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PubMed1994

Mapping of determinants of the host range for canine cells in the genome of canine parvovirus using canine parvovirus/mink enteritis virus chimeric viruses.

Feline panleukopenia virus (FPLV), mink enteritis virus (MEV) and canine parvovirus (CPV) are more than 98% similar in DNA and predicted amino acid sequences, but they show different host-cell specificities; CPV is able to replicate in canine cells in culture, whereas FPLV and MEV cannot or replicate only to a low titre. To map the genomic region responsible for the host range of CPV in vitro, CPV/MEV chimeric viruses were generated by transfecting infectious CPV/MEV chimeric plasmids into a cultured feline kidney cell line, and their host cell ranges were analysed. The 60 to 91 map units (m.u.) region of the CPV genome, which contains a part of the capsid protein (VP) gene encoding from amino acid 91 (in the VP2 sequence) to the carboxy terminus of VP protein, was required to impart the ability to replicate in canine cells to MEV, although the chimeric virus containing the 60 to 91 m.u. region of the CPV genome in the MEV background did not replicate in canine cells as efficiently as did CPV derived from the infectious plasmid of CPV. Not only the VP gene, but also a part of the NS gene of CPV were considered to participate in the full expression of the ability to replicate in canine cells. Within the 60 to 91 m.u. region, five of nine amino acid changes between MEV-Abashiri and CPV-Y1 were thought to be phylogenetically CPV-common; however, a recombinant virus containing all five amino acid changes of CPV in the MEV background replicated minimally in canine cells.

Animals↗
PubMed1992

kfrA gene of broad host range plasmid RK2 encodes a novel DNA-binding protein.

The korABF operon of broad host range IncP plasmid RK2 encodes proteins that coordinate expression of many other operons and that aid plasmid stability by providing at least part of a partitioning apparatus. The kfrA gene lies downstream from this operon and its transcription is repressed by all except one of the proteins encoded by this operon (KorA, KorFI, KorFII and KorB). We report here that transcription from the kfrA promoter is autoregulated by the kfrA gene product. We have purified KfrA, which is an acidic polypeptide of 308 amino acid residues, and show that it is a site-specific DNA-binding protein whose operator overlaps the primary kfrA promoter. Deletion analysis suggests that this activity is critically dependent on the N-terminal section of KfrA, which appears to contain an alpha-helix-beta-turn-alpha-helix motif. Circular dichroism confirmed the structural prediction that KfrA is almost entirely alpha-helical. The position of predicted turns suggests that, while amino acid residues 1 to 80 may form a globular domain of four or five helices, residues 80 to 280 of KfrA may adopt an extended coiled-coil domain containing a heptad repeat segment, which is probably responsible for formation of the multimers detected by crosslinking. The possibility that this unusual structure serves a second function, for example in providing a bridge to host structures required for plasmid partitioning, is discussed.

Amino Acid Sequence↗
PubMed1994

Cell surface receptor for ecotropic host-range mouse retroviruses: a cationic amino acid transporter.

The cell surface receptor for ecotropic host-range murine leukemia viruses is a sodium-independent transporter for essential cationic acids. Our evidence strongly identifies this receptor as the transporter system y+, which was previously characterized by transport assays. Mutational analysis indicates that transporter activity is not necessary for viral reception. Infection of cells with ecotropic retroviruses causes only a partial down-modulation of receptor expression on cell surfaces.

Amino Acid Sequence↗
PubMed1983

Deletion mapping of kil and kor functions in the trfA and trfB regions of broad host range plasmid RK2.

Figurski et al. (1982) have reported that certain loci on the broad host range plasmid RK2 (kil functions) can be cloned only in the presence of other trans-acting segments of the plasmid genome (kor functions). They have suggested that the presence of these functions may in part account for the structure of mini RK2 replicons which were constructed in order to define the regions of the plasmid which encode replication/maintenance functions (Thomas et al. 1980). We have therefore investigated the relationship between these two sets of kil and kor loci and the loci implicated in the replication/maintenance of RK2. We find that, whilst the three kil loci reported by Figurski et al. (1982) are absent from these derivatives, a fourth such locus (kilD) is closely linked to trfA, a gene essential for RK2 replication. The kilD locus was probably responsible for the inclusion in mini replicons of a segment of RK2 DNA which carries both korD and korA in addition to trfB, a gene defined by a temperature-sensitive maintenance defect, but which can be deleted leaving a functional RK2 replicon (Thomas 1981 b). The kilB locus is situated on the opposite side of kilD from trfA, all three loci lying within a 3.6 kb segment of RK2 DNA. The korA, korD and trfB functions all map within a 900 bp segment of DNA, while korB requires sequence information at least 1.5 kb from this segment.

DNA Replication↗
PubMed1987

A 38 base-pair segment of DNA is required in cis for conjugative mobilization of broad host-range plasmid R1162.

oriT, the region required in cis for conjugative mobilization of broad host-range plasmid R1162, has been localized to a 38 base-pair segment of DNA. The oriT DNA is also required for conjugation-dependent recombination. Point mutations at the HinPI cleavage site within oriT affect both mobilization and recombination, and the crossover location has been mapped to this site. An inverted repeat ten base-pairs from the recombination site is also involved in mobilization and recombination, and may be a recognition site for proteins involved in cleavage of the oriT DNA. The properties of conjugation-dependent recombination suggest that mobilization entails the formation of a linear intermediate that is transferred with both a unique origin and polarity.

Base Composition↗
PubMed1985

Broad-host-range plasmid vector for the in vitro construction of transcriptional/translational lac fusions.

A broad-host-range plasmid was constructed that allows the in vitro formation of beta-galactosidase fusions. DNA from the photosynthetic bacterium Rhodopseudomonas sphaeroides was cloned into this plasmid and a number of R. sphaeroides isolates were recovered that had varying levels of beta-galactosidase activity. beta-galactosidase antigenic activity from the fusion strains could be localized immunologically in polypeptides with an Mr of 120 000 or greater. Expression of beta-galactosidase activity under control of fusion derivatives was either very low or nonexistent in Escherichia coli relative to R. sphaeroides, indicating that R. sphaeroides promoters or translational start signals function poorly in E. coli.

Cell Compartmentation↗
PubMed2000

Use of green fluorescent protein color variants expressed on stable broad-host-range vectors to visualize rhizobia interacting with plants.

We developed two sets of broad-host-range vectors that drive expression of the green fluorescent protein (GFP) or color variants thereof (henceforth collectively called autofluorescent proteins [AFPs]) from the lac promoter. These two sets are based on different replicons that are maintained in a stable fashion in Escherichia coli and rhizobia. Using specific filter sets or a dedicated confocal laser scanning microscope setup in which emitted light is split into its color components through a prism, we were able to unambiguously identify bacteria expressing enhanced cyan fluorescent protein (ECFP) or enhanced yellow fluorescent protein (EYFP) in mixtures of the two. Clearly, these vectors will be valuable tools for competition, cohabitation, and rescue studies and will also allow the visualization of interactions between genetically marked bacteria in vivo. Here, we used these vectors to visualize the interaction between rhizobia and plants. Specifically, we found that progeny from different rhizobia can be found in the same nodule or even in the same infection thread. We also visualized movements of bacteroids within plant nodule cells.

Bacteriological Techniques↗
PubMed1987

DnaA protein is required for replication of the minimal replicon of the broad-host-range plasmid RK2 in Escherichia coli.

The minimal origin of replication of the broad-host-range plasmid RK2 has two potential recognition sequences for the DnaA protein of Escherichia coli. DNA transfer by transformation into a dnaA-null mutant of E. coli showed that DnaA protein is needed for replication or maintenance of mini-RK2. We isolated and purified DnaA protein as a chimeric protein, covalently attached to a piece of collagen and beta-galactosidase. The hybrid protein specifically bound to restriction fragments from the oriV region of RK2, which contained the two dnaA boxes. Deletion of the second dnaA box inactivated the origin and abolished the binding of the hybrid protein to the DNA fragment that had suffered the deletion. When the second dnaA box was replaced with an EcoRI linker of identical length, origin activity was restored. Binding experiments showed that the linker provided a weak dnaA box. An alternative explanation was that the linker restored proper spacing between sequences on either side of the deleted box, thus restoring origin activity.

Bacterial Proteins↗
PubMed1990

Partitioning of broad-host-range plasmid RP4 is a complex system involving site-specific recombination.

The broad-host-range plasmid RP4 encodes a highly efficient partitioning system (par) that was previously mapped within the 6.2-kb PstI C fragment. The essential functions were assigned to a region of 2.2 kb between fiwA and IS21 (IS8). On the basis of the nucleotide sequence data of the entire par locus and of in vitro and in vivo expression studies, three distinct loci encoding polypeptides of 9, 18, and 24 kDa were identified. Evidence for the expression of another polypeptide was found. A putative divergent promoter was localized in an intergenic region and is suggested to be responsible for transcription of these genes. It was found that the RP4 par region includes a function resolving plasmid dimers. The 24-kDa polypeptide is considered to function as a resolvase, since its predicted amino acid sequence shows homology to sequences of resolvases of the Tn3 family. Furthermore, palindromes present in the intergenic region containing the divergent promoter resemble repeat structures specific for res sites of Tn3-related transposons. However, it was found that dimer resolution itself was not sufficient for stabilization; additional functions, including the other two polypeptides, seemed to play an important role. These results suggested that RP4 contains a complex stabilization system involving resolution of plasmid dimers during cell division, thus ensuring the delivery of at least one copy to each daughter cell.

Amino Acid Sequence↗
PubMed1997

Prospects for altering host range for baculovirus bioinsecticides.

Advances in the understanding of baculovirus replication and the identification of genes that affect host range set the stage for constructing recombinant baculoviruses for specific past insects. The modification of baculovirus host specificity has recently been achieved by inserting or deleting genes that affect virus replication or cellular defenses.

Animals↗
PubMed1990

Genetic characterization of the stabilizing functions of a region of broad-host-range plasmid RK2.

One of the regions responsible for the stable inheritance of the broad-host-range plasmid RK2 is contained within the PstI C fragment, located from coordinates 30.8 to 37.0 kb (P.N. Saurugger, O. Hrabak, H. Schwab, and R.M. Lafferty, J. Biotechnol. 4:333-343, 1986). Genetic analysis of this 6.2-kb region demonstrated that no function was present that stabilized by selectively killing plasmid-free segregants. The sequence from 36.0 to 37.0 kb mediated a twofold increase in plasmid copy number, but this region was not required for stabilization activity. The PstI C fragment was shown to encode a multimer resolution system from 33.1 to 35.3 kb. The resolution cis-acting site was mapped to 140 bp, sequenced, and observed to contain two directly repeated sequences of 6 and 7 bases and two perfect inverted repeats of 6 and 8 bases. The trans-acting factor(s) was mapped and functionally determined to encode a resolvase capable of catalyzing recombination at high frequency between cis-acting sites in either direct or inverted orientation. Multimer resolution alone did not account for complete plasmid stabilization by the PstI C fragment, since removal of regions adjacent to the 35.3-kb border of the minimal mrs locus dramatically reduced stabilization. The minimal region required for complete stabilization, from 32.8 to 35.9 kb, was capable of fully stabilizing plasmids independently of the replicon or the recA proficiency of the host. Stabilization activity was also fully expressed in several diverse gram-negative bacteria, whereas the F plasmid par locus functioned only in Escherichia coli. On the basis of these observations, we conclude that under the growth conditions used, the minimal stabilization locus encodes both an mrs activity and a stabilization activity that has the properties of a par locus.

Azotobacter↗
PubMed1996

Construction of a broad-host-range promoter-probing vector and cloning of promoter fragments of Xanthomonas campestris.

A broad-host-range promoter-probing vector, pMY3 (8.0 kb), was constructed for cloning of DNA fragments containing promoter sequences of Xanthomonas campestris pv. campestris. This vector (pMY3) consists of the RK2 replicon, promoterless luxAB genes, the thr attenuator to block the transcription of RNA into the luxAB region, and multiple cloning sites for cloning of the fragment carrying promoter sequences. The feasibility of using pMY3 as a promoter-probing vector in both E. coli and Xc17 was demonstrated by using the lac promoter of E. coli, and the amy promoter of X. campestris in Xc17. Among the 63 promoter-containing fragments cloned from Xc17, only 9 were able to express in E. coli. It appears that X. campestris can recognize most E. coli type promoters, but, E. coli can recognize only a small portion of the X. campestris type promoters.

Cloning, Molecular↗
PubMed1995

In vivo and in vitro stability of the broad-host-range cloning vector pBBR1MCS in six Brucella species.

Previous studies have shown that the broad-host-range plasmid pBBR1MCS can be used for genetic complementation in Brucella abortus. To extend these observations, the in vivo and in vitro stability of pBBR1MCS was evaluated in the six currently recognized species of the genus Brucella. pBBR1MCS was readily introduced into all of the strains tested by electroporation and was stably maintained in broth cultures without antibiotic selection during five serial passages over a 10-day period. Furthermore, isolates of all six Brucella strains containing pBBR1MCS obtained from the spleens of BALB/c mice 1 week postinfection maintained the plasmid. Although pBBR1MCS maintains the mobilization locus present in the parental plasmid pBBR1CM, attempts to detect transfer of pBBR1MCS between Brucella strains by conjugation were unsuccessful. These results demonstrate the in vitro and in vivo stability of pBBR1MCS in Brucella spp. and reinforce the usefulness of this cloning vector for the genetic analysis of these organisms.

Animals↗
PubMed1987

The 20 bp, directly repeated DNA sequence of broad host range plasmid R1162 exerts incompatibility in vivo and inhibits R1162 DNA replication in vitro.

The broad host range plasmid R1162 contains a directly repeated, 20 bp DNA sequence in the region of the plasmid required in cis for replication and maintenance. This sequence has been chemically synthesized and cloned, and shown to be sufficient for expression of plasmid incompatibility. The sequence also inhibits replication of R1162 DNA in a cell-free system. The strengths of both these effects are determined by the number of direct repeats (DRs) present, and are also affected to similar degrees by different mutations within the repeated sequence. Several of the mutations were tested for their effect in cis on plasmid maintenance in the cell, and one was found to cause an increase in plasmid copy number. The results suggest that the direct repeats exert incompatibility by inhibiting DNA replication, presumably because they are the binding sites for a limiting essential protein.

Base Composition↗
PubMed1982

Development of broad-host-range vectors and gene banks: self-cloning of the Pseudomonas aeruginosa PAO chromosome.

A host-vector system for Pseudomonas aeruginosa PAO was developed. Scattered regions of the strain PAO chromosome were cloned by direct selection for complementation of auxotrophs or from a DNA gene bank which contains over 1,000 independently isolated chromosome-vector recombinant plasmids. The use of partially digested chromosomal DNA facilitated the selection of a variety of strain PAO chromosomal markers. The progenitor of the vector was a small, multicopy plasmid, pRO1600, found in a PAO strain which had acquired RP1 in a mating experiment. The bacterial host range that could be determined by transformation of vectors produced from pRO1600 resembles that for plasmid RP1. Two derivative plasmids were formed: pRO1613, for cloning DNA cleaved with restriction endonuclease PstI, and pRO1614, which was formed by deleting part of pRO1613 and fusion with plasmid pBR322. Plasmid pRO1614 utilizes known cloning sites within the tetracycline resistance region of pBR322.

Chromosomes, Bacterial↗
PubMed1998

Role of the parCBA operon of the broad-host-range plasmid RK2 in stable plasmid maintenance.

The par region of the stably maintained broad-host-range plasmid RK2 is organized as two divergent operons, parCBA and parDE, and a cis-acting site. parDE encodes a postsegregational killing system, and parCBA encodes a resolvase (ParA), a nuclease (ParB), and a protein of unknown function (ParC). The present study was undertaken to further delineate the role of the parCBA region in the stable maintenance of RK2 by first introducing precise deletions in the three genes and then assessing the abilities of the different constructs to stabilize RK2 in three strains of Escherichia coli and two strains of Pseudomonas aeruginosa. The intact parCBA operon was effective in stabilizing a conjugation-defective RK2 derivative in E. coli MC1061K and RR1 but was relatively ineffective in E. coli MV10Deltalac. In the two strains in which the parCBA operon was effective, deletions in parB, parC, or both parB and parC caused an approximately twofold reduction in the stabilizing ability of the operon, while a deletion in the parA gene resulted in a much greater loss of parCBA activity. For P. aeruginosa PAO1161Rifr, the parCBA operon provided little if any plasmid stability, but for P. aeruginosa PAC452Rifr, the RK2 plasmid was stabilized to a substantial extent by parCBA. With this latter strain, parA and res alone were sufficient for stabilization. The cer resolvase system of plasmid ColE1 and the loxP/Cre system of plasmid P1 were tested in comparison with the parCBA operon. We found that, not unlike what was previously observed with MC1061K, cer failed to stabilize the RK2 plasmid with par deletions in strain MV10Deltalac, but this multimer resolution system was effective in stabilizing the plasmid in strain RR1. The loxP/Cre system, on the other hand, was very effective in stabilizing the plasmid in all three E. coli strains. These observations indicate that the parA gene, along with its res site, exhibits a significant level of plasmid stabilization in the absence of the parC and parB genes but that in at least one E. coli strain, all three genes are required for maximum stabilization. It cannot be determined from these results whether or not the stabilization effects seen with parCBA or the cer and loxP/Cre systems are strictly due to a reduction in the level of RK2 dimers and an increase in the number of plasmid monomer units or if these systems play a role in a more complex process of plasmid stabilization that requires as an essential step the resolution of plasmid dimers.

Bacterial Proteins↗
PubMed2004

Modular broad-host-range expression vectors for single-protein and protein complex purification.

A set of modular broad-host-range expression vectors with various affinity tags (six-His-tag, FLAG-tag, Strep-tag II, T7-tag) was created. The complete nucleotide sequences of the vectors are known, and these small vectors can be mobilized by conjugation. They are useful in the purification of proteins and protein complexes from gram-negative bacterial species. The plasmids were easily customized for Thiocapsa roseopersicina, Rhodobacter capsulatus, and Methylococcus capsulatus by inserting an appropriate promoter. These examples demonstrate the versatility and flexibility of the vectors. The constructs harbor the T7 promoter for easy overproduction of the desired protein in an appropriate Escherichia coli host. The vectors were useful in purifying different proteins from T. roseopersicina. The FLAG-tag-Strep-tag II combination was utilized for isolation of the HynL-HypC2 protein complex involved in hydrogenase maturation. These tools should be useful for protein purification and for studying protein-protein interactions in a range of bacterial species.

Bacterial Proteins↗
PubMed2000

Pm promoter expression mutants and their use in broad-host-range RK2 plasmid vectors.

By coupling the Pm/xylS promoter system to minimal replicons of the broad-host-range plasmid RK2 we recently showed that such vectors are useful for both high- and low-level inducible expression of cloned genes in gram-negative bacteria. In this report, we extend this potential by identifying point mutations in or near the -10 transcriptional region of Pm. Point mutations leading to gene-independent enhancements of expression levels of the induced state or reduced background expression levels were identified using Escherichia coli as a host. By combining these mutations an additive effect in expression levels from the constructed Pm was observed. The highest induced expression level was obtained by inserting an E. coli consensus sigma70 - 10 recognition region. Most of the remaining activities in the reduced-background mutations appeared to originate from a transcriptional start site other than Pm. The effects of some of these mutations were also analyzed in Pseudomonas aeruginosa and were found to act similarly, but less pronounced in this host.

Bacterial Proteins↗