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PubMed1992

Analysis of mutations in trfA, the replication initiation gene of the broad-host-range plasmid RK2.

Plasmids with mutations in trfA, the gene encoding the replication initiation protein of the broad-host-range plasmid RK2, were isolated and characterized. Mutants identified from a nitrosoguanidine bank were defective in supporting the replication of a wild-type RK2 origin in Escherichia coli. Most of the mutations were clustered in a region of trfA corresponding to the carboxy-terminal quarter of the TrfA protein. 5' and 3' deletion mutants of trfA were also constructed. A C-terminal deletion of three amino acids of the Tr A protein was completely nonfunctional for RK2 replication. However, a deletion of 25 amino acids from the start of the 33-kDa TrfA protein was still competent for replication. Further characterization of the point and deletion trfA mutants in vivo revealed that a subset was capable of supporting RK2 replication in other gram-negative bacteria, including Pseudomonas putida, Agrobacterium tumefaciens, and Azotobacter vinelandii. Selected mutant TrfA proteins were partially purified and characterized in vitro. Velocity sedimentation analysis of these partially purified TrfA proteins indicated that the wild-type protein and all mutant TrfA proteins examined exist as dimers in solution. Results from in vitro replication assays corroborated the experimental findings in vivo. Gel retardation results clearly indicated that the point mutant TrfA-33:151S, which was completely defective in replication of an RK2 origin in all of the bacterial hosts tested in vivo, and a carboxy-terminal deletion mutant, TrfA-33:C delta 305, were not able to bind iterons in vitro. In addition to the partially defective or could not be distinguished from the wild-type protein in binding to the origin region. The mutant proteins with apparently normal DNA-binding activity in vitro either were inactive in all four gram-negative bacteria tested or exhibited differences in functionality depending on the host organism. These mutant TrfA proteins may be altered in the ability to interact with the replication proteins of the specific host bacterium.

Agrobacterium tumefaciens↗
PubMed1985

Copy number of the broad host-range plasmid R1162 is determined by the amounts of essential plasmid-encoded proteins.

DNA of the broad host-range plasmid R1162 contains a 1700 base-pair segment essential for plasmid maintenance. This region, RepI, consists of two cotranscribed genes encoding polypeptides with molecular weights of 29,000 and 31,000. Fusion of RepI to the strong tac promoter results in greatly increased amounts of at least one of these polypeptides. In trans, this construction has two other properties: it can raise the copy number of R1162, and it can protect this plasmid from loss due to incompatibility. Both effects require intact RepI genes. These properties of the RepI region, along with those of an origin-linked region described earlier, are discussed with respect to current models for control of plasmid copy number.

Adenosine Triphosphatases↗
PubMed1976

Generation in vitro of deletions in the broad host range plasmid RK2 using phage Mu insertions and a restriction endonuclease.

Several non-lethal deletions of the broad host range plasmid RK2 (molecular weight of 37.6 . 10(6) have been produced in vitro. The method employed relied on the single HindIII restriction nuclease site in RK2 and the ability of phage Mu to insert and thereby add new HindIII restriction sites at various positions in the plasmid. The deleted plasmids have in each case lost kanamycin (Km) resistance, and in two cases are defective in self-transmissibility. The method used to reduce the size of the RK2 plasmid also results in the cloning of each of the two ends of the Mu phage DNA on the plasmid derivatives.

Ampicillin↗
PubMed1980

Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium meliloti.

A broad host range cloning vehicle that can be mobilized at high frequency into Gram-negative bacteria has been constructed from the naturally occurring antibiotic resistance plasmid RK2. The vehicle is 20 kilobase pairs in size, encodes tetracycline resistance, and contains two single restriction enzyme sites suitable for cloning. Mobilization is effected by a helper plasmid consisting of the RK2 transfer genes linked to a ColE1 replicon. By use of this plasmid vehicle, a gene bank of the DNA from a wild-type strain of Rhizobium meliloti has been constructed and established in Escherichia coli. One of the hybrid plasmids in the bank contains a DNA insert of approximately 26 kilobase pairs which has homology to the nitrogenase structural gene region of Klebsiella pneumoniae.

Cloning, Molecular↗
PubMed1986

Complementation of mutations in Escherichia coli and Bordetella pertussis by B. pertussis DNA cloned in a broad-host-range cosmid vector.

A gene library of Bordetella pertussis DNA was constructed in Escherichia coli using the broad-host-range cosmid vector pLAFR1. The average insert size was 24.9 kb. From 500 members of the gene library, clones were identified which complemented trpE, glnA and Thr- mutations in E. coli but none which complemented trpD, trpC, trpB, trpA, proA or Leu- mutations. Four clones were identified which complemented trpE in E. coli. Anthranilate synthase activity was detected in a trpE strain only when it harboured a plasmid from one of these clones; activity was repressed when tryptophan was included in the growth medium. Two clones were identified which complemented glnA of E. coli. A recombinant plasmid from one of these clones also restored some of the nitrogen acquisition functions of glnG and glnL in E. coli. Expression of several B. pertussis virulence-associated products (haemolysin, heat-labile toxin, adenylate cyclase, filamentous haemagglutinin, and the cell-envelope polypeptide of Mr 30,000) was not detected in 500 independent clones. However, by transferring the recombinant plasmids to a mutant of B. pertussis deficient in haemolysin and adenylate cyclase, a plasmid was identified which restored both these activities.

Anthranilate Synthase↗
PubMed2006

Transformation of rhizobia with broad-host-range plasmids by using a freeze-thaw method.

Several species of rhizobia were successfully transformed with broad-host-range plasmids of different replicons by using a modified freeze-thaw method. A generic binary vector (pPZP211) was maintained in Mesorhizobium loti without selection and stably inherited during nodulation. The method could extend the potential of rhizobia as a vehicle for plant transformation.

Alphaproteobacteria↗
PubMed1990

Construction of broad-host-range plasmid vectors for easy visible selection and analysis of promoters.

We have constructed a series of broad-host-range plasmids which use "visual screens" to detect promoter activity. These plasmids contain the pMB1 and pRO1600 origins of replication and are capable of replicating in a wide range of gram-negative bacteria. The genes encoding beta-galactosidase and alkaline phosphatase from Escherichia coli and bacterial luciferase from Vibrio harveyi supply the promoterless indicator genes. The constructs were tested in E. coli and Pseudomonas aeruginosa.

Alkaline Phosphatase↗
PubMed1998

Diversity of the limited-host range iaaH gene of Agrobacterium vitis strain Ag162.

The indole-3-acetamide hydrolase gene (iaaH) of the limited-host range strain AG162, a biotype III strain of Agrobacterium tumefaciens has been the subject of several studies and reviews, but its primary structure has not been previously reported. In the course of our own work we found that this gene hybridizes only weakly to a nucleic acid probe corresponding to the iaaH gene from a biotype I strain of A. tumefaciens. Analysis of the primary structure of the Ag162 iaaH gene revealed that it is diverse from biotype I iaaH genes and, surprisingly, also from the iaaH genes of previously characterized biotype III Agrobacterium strains.

Amidohydrolases↗
PubMed1996

Host range restriction of parainfluenza virus growth occurs at the level of virus genome replication.

To illuminate the molecular basis for host range restriction of parainfluenza virus replication, we have examined the types of virus macromolecules produced during abortive infection of nonpermissive MDBK cells with human parainfluenza virus type 1 (hPIV1). While these cells do not support production of hPIV1 virus, they can be infected by hPIV1 as evidenced by accumulation of intracellular viral NP and HN proteins. HPIV1 is also able to drive transcription of a synthetic analog of Sendai virus (SV) genome RNA transfected into virus-infected MDBK cells. In contrast to transcription, hPIV1 genome replication does not occur in MDBK cells. Intracellular full-length genome RNA was detected only in trace amounts 2 days after infection, and was undetectable 4 days after infection. Full-length antigenome (+) sense RNA was not detectable. Nucleocapsid complexes failed to accumulate in the cytoplasm of nonpermissive cells, and no detectable nucleocapsids were released into the medium as virus particles. The data indicate that defective vRNA synthesis and/or nucleocapsid formation is responsible for the inability of hPIV1 to grow in MDBK cells. Our data also show that hPIV1 is capable of providing all helper functions for packaging SV synthetic genome analogs into infectious particles, but these SV-specific RNAs encapsidated with hPIV1 proteins are in turn not replicated by SV proteins. These results suggest that functional protein-protein interactions between parainfluenza virus strains have more stringent requirements than do protein-RNA interactions.

Animals↗
PubMed1984

Transcription in the trfA region of broad host range plasmid RK2 is regulated by trfB and korB.

Transcription at various points in the trf A region of broad host range plasmid RK2 has been analysed by measuring expression of the galK gene inserted at EcoRI sites introduced previously by TB1723 transposition mutagenesis. Rightward transcription (anti-clockwise on RK2) probably from a single promoter, proceeds across two open reading frames coding for a 13 kD polypeptide of unknown function, and the trf A gene, which provides a protein(s) essential for plasmid replication. This transcription is not auto-regulated by the products of either open reading frame and is also not subject to significant attenuation prior to the end of the trfA open reading frame. Leftward transcription appears to be directed by at least two well separated promoters, the more leftward being three to four times stronger than the more rightward. Rightward, but not leftward, transcription is repressed about 9-fold by the trfB locus of RK2 alone (so far not separable from the loci korA and korD) in trans while the combination of the korB and trfB loci in trans represses both rightward transcription (about 100-fold) and leftward transcription (the stronger activity by 10 to 15-fold). Regulation of these operons is therefore qualitatively different. The kilD locus in the trfA region, which is suppressed by korD (trfB) is thus probably part of the rightward (trfA) operon, while leftward transcription may represent the start of an operon containing kilB. The results suggest that RK2kor loci act by repressing transcription of kil loci and that the kil and kor control circuits may be part of an interlocking system of RK2 genes involved in replication and stable maintenance.

Chromosome Mapping↗
PubMed1990

A multipurpose broad host range cloning vector and its use to characterise an extracellular protease gene of Xanthomonas campestris pathovar campestris.

A multipurpose broad host range plasmid, pIJ3200, was constructed by inserting the polylinker-containing 445 bp PvuII fragment of Bluescript M13 into the EcoRI site of the cosmid pLAFR1. Using this vector a protease gene of Xanthomonas campestris pathovar campestris, previously cloned in the recombinant plasmid pIJ3070, was located by deletion to a 2.2 kb DNA region. Sequencing of the protease gene revealed an open reading frame encoding a 580 amino acid polypeptide with molecular weight of 57,000. The deduced amino acid sequence showed strong homology with the subtilisin family of serine proteases. This, together with its sensitivity to inhibition by phenylmethylsulphonyl fluoride, suggests that the enzyme belongs to this family of proteases.

Amino Acid Sequence↗
PubMed1990

Electroporation and expression of the broad host-range plasmid pRK2501 in Azotobacter vinelandii.

Azotobacter vinelandii cells were transformed via high-voltage electroporation, with the broad host-range plasmid pRK2501. The number of transformants was dependent on the applied voltage, capacitance, and recovery procedure after electroporation. For example, Log, 4.44 transformants microgram-1 DNA were recovered in the A. vinelandii cell suspension electroporated at 1500 V and 25 microF capacitance (time constant 29.0 ms) and recovered on LB agar amended with 0.5 microgram/ml-1 kanamycin (pRK2501 encodes for both kanamycin and tetracycline resistance). Electroporation at 2500 V and capacitance settings of 25 and 3 microF did not produce any transformants. Cell survival was also poor at high voltages. A. vinelandii transformants were not recovered on N-free agar medium. In addition, no viable cells were recovered on N-free agar after electroporation at 2500 V, 25 microF; 2500 V, 3 microF; and 1500 V, 25 microF. Electroporation may be a useful method to genetically transform Azotobacter species for use in physiological and/or genetic studies.

Azotobacter↗
PubMed1987

Mode of insertion of the broad-host-range plasmid RP4 and its derivatives into the chromosome of Myxococcus xanthus.

The mode of insertion of the broad-host-range plasmid RP4 into the chromosome of Myxococcus xanthus strain DZ1 has been analyzed. The plasmid integrated in numerous sites of the chromosome and generated insertional mutations. There is a hot spot of integration located between 31.5 and 34.5 kb clockwise from the EcoRI site of the plasmid. In the absence of this segment the insertion can, however, take place, but much less efficiently. The presence of transposable elements on the plasmid decreases severely the insertion frequency. Once integrated, RP4 could be transferred back to Escherichia coli, either by precise excision or with a segment of the Myxococcus chromosome. The role of site-specific recombination in RP4 integration is discussed.

Chromosomes, Bacterial↗
PubMed1991

Elimination of broad-host range plasmid vectors in Escherichia coli by curing agents.

A comparative study was made of the susceptibility of broad-host range vector plasmids belonging to Inc P1 and Q groups in Escherichia coli to various curing agents. Plumbagin and SDS eliminated RP4 (Inc P1 group) plasmid whereas pKT231 (Inc Q) and pRK2013 (having ColE1 replicon) were eliminated by hexamine ruthenium (III) chloride, alpha-santonin, coumermycin A1 and cis-dichloro diamine platinum (II). The curing activity of these agents was specific.

Aminocoumarins↗
PubMed1995

An enhanced broad-host-range vector for gram-negative bacteria: avoiding tetracycline phototoxicity during the growth of photosynthetic bacteria.

A mobilizable, broad-host-range (bhr) plasmid was derived from the widely used IncP1 vector pRK415. The new vector, pRKD418, contains an additional resistance gene and an enlarged multiple cloning site (MCS) region. The optimal growth of pRK415-containing bacteria under photosynthetic conditions generally requires the use of optical filters to protect the selective antibiotic tetracycline (Tc) from photooxidation with the resulting production of toxic photoproducts; pRK415 is not stably maintained in the absence of selective pressure. The addition of a trimethoprim-resistant dihydrofolate reductase-encoding gene provided for optimal photosynthetic growth in the presence of a selective antibiotic without any special apparatus. The presence of an antibiotic marker not found in commonly used cloning vectors in many cases facilitates the subcloning of inserts into the bhr plasmid. The new MCS region provides further cloning flexibility with at least sixteen available restriction sites. Easily constructed derivative plasmids, exemplified by pRKD418KmE, provide a convenient screening procedure for the detection of recombinants during subcloning.

Base Sequence↗
PubMed1995

Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes.

Four new antibiotic-resistant derivatives of the broad-host-range (bhr) cloning vector pBBR1MCS have been constructed. These new plasmids have several advantages over many of the currently available bhr vectors in that: (i) they are relatively small (< 5.3 kb), (ii) they possess an extended multiple cloning site (MCS), (iii) they allow direct selection of recombinant plasmid molecules in Escherichia coli via disruption of the LacZ alpha peptide, (iv) they are mobilizable when the RK2 transfer functions are provided in trans and (v) they are compatible with IncP, IncQ and IncW group plasmids, as well as with ColE1- and P15a-based replicons.

Bacteria↗
PubMed1993

A base-paired hairpin structure essential for the functional priming signal for DNA replication of the broad host range plasmid RSF1010.

The two single-strand DNA initiation signals, ssiA(RSF1010) and ssiB(RSF1010) of the broad host-range plasmid RSF1010 contain proposed stem-loop structures. Nine single base-change mutations in the stem of the ssiA structure, each of which destroyed a relevant base pairing, damaged the ssiA activity. A second single-base change was introduced into each of the nine ssiA mutants in such a way that the base pairing was restored. Only three out of nine second base changes that restored the base pairing restored the ssiA activity up to the wild-type level. Thus, the three are intramolecular suppressors. The results strongly suggested that, in the area of the stem of ssiA where the suppressor mutations fell, base pairing was the most important structural parameter for the ssiA activity. By contrast, it is most probable that, in the other part of the stem of ssiA, both base-pairing and the intrinsic base sequence were the major determinants of the ssiA activity.

Base Sequence↗
PubMed1995

Characterization of the basic replicon of pCM1, a narrow-host-range plasmid from the moderate halophile Chromohalobacter marismortui.

The moderately halophilic bacterium Chromohalobacter marismortui contains a 17.5-kb narrow-host-range plasmid, pCM1, which shows interesting properties for the development of cloning vectors for the genetic manipulation of this important group of extremophiles. Plasmid pCM1 can stably replicate and is maintained in most gram-negative moderate halophiles tested. The replication origin has been identified and sequenced, and the minimal pCM1 replicon has been localized to a 1,600-bp region which includes two functionally discrete regions, the oriV region and the repA gene. oriV, located on a 700-bp fragment, contains four iterons 20 bp in length adjacent to a DnaA box that is dispensable but required for efficient replication of pCM1, and it requires trans-acting functions. The repA gene, which encodes a replication protein of 289 residues, is similar to the replication proteins of other gram-negative bacteria.

Amino Acid Sequence↗