of pT181, pSN2 and pE194 plasmids initiated at mult

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.tb02377.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY