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Biomedical subjects

E Lanka

Publications and source records attributed to E Lanka.

At least 73 records · Page 4Linked to original sources
PubMed1990

Covalent association of the traI gene product of plasmid RP4 with the 5'-terminal nucleotide at the relaxation nick site.

Formation of relaxosomes is the first step in the initiation of transfer DNA replication during bacterial conjugation. This nucleoprotein complex contains all components capable of introducing a site- and strand-specific nick at a cognate transfer origin (oriT) on supercoiled plasmid DNA, thus providing the substrate for generation of the strand to be transferred. Characterization of the terminal nucleotides at the oriT nick site revealed that relaxation occurs by hydrolysis of a single phosphodiester bond between a 2'-deoxyguanosyl and a 2'-deoxycytidyl residue. The relaxation nick site and a 19-base pair invert repeat sequence that is recognized by asymmetric binding of the RP4 TraJ protein are interspaced by 8 base pairs. The nicking reaction results in covalent attachment of the RP4 TraI protein to the 5'-terminal 2'-deoxycytidyl residue of the cleaved strand. The arrangement of the TraJ binding site and the relaxation nick site on the same side of the DNA double helix suggests that protein-protein interactions between TraJ and TraI are a prerequisite for oriT specific nicking. In accordance with the current model of transfer DNA replication, the 3' end remains accessible for primer extension by DNA polymerase I, enabling replacement strand synthesis in the donor cell by a rolling circle-type mechanism.

Bacterial Proteins↗
PubMed1990

Identification of a seventh operon on plasmid RK2 regulated by the korA gene product.

Broad-host-range IncP plasmids possess a series of operons involved in plasmid maintenance, whose expression is coordinated by a series of regulators, most of which are encoded in a central regulatory operon. The nucleotide sequence of a new monocistronic operon located between coordinates 55.0 and 56.0 kb on the genome of the IncP alpha plasmids RK2 and RP4 is presented. The operon encodes a 34 kDa protein which has a net negative charge. Transcription of the operon, designated by us kfrA (korF-regulated), is repressed not only by the product of the previously described korA gene but also by the product of a gene which we have designated korF and which has not been described previously. The korF gene is encoded downstream from korB within the key korA/korB regulatory operon. We propose that K or F binds to a novel inverted repeat overlapping the promoter for the kfrA operon.

Amino Acid Sequence↗
PubMed1990

In vitro assembly of relaxosomes at the transfer origin of plasmid RP4.

During initiation of conjugative transfer of DNA containing the transfer origin (oriT) of the promiscuous plasmid RP4, the proteins TraI, TraJ, and TraH interact and assemble a specialized nucleoprotein complex (the relaxosome) at oriT. The structure can be visualized on electron micrographs. Site- and strand-specific nicking at the transfer origin in vitro is dependent on the proteins TraI and TraJ and on Mg2+ ions. Substrate specificity is directed exclusively towards the cognate transfer origin: the RP4-specified TraJ protein cannot recognize the closely related oriT of plasmid R751. After nicking, TraI protein remains attached to the 5'-terminal 2'-deoxycytidyl residue at the nic site [Pansegrau, W., Ziegelin, G. & Lanka, E. (1990) J. Biol. Chem. 265, 10637-10644]. Nicking and relaxosome formation require supercoiled DNA. Thus, a complicated structure involving multiple plasmid-specified proteins and a defined region of DNA must be formed at the transfer origin to prepare the plasmid for generating the single strand to be transferred.

Amino Acid Sequence↗
PubMed1989

TraJ protein of plasmid RP4 binds to a 19-base pair invert sequence repetition within the transfer origin.

Transfer of plasmid RP4 during bacterial conjugation requires the plasmid-encoded TraJ protein, which binds to the transfer origin (Fürste, J. P., Pansegrau, W., Ziegelin, G., Kröger, M., and Lanka, E. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 1771-1775). As indicated by traJ mutants, the TraJ protein is a constituent of the relaxosome, the initiation complex of transfer DNA replication. The traJ gene maps adjacent to the transfer origin (oriT). The structural gene consists of a 372-base pair sequence encoding a polypeptide of 122 amino acids (13,282 Da). TraJ was purified from an Escherichia coli strain overproducing the protein. DNA footprinting experiments involving DNase I demonstrated that the purified protein binds to the right arm of a 19-base pair inverted repeat within oriT. Hydroxyl radical footprints of the DNA-protein complex revealed that TraJ protein is bound to only one side of the DNA helix.

Amino Acid Sequence↗
PubMed1989

Conjugative transfer of promiscuous IncP plasmids: interaction of plasmid-encoded products with the transfer origin.

To characterize protein-DNA interactions involved in the initiation of conjugative transfer replication, we isolated and sequenced the transfer origins (oriT) of the promiscuous IncP plasmids RP4 and R751. The central initiating event at the transfer origin of a conjugative plasmid is the cleavage at a unique site (nic) of the strand to be transferred to a recipient cell. This process can be triggered after the assembly of "relaxosomes" (plasmid DNA-protein relaxation complexes), requiring plasmid-encoded gene products. We analyzed the nicking reaction for plasmid RP4 and demonstrated that one of the plasmid strands is specifically cleaved within oriT. The fully functional oriT of RP4 represents an intergenic DNA region of approximately 350 base pairs. Dissection of oriT revealed that a portion carrying nic and symmetric sequence repeats determines oriT specificity. This part of oriT is contiguous to a region that is essential for efficient mobilization of oriT plasmids. In addition, oriT contains potential promoter sites allowing divergent transcription of two operons flanking oriT. We over-produced gene products and, from analyzing the products of defined deletion mutants, deduced the gene arrangements. Formation of RP4 relaxosomes is likely to depend on the presence of at least two plasmid-encoded components, which act in trans. Corresponding genes map on one side of oriT. Purification of the traJ product revealed it to be an 11-kDa polypeptide that binds to oriT DNA in vitro. The protein recognizes the part of oriT that is responsible for oriT specificity.

Base Sequence↗
PubMed1989

Mutagenesis of the Tra1 core region of RK2 by using Tn5: identification of plasmid-specific transfer genes.

The conjugation system of the IncP alpha plasmid RK2/RP4 is encoded by transfer regions designated Tra1, Tra2, and Tra3. The Tra1 core region, cloned on plasmid pDG4 delta 22, consists of the origin of transfer (oriT) and 2.6 kilobases of flanking DNA providing IncP alpha plasmid-specific functions that allow pDG4 delta 22 to be mobilized by the heterologous IncP beta plasmid R751. Tn5 insertions in pDG4 delta 22 define a minimal 2.2-kilobase region required for plasmid-specific transfer of oriT. The Tra1 core contains the traJ and traK genes as well as an 18-kilodalton open reading frame downstream of traJ. The traJ and traK genes were shown to be required for transfer by complementation of inserts within these genes. Genetic evidence for the role of the 18-kilodalton open reading frame in transfer was obtained, although this protein has not been detected in cell lysates. These studies indicate that at least three transfer proteins are involved in plasmid-specific interactions at oriT.

Bacterial Proteins↗
PubMed1988

The origin of conjugative IncP plasmid transfer: interaction with plasmid-encoded products and the nucleotide sequence at the relaxation site.

To characterize protein-DNA interactions involved in the initiation of conjugative transfer replication we isolated and dissected the transfer origins (oriT) of the promiscuous IncP plasmids RP4 and R751. Essential features of oriT are conserved: symmetric sequence repeats, the nic site and a pair of potential promoter sites that allow for divergent transcription of two tra operons. The relaxation nick and the end of a 19 bp inverted repeat are interspaced by eight basepairs. The 5'-terminal nucleotide at the nick is modified by an alkali-resistant residue and the 3'-nucleotide is accessible to extension by DNA polymerase I. Transfer gene products essential for the formation of the initiation complex (relaxosome) of conjugative DNA synthesis map adjacent to oriT. Two of these products, TraJ and TraK confer specificity to their homologous oriT exclusively. Proteins TraJ and TraK are the only components of the RP4 and R751 transfer machinery which cannot be interchanged. TraJ and at least two additional plasmid-encoded products are necessary for specific relaxation. The purified TraJ protein of RP4 possesses oriT-binding ability. The recognition sequence contains a palindromic sequence located within the right arm of the 19 bp inverted repeat. The TraJ binding site and the nic site are located on one side of the DNA double helix. We presume that this nucleoprotein structure is the initial complex in the pathway to the assembly of functional relaxosomes.

Bacterial Proteins↗
PubMed1988

Replication of mini RK2 plasmid in extracts of Escherichia coli requires plasmid-encoded protein TrfA and host-encoded proteins DnaA, B, G DNA gyrase and DNA polymerase III.

Soluble extracts of Escherichia coli capable of carrying out replication of the mini-RK2 derivative pCT461 have been prepared from cells carrying this plasmid or from plasmid-free bacteria. The latter are dependent upon exogenously added plasmid-encoded replication protein (TrfA) and require additional DnaA protein for optimum activity. This dependence upon DnaA was confirmed by the failure of DnaA-deficient cell extracts to support replication of pCT461 in the absence of added DnaA protein. Replication is unidirectional and begins at or near oriV, the vegetative replication origin of RK2. DNase I protection studies with purified TrfA indicate that this protein acts by binding to short (17 base-pairs) directly repeated DNA sequences present in oriV. The in vitro replication is resistant to rifampicin but can be abolished by antibodies against DnaG protein (E. coli primase) or DnaB protein (helicase) and by DNA gyrase inhibitors. Inhibition by arabinosyl-CTP suggests that DNA polymerase III is responsible for elongation of nascent DNA strands. These results are discussed in relation to the mechanism of RK2 replication and in the context of the host range of the plasmid.

Bacterial Proteins↗
PubMed1987

Nucleotide sequence of the kanamycin resistance determinant of plasmid RP4: homology to other aminoglycoside 3'-phosphotransferases.

The kanamycin resistance determinant of the broad-host-range plasmid RP4 encodes an aminoglycoside 3'-phosphotransferase of type I. The nucleotide sequence of the kanamycin resistance gene (Kmr) and the right end of the insertion element IS8 of plasmid RP4 has been determined. The gene (816 bp) is located between IS8 and the region (Tra 1) encoding plasmid factors mediating bacterial conjugation. Kmr and Tra 1 are transcribed toward each other. The nucleotide sequence has been compared to five related aphA genes originating from gram-negative and gram-positive organisms and from antibiotic producers. Among these that of Tn903 shares the highest degree of similarity (60%) with the RP4 gene. Significant similarities were also detected between the amino acid sequences of the six enzymes. The C-terminal domains of six different aminoglycoside 3'-phosphotransferases (APH(3'] are highly conserved. They are substantially similar to segments of a variety of enzymes using ATP as cofactor. The role of the C-terminal sequences of APH(3') as potential domains for ATP recognition and binding is discussed.

Base Sequence↗
PubMed1986

The involvement of host replication proteins and of specific origin sequences in the in vitro replication of miniplasmid R1 DNA.

The in vitro replication of R1 miniplasmid promoted by purified preparations of the plasmid encoded RepA protein in cell extracts of E. coli is resistant to rifampicin and can be completely inhibited by antibodies against DnaG, the primase of the cell, as well as by antibodies against proteins DnaB and SSB. R1 replication is abolished in extracts deficient in the DnaA protein. This deficiency is efficiently complemented by purified preparations of the DnaA protein. The in vitro replication of plasmid R1 is also abolished in DnaC deficient extracts and by a 10 bp deletion (nucleotides 1463-1472) within the minimal origin region. These data indicate the requirement of the DnaA, DnaB, DnaC, DnaG and SSB replication proteins of the host, as well as of specific oriR1 sequences for the RepA dependent replication of plasmid R1. The implications of these results for the initiation of R1 replication are discussed.

Bacterial Proteins↗
PubMed1986

Molecular cloning of the plasmid RP4 primase region in a multi-host-range tacP expression vector.

Plasmid RP4 primase was overproduced by utilizing autoregulated high-level expression vector systems in Escherichia coli and in four other Gram-negative bacterial species. Analysis of the products in E. coli revealed that in addition to the two primase polypeptides of 118 and 80 kDa the pri region of RP4 encodes two smaller proteins of 16.5 and 8.6 kDa. The transcript for the four RP4-specified products is polycistronic. The vector system used in E. coli is based on the plasmid pKK223-3 (Brosius and Holy, 1984), a ColE1-type replicon which contains a polylinker sequence flanked on one side by the controllable tac promoter and on the other side by two strong transcriptional terminators. The gene for the lac repressor (lacIQ) was inserted to render the use of the plasmid independent from repressor-overproducing strains. The gene cartridge essential for high-level expression and selection was combined with the RSF1010 replicon to generate a vector plasmid functioning in a wide variety of Gram-negative hosts. The versatility of the vector family was extended by constructing derivatives that contain the polylinker in inverted orientation relative to the tac promoter. Therefore, the orientation of the cloned fragment can be chosen by 'forced cloning' into the appropriately selected vector.

Cloning, Molecular↗
PubMed1986

Molecular cloning into Tn5 and integration in the Pseudomonas aeruginosa chromosome: a tool for heterologous gene expression.

The DNA primase gene of the promiscuous IncP-1 conjugative plasmid RP1, encoding two polypeptides of 118 and 80 kDa, was inserted into the transposon Tn5 in Escherichia coli. The derivative transposon, Tn2523, was then transposed to a temperature-sensitive replication mutant of the promiscuous IncP-1 conjugative plasmid R68 at permissive temperature and the plasmid transferred to Pseudomonas aeruginosa strain PAO. The latter strain was then grown at non-permissive temperature to identify transposition of Tn2523 into the P. aeruginosa chromosome. Immunological and enzymic analysis showed the expression of functional primase polypeptides in the constructed P. aeruginosa strain. This strain also restored wild-type conjugational transfer proficiency, by complementation, to mutants of the IncP-1 plasmid R18 affected in transfer from P. aeruginosa to P. stutzeri or to Acinetobacter calcoaceticus due to transposon Tn7 insertion mutations in the primase gene. This strategy of cloning into a transposon and integration into the bacterial chromosome should facilitate genetic manipulation and studies of gene expression in a range of Gram-negative bacteria.

Chromosomes, Bacterial↗
PubMed1985

Conserved regions at the DNA primase locus of IncP alpha and IncP beta plasmids.

Genes specifying DNA primases (pri) are common in all IncP plasmids examined so far. These plasmids suppress the thermosensitive character of the Escherichia coli dnaG3 mutation. The mechanism of suppression appears to be identical to that known for RP4 and IncI alpha plasmids. The DNA primases of both these plasmid types can substitute for the dnaG protein in chromosomal DNA replication. The pri genes of the alpha and beta subgroup of IncP plasmids are related to each other as judged from Southern hybridization and immunological data. Extensive DNA and protein sequence homology has been detected although the gene products of the alpha and beta subgroups exhibit substantial differences in size. The arrangement of overlapping genes at the pri locus of IncP alpha plasmids also appears to be present in the IncP beta group.

DNA Primase↗
PubMed1985

F factor inhibition of conjugal transfer of broad-host-range plasmid RP4: requirement for the protein product of pif operon regulatory gene pifC.

By the use of deletions, point mutations, and gene fusions, we show that the protein product of the F factor pifC gene is responsible for F factor inhibition of plasmid RP4 conjugal transfer. Deletion analysis of pif sequences carried by pSC101-F chimeric plasmids demonstrated that removal of all or part of the pifC coding sequence greatly decreased or abolished the ability of these plasmids to inhibit RP4 transfer. Amber mutations in the pifC gene eliminated inhibition in an Su- host strain but not in and Su+ (supF) host. Plasmids carrying nonpolar pifC mutations did not decrease the efficiency of RP4 transfer when present in trans. Whereas pifC+ plasmids inhibited RP4 transfer, the presence of RP4 in the same cell as F' lac increased F'lac Pif activity approximately 1,000-fold. This effect most likely resulted from the binding of the pifC product to RP4 DNA and concomitant derepression of the F factor pif operon. PifC inhibited trans mobilization of pMS204, a nonconjugative plasmid carrying the RP4 oriT locus, by the RP1 derivative pUB307. pMS204 had no trans effect on pif operon expression, whereas pUB307 increased F'lac Pif expression, as did RP4. Our results suggest that the pifC product inhibits expression of one or more RP4 genes, the products of which are required for conjugal transfer of RP4 and are required in trans for mobilization of nonconjugal RP4 oriT containing plasmids.

Base Sequence↗
PubMed1984

Plasmid RP4 encodes two forms of a DNA primase.

The pri gene locus of the conjugative broad host range plasmid RP4 maps between coordinates 40.3 and 43.5 and encodes two antigenically related forms of a DNA primase with a molecular mass of 118 and 80 kDa (kilodalton). Genesis of these two products has been examined using Pri+-recombinant plasmids. As shown by deletion analysis, the primase polypeptides are tow separate translation products which arise from an in-phase overlapping gene arrangement. It is suggested that transcription of a set of RP4 genes including the pri gene starts at a promoter site within the Tra1 region. In vivo, RP4 mutant primase can apparently substitute for Escherichia coli primase as demonstrated by measuring suppression of the dnaG3 (ts) mutant.

Bacterial Proteins↗
PubMed1984

Insertion mutations in the promiscuous IncP-1 plasmid R18 which affect its host range between Pseudomonas species.

Fifty-one host range mutants of the promiscuous plasmid R18 were isolated by Tn7 insertion mutagenesis by using Pseudomonas aeruginosa as the permissive, and P. stutzeri as the nonpermissive, host. Endonuclease cleavage mapping of 40/51 mutants showed that 37 mutations mapped to kilobase coordinates 40.3-43.8 in the two overlapping genes encoding plasmid DNA primase. Thus by this procedure it has been possible readily to isolate a large number of primase mutants. The majority of these mutations mapped to the overlapping DNA whereas a few also mapped to the nonoverlap region encoding the larger 118-kDa polypeptide. Among these mutants were four which had long deletions within the overlapping segment and extending to varying lengths anticlockwise of it. The genetic defect in these mutants has been correlated with greatly reduced in vitro primase enzyme activity. The primase mutations drastically affected the mutant's ability to mobilize a nonconjugative, wide-host-range IncP-4(Q) plasmid from P. aeruginosa to P. stutzeri although mobilization within P. aeruginosa was affected to a lesser degree. Other insertion mutations were mapped to the regions of plasmid origin of transfer (oriT) and origin of replication (oriV), but their physical location was different to previously identified similar mutations obtained using Escherichia coli as the nonpermissive host. Their physically distinct locations were correlated with differences in their transmissibility from P. aeruginosa into enteric bacterial species and into other Pseudomonas species.

Chromosome Mapping↗