Biomedical subjects

C I Kado

Publications and source records attributed to C I Kado.

At least 55 records · Page 3Linked to original sources

PubMed1991

Mapping of the ros virulence regulatory gene of A. tumefaciens.

Virulence functions associated with the oncogenicity of Agrobacterium tumefaciens are encoded by vir genes contained in six major operons located on the Ti plasmid. The virC and virD operons encode functions responsible for host range and T-intermediate processing. These two operons are regulated positively by the product of virG and negatively by the product of the chromosomal gene ros, which encodes a 15.5 kDa repressor. To determine the location of the ros gene we have constructed A. tumefaciens HFR strains, using transposon Tn5mob to mobilize the ros locus, and used them to map the location of ros relative to auxotrophic loci. Tight linkage was found between ros, his-34 and his-19. A linkage map is presented showing the location of ros relative to other known chromosomal genes associated with virulence functions.

Bacterial Proteins↗

PubMed1991

The osa gene of pSa encodes a 21.1-kilodalton protein that suppresses Agrobacterium tumefaciens oncogenicity.

The incompatibility group W plasmid pSa suppresses Agrobacterium tumefaciens oncogenicity (J. Loper and C. Kado, J. Bacteriol. 139:591-596, 1979). The oncogenic suppressive activity was localized to a 3.1-kb region of pSa by Tn5 mutagenesis and deletion analysis. Within this fragment, a 1.1-kb subclone bearing oncogenic suppressive activity was subjected to further characterization. Nucleotide sequencing of the 1.1-kb fragment revealed a 570-bp open reading frame (ORF1) that has a coding capacity for a protein of 21.1 kDa. Sequencing of flanking regions revealed a second ORF (ORF2) located 3 bp upstream of ORF1, with a coding capacity for a protein of 22.8 kDa. Gene fusions of these ORFs to a T7 phi 10 expression system in Escherichia coli resulted in the synthesis of polypeptides of the predicted sizes. An E. coli promoter consensus sequence was not found in the expected positions in the region preceding ORF1. However, several sequences with similarity to the consensus -10 sequence of the A. tumefaciens vir gene promoters were found upstream of ORF1. Potential translational start signals are upstream of ORF1 and ORF2. These sequences showed no significant similarity at the nucleotide or amino acid levels with those in available data bases. However, the C-terminal portion of the ORF1 protein is rich in hydrophobic residues. Perhaps oncogenicity suppression is effected by an association of this protein with the Agrobacterium membrane such that T-DNA transfer is blocked.

Amino Acid Sequence↗

PubMed1991

The virC and virD operons of the Agrobacterium Ti plasmid are regulated by the ros chromosomal gene: analysis of the cloned ros gene.

The ros chromosomal gene is present in octopine and nopaline strains of Agrobacterium tumefaciens as well as in Rhizobium meliloti. This gene encodes a 15.5-kDa protein that specifically represses the virC and virD operons in the virulence region of the Ti plasmid. The ros gene was cloned from a genomic bank by electroporation and complementation in Agrobacterium cells. Reporter fusion to the ros gene indicates that the level of transcription is controlled in part by autoregulation. A consensus inverted repeat sequence present in the ros promoter and in the virC and virD promoters of pTiC58, pTiA6, and pRiA4b suggests that a specific Ros binding site exists in these promoters. In the virC and virD promoter region, this binding site is within a cluster of vir box consensus sequences in which the VirG protein binds. This suggests possible binding competition between Ros and VirG at the virC and virD promoters. That the Ros protein binds DNA is suggested by the presence of a 'zinc finger' consensus sequence in the protein.

Amino Acid Sequence↗

PubMed1990

VirD2 gene product from the nopaline plasmid pTiC58 has at least two activities required for virulence.

Virulence genes virD1 and virD2 are required for T-DNA processing in Agrobacterium tumefaciens. The regions within virD2 contributing to T-DNA processing and virulence were investigated. Some insertional mutations in virD2 prevented T-DNA border endonucleolytic cleavage and produced an avirulent phenotype. However, a non-polar insertion immediately after bp 684 of the 1344 bp open reading frame of virD2 did not inhibit endonucleolytic cleavage but still caused a loss of virulence. This suggested that in addition to T-DNA border cleaving activity, the VirD2 protein has another virulence function which resides in the C-terminal half of the protein. Comparative nucleotide sequence analyses of virD2 showed that the first 684 bp were 81% homologous to virD2 of an octopine Ti plasmid whereas the remaining 660 bp were only 44% homologous. A plasmid containing the virD region from octopine Ti plasmid could restore both virulence and processing to a nopaline virD2 mutant. No complementation resulted when a nopaline virD2 clone containing a region similar to eukaryotic nuclear envelope transport sequences was deleted from the 3' end. These results suggest that virD1 and only the first half of virD2 are required to encode for the T-DNA processing endonuclease, and that the 3'-half of virD2 encodes a function separate from endonuclease activity that is required for virulence.

Amino Acid Sequence↗

PubMed1990

Small, stable shuttle vectors for use in Xanthomonas.

Plasmids from three broad-host-range (bhr) incompatibility groups (Inc) were evaluated for use as cloning vectors in Xanthomonas campestris pv. malvacearum (Xcm), the causal agent of bacterial blight of cotton. The IncP vectors pLAFR3 and pVK102 could not be introduced into Xcm at a significant frequency (less than 1 x 10(-10] and IncQ vectors such as pKT210 were unstable in their maintenance and tended to delete cloned inserts. IncW vectors such as pSa747 also were lost readily from Xcm in the absence of selection pressure. We constructed two plasmids, pUFR027 and a cosmid derivative, pUFR034, which have proven useful as cloning vectors in Xcm and other xanthomonads. They contain the pSa origin of DNA replication, the partition locus parA from the Agrobacterium plasmid pTAR, a neomycin-resistance selection marker, and alacZ alpha cassette with cloning sites. pUFR027 is 9.3 kb, and pUFR034 is 8.7 kb in size. They can be mobilized by conjugation into Xcm at a frequency of approx. 1 x 10(-6) per recipient and are maintained stably (greater than 95% retention over 36 generations without selection pressure) in both broth culture and in planta. The plasmids were introduced and maintained stably in X. citri, and in X. campestris pathovars campestris, citrumelo, vesicatoria and translucens, and were moderately stable in X. phaseoli. No effects of the plasmids on pathogenicity have been observed.

Chromosome Mapping↗

PubMed1990

Agrobacterium-mediated plant transformation by novel mini-T vectors in conjunction with a high-copy vir region helper plasmid.

A new binary vector system for Agrobacterium-mediated plant transformation was developed. A set of four mini-T vectors comprised of T-DNA border sequences from nopaline-type Ti-plasmid pTiC58 flanking a chimaeric hygromycin-resistance gene for selection of transformants and up to eight unique restriction sites for cloning foreign DNA was constructed on a broad-host replicon containing the oriV of plasmid pSa. In two of the constructs these multiple cloning sites are flanked by a strong promoter to activate transcription of inserted DNA in planta. High-efficiency transformation was prompted by a high-copy, stable virulence helper plasmid pUCD2614, which contains a cloned virulence region of pTiC58 and tandem copies of the par locus of plasmid pTAR. Southern blot hybridization and genetic analyses of the progeny of transformed plants showed that the hygromycin resistance gene was stably inherited.

Cloning, Molecular↗

PubMed1990

Molecular characterization of the vir regulon of Agrobacterium tumefaciens: complete nucleotide sequence and gene organization of the 28.63-kbp regulon cloned as a single unit.

The entire vir regulon of Agrobacterium tumefaciens was subcloned and the complete 28.6-kbp nucleotide sequence was determined. The regulon was cloned as a single unit into two replicons, one of which replicates at a high copy number in this bacterium, and a second which has broad-host-range features to replicate in other Gram-negative bacteria. These vir region plasmids are able to confer in trans the processing and transfer activities on a second plasmid containing the T-DNA. In the high copy number vir region plasmid pUCD2614, a moderate increase in basal vir gene expression was observed as judged by virE::cat fusion expression assays relative to the wild-type control plasmid. Furthermore, higher efficiencies of tobacco leaf disk transformation were observed than with the widely used vir helper plasmid pAL4404. The nucleotide sequence studies showed that the vir region consists of 28,631 bp comprising 24 open reading frames which encode proteins involved in tumorigenicity. Two open reading frames not previously characterized, virH and ORF5, were uncovered within the virD/virE intervening spacer region. Together these studies more completely characterize the structure and function of the vir regulon.

Base Sequence↗

PubMed1990

Specific binding of VirG to the vir box requires a C-terminal domain and exhibits a minimum concentration threshold.

The positive regulatory protein VirG from the virulence region of the Ti plasmid of Agrobacterium tumefaciens was first demonstrated to possess DNA-binding capabilities using chromatographically purified protein and in vitro assays (Powell et al., 1989). This paper is an extension of that research and presents evidence on the in vivo DNA-binding properties of VirG using a transcription interference assay. VirG protein bound specifically to a 'vir box' response element and repressed transcription of a lacZ reporter gene, but increased transcription in the absence of a vir box. A biphasic response in specific DNA-binding was observed upon increasing virG expression, suggesting that specific binding was co-operatively affected by protein concentration. Certain TrpE'-'VirG hybrid proteins also bound the vir box, but required sequences distal to amino acid Arg-118 of the VirG polypeptide. These data further localize a DNA-binding domain within VirG, and support a modified model for the regulation of virulence genes in which transphosphorylation by the coregulator VirA functions to stabilize specific DNA-binding by low concentrations of VirG, resulting in gene activation. Otherwise, at high concentrations, VirG promotes expression of the virulence regulon without assistance from VirA as was shown previously (Rogowsky et al., 1987).

Amino Acid Sequence↗

PubMed1990

Characterization of the virB operon of an Agrobacterium tumefaciens Ti plasmid: nucleotide sequence and protein analysis.

The virulence regulon of the Agrobacterium tumefaciens TiC58 plasmid is composed of six operons, virA, virB, virG, virC, virD and virE, which direct the transfer of T-DNA into plant cells. The 9.5 kbp virB operon is the largest of these operons and its entire nucleotide sequence was determined and found to contain eleven open reading frames (ORFs). Gene fusions of each VirB ORF to T7 phi 10 were made and overexpressed in Escherichia coli to confirm that they encode proteins of predicted size. Hydrophobic analysis of these peptide sequences revealed nine proteins that contain hydrophobic spanning regions including signal-peptide-like sequences. These data suggest that the majority of VirB proteins may associate with bacterial cell membranes, while the two additional proteins possess a potential ATP-binding site. Strong homologies in amino acid sequences were observed between nopaline- and octopine-type plasmids. Specific differences in amino acid sequence encoded by VirB ORFs of nopaline and octopine Ti plasmid and a functional role of the gene products are discussed.

Adenosine Triphosphate↗

PubMed1990

Phenotypic Switching Affecting Chemotaxis, Xanthan Production, and Virulence in Xanthomonas campestris.

The chemotaxis towards sucrose and yeast extract of nine strains of Xanthomonas campestris representing pathovars campestris, armoraciae, translucens, vesicatoria, and pelargonii was analyzed by using swarm plates. Unexpectedly, each of these strains formed small or reduced swarms typical of nonmotile or nonchemotactic bacteria. With time, however, chemotactic cells appeared on the swarm plates as blebs of bacteria. These cells were strongly chemotactic and were concomitantly deficient in exopolysaccharide production. The switch from the wild type (exopolysaccharide producing and nonchemotactic) to the swarmer type (exopolysaccharide deficient and chemotactic) appeared irreversible ex planta in bacteriological medium. However, in radish leaves swarmer-type strains of X. campestris pv. campestris were able to revert to the wild type. Swarmer-type derivatives of two X. campestris pv. campestris wild-type isolates showed reduced virulence and growth in the host plants cauliflower and radish. However, exocellular complementation of X. campestris pv. campestris Hrp (nonpathogenic) mutant was achieved by coinoculation with a swarmer-type strain.

Journal Article↗

PubMed1990

VirA, a coregulator of Ti-specified virulence genes, is phosphorylated in vitro.

High-level expression of a chimeric virA gene was obtained by replacing the first 524 codons of virA with the first half of trpE. The encoded fusion protein was isolated and found to exhibit autokinase activity. Therefore, a kinase domain is in the C-terminal portion of VirA, and protein phosphorylation may be an important feature of VirA function.

Bacterial Proteins↗

PubMed1990

Virulence genes promote conjugative transfer of the Ti plasmid between Agrobacterium strains.

Certain virulence region operons of the Agrobacterium tumefaciens Ti plasmid promoted conjugative Ti plasmid transfer. Mutations in the vir region of pTiC58 inhibited conjugative plasmid transfer between A. tumefaciens strains. Mutations in virA, virG, 5' virB, and virE had the greatest effect on plasmid transfer, and mutations in virC had no effect. Transfer inhibition in vir mutants occurred in the presence or absence of acetosyringone.

Conjugation, Genetic↗

PubMed1990

A plant-inducible gene of Xanthomonas campestris pv. campestris encodes an exocellular component required for growth in the host and hypersensitivity on nonhosts.

Using Tn4431, a transposon that allows transcriptional fusions to a promoterless luciferase (lux) operon, we have isolated a nonpathogenic mutant of Xanthomonas campestris pv. campestris, i.e., JS111, that does not incite any of the black rot symptoms on all tested cruciferous host plants (J. J. Shaw, L. G. Settles, and C. I. Kado, Mol. Plant Microbe Interact. 1:39-45, 1988). In the study reported here, we determined that in contrast to the wild-type strain, JS111 is unable to induce a hypersensitive necrotic response on nonhost plants such as datura, tomato, and cucumber, suggesting that JS111 is a nonpathogenic, nonhypersensitive Hrp mutant. JS111 displayed culture growth rates, exopolysaccharide production, and protease, pectate lysase, cellulase, amylase, and phosphatase activities comparable to those of the wild-type strain. However, the growth of JS111 in host leaves was markedly attenuated. Coinoculation of JS111 with the wild-type strain in cauliflower or radish leaves rescued the growth deficiency of the mutant to normal levels. The locus mutated in JS111 was cloned and named hrpXc, and transcriptional and genetic complementation analyses of the hrpXc locus were conducted. The regulation of hrpXc expression was also investigated in vitro and in planta, using fusions to a lux or chloramphenicol acetyltransferase reporter gene. The hrpXc gene was found to be strongly induced in radish leaves. This is the first report and analysis of a hrp locus from a Xanthomonas species.

Chromosome Mapping↗

PubMed1990

[Demonstration of 3 functional domains responsible for a kinase activity in VirA, a transmembrane sensory protein encoded by the Ti plasmid of Agrobacterium tumefaciens].

High-level expression of chimeric virA genes were obtained by replacing the first codons of virA with the first codons of trpE. The fusion proteins encoded by these constructs were partially purified and one of them exhibited autokinase activity. Therefore, protein phosphorylation may be an important feature of VirA function. This allowed us to define the existence of three functional domains inside VirA.

Bacterial Proteins↗

PubMed1989

A translational enhancer derived from tobacco mosaic virus is functionally equivalent to a Shine-Dalgarno sequence.

When present at the 5' end of mRNAs, the untranslated leader sequence (omega) of tobacco mosaic virus RNA significantly enhances translation in eukaryotes and prokaryotes. We have tested a deletion derivative of the omega sequence, omega delta 3, for its enhancing ability on gene constructs in which the ribosomal binding site was either present or deleted, in several Gram-negative bacterial species including Escherichia coli, Agrobacterium tumefaciens, Xanthomonas campestris pv. vitians, Erwinia amylovora, and Salmonella typhimurium. In vivo production of chloramphenicol acetyltransferase from a gene construct lacking its native ribosomal binding site was enhanced 40- to 120-fold by the presence of omega delta 3. Similar levels of enhancement (30- to 240-fold) were observed when the gene encoding beta-glucuronidase was tested. With a chloramphenicol acetyltransferase construct containing a ribosomal binding site, enhancement was markedly less, between 1- and 3.8-fold. Omega delta 3 appeared to enhance translation independent of its position upstream of the AUG codon used for initiation.

Amino Acid Sequence↗

PubMed1989

High levels of double-stranded transferred DNA (T-DNA) processing from an intact nopaline Ti plasmid.

To obtain bacterial-mediated oncogenic transformation of plants, the transferred DNA (T-DNA) of the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens is transferred to its plant host cells during infection. The initial phases of transformation involve the processing of the T-DNA in the bacterial cell after induction of the vir genes located on the Ti plasmid. The kinetics and conditions of this processing were examined and upon induction with acetosyringone up to 40% of the left and right borders of the T-DNA were cleaved. This cleavage was dependent upon virA, virG, and VirD and was rec-independent. Processed T-DNA was observed within 30 min after induction and was delayed by an increased concentration of phosphate in the induction medium. When DNA was isolated in the absence of protease treatment, the DNA fragment corresponding to the left side of the cut at both the left and right border region exhibited gel retardation, suggesting one or more "pilot" proteins may be involved in T-DNA transfer. Although the relative abundance of a processed product does not necessarily imply relative importance, the preponderance of double-stranded cleavage products suggests that double-stranded T-DNA should be considered as a possible intermediate in T-DNA transfer.

Arginine↗

PubMed1989

virG of Agrobacterium tumefaciens plasmid pTiC58 encodes a DNA-binding protein.

Virulence genes of the Agrobacterium tumefaciens Ti plasmid are positively regulated by the products of virA and virG. To study the DNA-binding properties of the VirG protein, a translational fusion between virG and the trpE gene of Escherichia coli was constructed, and antiserum was raised against the encoded fusion protein. Using this antiserum, a protein of Mr congruent to 29,000, a size similar to that calculated from the virG nucleotide sequence, was detected in an E. coli strain harbouring a virG expression vector. Both the virG protein and the fusion protein were found, by filter-binding and gel retardation analyses, to bind DNA nonspecifically. These data support an existing model for the two-component regulatory systems of bacteria.

Antibodies, Bacterial↗

PubMed1989

Characterization of the virA virulence gene of the nopaline plasmid, pTiC58, of Agrobacterium tumefaciens.

We have determined the complete nucleotide sequence of a 4.8 kilobase fragment encompassing the virA locus of the nopaline-type plasmid, pTiC58, of Agrobacterium tumefaciens. virA is composed of a single open reading frame of 2499 nucleotides, capable of encoding a protein of 91.3 kiloDaltons. A trpE::virA gene fusion was used to confirm the reading frame of virA. High nucleotide and amino acid sequence homologies were observed between pTiC58 virA and the virA sequences of three octopine-type plasmids. Strong homologies in amino acid sequence were observed between pTiC58 VirA and seven bacterial proteins which control various regulons. Two hydrophobic domains within VirA are also consistent with a model in which VirA acts as a membrane-bound sensor of plant signal molecules.

Amino Acid Sequence↗