Reexamining the role of the accessory plasmid pAtC58 in the virulence of Agrobacterium tumefaciens strain C58

Isogenic strains of Agrobacterium tumefaciens carrying pTiC58, pAtC58, or both were constructed and assayed semiquantitatively and quantitatively for virulence and vir gene expression to study the effect of the large 542-kb accessory plasmid, pAtC58, on virulence. Earlier studies indicate that the att (attachment) genes of A. tumefaciens are crucial in the ability of this soil phytopathogen to infect susceptible host plants. Mutations in many att genes, notably attR and attD, rendered the strain avirulent. These genes are located on pAtC58. Previous work also has shown that derivatives of the wild-type strain C58 cured of pAtC58 are virulent as determined by qualitative virulence assays and, hence, pAtC58 was described as nonessential for virulence. We show here that the absence of pAtC58 in pTiC58-containing strains results in reduced virulence but that disruption of the attR gene does not result in avirulence or a reduction in virulence. Our studies indicate that pAtC58 has a positive effect on vir gene induction as revealed by immunoblot analysis of Vir proteins and expression of a PvirB::lacZ fusion.     

Phase variants of Bordetella bronchiseptica arise by spontaneous deletions in the vir locus

Bordetella bronchiseptica is a common respiratory tract pathogen of many mammalian species. Nucleotide sequences from the locus involved in coordinate regulation of B. pertussis virulence factors, vir, were shown to have a high degree of homology to chromosomal DNA from virulent (Vir+) and avirulent (Vir-) strains of B. bronchiseptica. Small deletions, 50 bp to 500 bp, within the vir locus were found in some of the Vir- phase variants. The vir locus and the adjacent 5' portion of the fhaB structural gene were cloned from the parental Vir+ B. bronchiseptica strain on a 23.5 kb BamHI fragment. Restriction enzyme mapping of the cloned B. bronchiseptica vir locus revealed similarities with and differences from the previously cloned B. pertussis vir locus. The cloned B. bronchiseptica vir locus complemented spontaneous Vir- variants of Bordetella pertussis and B. bronchiseptica as well as vir::Tn5 mutants of B. pertussis. Comparison of various functions of the vir loci of B. bronchiseptica and B. pertussis revealed some interesting differences in the coordinate regulation of virulence factors.     

Additional virulence genes and sonication enhance <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated loblolly pine transformation

Additional virulence (vir) genes in Agrobacterium tumefaciens and sonication were investigated for their impact on transformation efficiency in loblolly pine (Pinus taeda L.). Mature zygotic embryos of loblolly pine were co-cultivated with disarmed A. tumefaciens strain EHA105 containing either plasmid vector pCAMBIA1301 or vector pCAMBIA1301 with an additional 15.8-kb fragment carrying extra copies of the Vir B, Vir C, and Vir G regions from the supervirulent plasmid pTOK47. pCAMBIA1301 contains hygromycin resistance and the beta-glucuronidase (GUS) reporter gene. Expression of GUS was observed after 3-6 days of co-cultivation, with peak expression at approximately 21 days. The highest numbers of GUS-expressing areas were visible up to 21 days after co-cultivation, declining rapidly thereafter. Both transient and stable transformation efficiencies increased when the explants were sonicated before co-cultivation and/or the additional virB, virC, and virG genes were included with the pCAMBIA1301 plasmid T-DNA. Use of the plasmid with additional vir genes and sonication dramatically enhanced the efficiency of Agrobacterium-mediated gene transfer not only in transient expression but also in the recovery of hygromycin-resistant lines. Stably transformed cultures and transgenic plants were produced from embryos transformed with A. tumefaciens EHA105 carrying pCAMBIA1301 or pCAMBIA1301+pTOK47 in the three families of loblolly pine. The presence of the introduced GUS and hygromycin phosphotransferase genes in the transgenic plants was confirmed by polymerase chain reaction and Southern hybridization analyses.     

Frameshift mutations in the bacteriophage Mu repressor gene can confer a trans-dominant virulent phenotype to the phage.

Virulent mutations in the bacteriophage Mu repressor gene were isolated and characterized. Recombination and DNA sequence analysis have revealed that virulence is due to unusual frameshift mutations which change several C-terminal amino acids. The vir mutations are in the same repressor region as the sts amber mutations which, by eliminating several C-terminal amino acids, suppress thermosensitivity of repressor binding to the operators by its N-terminal domain (J. L. Vogel, N. P. Higgins, L. Desmet, V. Geuskens, and A. Toussaint, unpublished data). Vir repressors bind Mu operators very poorly. Thus the Mu repressor C terminus, either by itself or in conjunction with other phage or host proteins, tunes the DNA-binding properties at the repressor N terminus.     

Phosphoenolpyruvate carboxykinase is an acid-induced, chromosomally encoded virulence factor in Agrobacterium tumefaciens

The pckA gene, encoding phosphoenolpyruvate carboxykinase, catalyzes the reversible decarboxylation and phosphorylation of oxaloacetate to form phosphoenolpyruvate. Located on the circular chromosome of Agrobacterium, this locus is adjacent to the loci chvG and chvI, encoding a two-component regulatory system that has been shown to be important in virulence. Using a reporter gene fusion, studies showed that the pckA gene is induced by acidic pH but not by acetosyringone. This acid induction is regulated by the chvG-chvI regulatory system, which controls acid-inducible genes. A pckA mutant had no demonstrable PckA enzyme activity and grew on AB minimal medium with glucose but did not grow on the same medium with succinate as the sole carbon source and was more inhibited in its growth than the wild-type strain by an acidic environment. A pckA mutant was highly attenuated in tumor-inducing ability on tobacco leaf disks and was severely attenuated in vir gene expression. Although vir gene induction was completely restored when a constitutive virG gene was introduced into the mutant strain, virulence was only partially restored. These results suggest that avirulence may be due to a combination of the inhibition of this mutant in the acidic plant wound environment and the poor induction of the vir genes.     

Hypovirulence-associated traits induced by a mycovirus of Cryphonectria parasitica are mimicked by targeted inactivation of a host gene.

Expression of the Vir2 gene of Cryphonectria parasitica is down-regulated in strains of the fungus containing a double-stranded RNA genetic element that reduces fungal virulence (W. A. Powell and N. K. Van Alfen, Mol. Cell. Biol. 7:3688-3693, 1987). We have sequenced the Vir2 gene and characterized its structure; the mRNA contains a short open reading frame whose product has structural similarities to several fungal pheromones. A null mutant was constructed by homologous recombination to determine the function of the Vir2 gene and whether its disruption resulted in any of the altered phenotypes exhibited by many hypovirulent strains, such as reductions in virulence, pigmentation, and sporulation. The Vir2 null mutant (18dm) exhibited a wild-type phenotype with respect to gross colony morphology, growth rate, pigmentation, asexual spore viability, and virulence in apple fruit and chestnut trees. However, numbers of asexual fruiting bodies (pycnidia) and conidia were reduced significantly in comparison with the wild-type strain EP155/2. In sexual crosses of 18dm with a wild-type strain of the opposite mating type, perithecia (sexual fruiting bodies) developed but were barren. Deletion of the Vir2 gene results in a phenotype that mimics that of many double-stranded-RNA-containing hypovirulent strains; i.e., the null mutant exhibits significant reductions in asexual sporulation and pycinidum production as well as impaired sexual crossing ability. To our knowledge, this is the first report of the partial reproduction of a virus-induced phenotype by deletion of a virus-perturbed host gene.     

Molecular subtyping of group A streptococcal strains based on virulence regulon polymorphism

A total of 64 Streptococcus pyogenes isolates included in a previous study (Shundi et al., 2000) were further analyzed by Vir typing. Vir typing is based on restriction fragment length polymorphism (RFLP) analysis of a 4- to 8-kb pathogenicity island (vir regulon) encoding emm and other virulence genes. As all our isolates contained a single vir regulon, the stoichiometric yield of restriction fragments avoided ambiguities in interpretation of results. By using both HaeIII and HinfI restriction endonucleases to generate RFLP profiles, the 64 GAS isolates were distributed among 22 Hae- and 26 Hinf-Vir types respectively.     

Two trans-acting regulatory genes (vir and mod) control antigenic modulation in Bordetella pertussis.

Expression of virulence factors by Bordetella pertussis is altered by environmental signals (antigenic modulation) and is dependent on an activator encoded by a gene called vir. We have used TnphoA (Tn5 IS50L::phoA) gene fusions to define two sets of genes whose expression is either activated (vag loci) or repressed (vrg loci) by modulation signals. Both groups of genes appear to be regulated by the vir gene product in that, in the absence of modulators, null mutations in vir lead to the repression of vag gene fusions and derepression of vrg gene fusions. Mutants of B. pertussis were isolated that constitutively express virulence factors in the presence of the modulator MgSO4, nicotinic acid, or low incubation temperature. We designate the gene that carries such mutations mod (modulation) and have characterized one (mod-1) of these mod constitutive mutations. A method was developed for the insertional inactivation of the vir gene by using the integration of a suicide replicon. Inactivation of the vir gene in the mod-1 mutant, followed by transcomplementation with the cloned wild-type vir gene, gives the Mod-1 constitutive phenotype, showing that the mod-1 mutation defines a gene distinct from vir. The gene carrying the mod-1 mutation is linked to vir and was cloned on a recombinant cosmid (pLAF-C1) which transcomplements the vir-1::Tn5 mutation in B. pertussis 347. Introduction of pLAF-C1 into vir mutant and vir+ B. pertussis strains also gives the Mod-1 constitutive phenotype, indicating that mod-1 is a dominant allele. These data suggest that the mod gene product could have sensory functions for the environmental signals that affect the expression of vir-regulated genes of B. pertussis. The mod constitutive strains and plasmids described here also have applications in pertussis vaccine development.     

Proteomic analysis of Agrobacterium tumefaciens response to the Vir gene inducer acetosyringone

Agrobacterium tumefaciens causes crown gall disease in a wide range of plants by transforming plants through the transfer and integration of its transferred DNA (T-DNA) into the host genome. In the present study, we used two-dimensional gel electrophoresis to examine the protein expression profiles of A. tumefaciens in response to the phenolic compound acetosyringone (AS), a known plant-released virulence (vir) gene inducer. Using mass spectrometry, we identified 11 proteins consisting of 9 known AS-induced Vir proteins and 2 newly discovered AS-induced proteins, an unknown protein Y4mC (Atu6162) and a small heat shock protein HspL (Atu3887). Further expression analysis revealed that the AS-induced expression of Y4mC and HspL is regulated by the VirA/VirG two-component system. This report presents the first proteomics study successfully identifying both known and new AS-induced proteins that are implicated in Agrobacterium virulence.     

Interaction of the virulence protein VirF of Agrobacterium tumefaciens with plant homologs of the yeast Skp1 protein

The infection of plants by Agrobacterium tumefaciens leads to the formation of crown gall tumors due to the transfer of a nucleoprotein complex into plant cells that is mediated by the virulence (vir) region-encoded transport system (reviewed in [1-5]). In addition, A. tumefaciens secretes the Vir proteins, VirE2 and VirF, directly into plant cells via the same VirB/VirD4 transport system [6], and both assist there in the transformation of normal cells into tumor cells. The function of the 22 kDa VirF protein is not clear. Deletion of the virF gene in A. tumefaciens leads to diminished virulence [7, 8] and can be complemented by the expression of the virF gene in the host plant. This finding indicates that VirF functions within the plant cell [8]. Here, we report that the VirF protein is the first prokaryotic protein with an F box by which it can interact with plant homologs of the yeast Skp1 protein. The presence of the F box turned out to be essential for the biological function of VirF. F box proteins and Skp1p are both subunits of a class of E3 ubiquitin ligases referred to as SCF complexes. Thus, VirF may be involved in the targeted proteolysis of specific host proteins in early stages of the transformation process.     

Factors and markers of virulence in Escherichia coli from human septicemia

A lethal and necrotic factor which causes cell multinucleation in HeLa cell cultures has previously been shown to be coded by the Vir plasmid of Escherichia coli. Using an absorbed rabbit antiserum which neutralized the Vir toxic properties, we have compared the SDS-PAGE immunoblots from laboratory and field strains which either produce or do not produce Vir toxicity. A single band of 110 kDa was found to be specifically associated with vir toxicity in E. coli strains. This antiserum also recognized the 115 kDa protein band which was previously identified as the cytotoxic necretozing factor (CNF) of certain E. coli strains. These results suggest that the toxin coded by the Vir plasmid is a protein of 110 kDa distinct from, but immunologically related to CNF.     

Conditionally replicating plasmid vectors that can integrate into the Klebsiella pneumoniae chromosome via bacteriophage P4 site-specific recombination.

P4 is a satellite phage of P2 and is dependent on phage P2 gene products for virion assembly and cell lysis. Previously, we showed that a virulent mutant of phage P4 (P4 vir1) could be used as a multicopy, autonomously replicating plasmid vector in Escherichia coli and Klebsiella pneumoniae in the absence of the P2 helper. In addition to establishing lysogeny as a self-replicating plasmid, it has been shown that P4 can also lysogenize E. coli via site-specific integration into the host chromosome. In this study, we show that P4 also integrates into the K. pneumoniae chromosome at a specific site. In contrast to that in E. coli, however, site-specific integration in K. pneumoniae does not require the int gene of P4. We utilized the alternative modes of P4 lysogenization (plasmid replication or integration) to construct cloning vectors derived from P4 vir1 that could exist in either lysogenic mode, depending on the host strain used. These vectors carry an amber mutation in the DNA primase gene alpha, which blocks DNA replication in an Su- host and allows the selection of lysogenic strains with integrated prophages. In contrast, these vectors can be propagated as plasmids in an Su+ host where replication is allowed. To demonstrate the utility of this type of vector, we show that certain nitrogen fixation (nif) genes of K. pneumoniae, which otherwise inhibit nif gene expression when present on multicopy plasmids, do not exhibit inhibitory effects when introduced as merodiploids via P4 site-specific integration.     

Adhesive properties associated with the Vir plasmid: a transmissible pathogenic characteristic associated with strains of invasive Escherichia coli

Escherichia coli strain S5 (O15:K+:H21) isolated from a septicaemic lamb and previously shown to possess a virulence plasmid, Vir, attached in vitro to calf epithelial tissue from the ileum, oesophagus and trachea in the presence of 0.5% (w/v) D-mannose. The Vir+ recombinant strains 711v and H209av, which had received the Vir plasmid(s) from strain S5, also attached to these epithelia but the parent strains 711 and H209a without the Vir plasmid were non-adhesive. The attachment of the Vir+ strain 711v to intestinal brush borders was inhibited by antiserum to live Vir+ strain H209av but not by antiserum to strain H209a lacking Vir. No adherence occurred with Vir+ organisms grown at 18 degrees C or after heating at 65 degrees C. Adhesion was unaffected by 0.5% (w/v) formaldehyde. Glucosamine, mannosamine, their N-acetyl derivatives and wheat germ lectin each inhibited attachment of Vir+ strain 711v to brush border epithelia.     

单、双子叶植物的代谢物调节农杆菌Vir区基因表达的研究

本文研究了六种植物（三种单子叶植物,三种双子叶植物）愈伤组织的 渗出物和抽提物对农杆菌Vir基因表达的调节作用,其调节水平植物的不同而明显不同,但单,双子叶植物的代谢物对Vir基因表达的调节作用并非截然分开,即使在双子叶植物（如大豆）的抽提物与渗出物中也存在着抑制Vir基因表达的因子,而在单子叶植物（如玉米等）的抽提物与渗出物中也存在着促进Vir基因表达的调节因子,Vir位点的调节反应随渗出物与抽提物的种类不同而明显不同,不同Vir位点对同类渗出物或抽提物的反应也不同,渗出物对Vir基因表达的正调节效应优于抽提物,植物渗出物与AS对Vir区基因表达的调节并不表现简单的累加效应或协同作用,相反,在渗出物中还存在着不同程度阻抑AS对Vir基因表达正调节的因子。     

The regulatory VirA protein of Agrobacterium tumefaciens does not function at elevated temperatures.

Previous studies have shown that Agrobacterium tumefaciens causes tumors on plants only at temperatures below 32 degrees C, and virulence gene expression is specifically inhibited at temperatures above 32 degrees C. We show here that this effect persists even when the virA and virG loci are expressed under the control of a lac promoter whose activity is temperature independent. This finding suggests that one or more steps in the signal transduction process mediated by the VirA and VirG proteins are temperature sensitive. Both the autophosphorylation of VirA and the subsequent transfer of phosphate to VirG are shown to be sensitive to high temperatures (> 32 degrees C), and this correlates with the reduced vir gene expression observed at these temperatures. At temperatures of 32 degrees C and higher, the VirA molecule undergoes a reversible inactivation while the VirG molecule is not affected. vir gene induction is temperature sensitive in an acetosyringone-independent virA mutant background but not in a virG constitutive mutant which is virA and acetosyringone independent. These observations all support the notion that the VirA protein is responsible for the thermosensitivity of vir gene expression. However, an Agrobacterium strain containing a constitutive virG locus still cannot cause tumors on Kalanchoe plants at 32 degrees C. This strain induces normal-size tumors at temperatures up to 30 degrees C, whereas the wild-type Agrobacterium strain produces almost no tumors at 30 degrees C. These results suggest that at temperatures above 32 degrees C, the plant becomes more resistant to infection by A. tumefaciens and/or functions of some other vir gene products are lost in spite of their normal levels of expression.     

ChvD, a chromosomally encoded ATP-binding cassette transporter-homologous protein involved in regulation of virulence gene expression in Agrobacterium tumefaciens

A yeast two-hybrid screen searching for chromosomally encoded proteins that interact with the Agrobacterium tumefaciens VirB8 protein was carried out. This screen identified an interaction candidate homologous to the partial sequence of a gene that had previously been identified in a transposon screen as a potential regulator of virG expression, chvD. In this report, the cloning of the entire chvD gene is described and the gene is sequenced and characterized. Insertion of a promoterless lacZ gene into the chvD locus greatly attenuated virulence and vir gene expression. Compared to that of the wild-type strain, growth of the chvD mutant was reduced in rich, but not minimal, medium. Expression of chvD, as monitored by expression of beta-galactosidase activity from the chvD-lacZ fusion, occurred in both rich and minimal media as well as under conditions that induce virulence gene expression. The ChvD protein is highly homologous to a family of ATP-binding cassette transporters involved in antibiotic export from bacteria and has two complete Walker box motifs. Molecular genetic analysis demonstrated that disruption of either Walker A box, singly, does not inactivate this protein's effect on virulence but that mutations in both Walker A boxes renders it incapable of complementing a chvD mutant strain. Constitutive expression of virG in the chvD mutant strain restored virulence, supporting the hypothesis that ChvD controls virulence through effects on virG expression.     

Citrate synthase mutants of Agrobacterium are attenuated in virulence and display reduced vir gene induction

A citrate synthase (CS) deletion mutant of Agrobacterium tumefaciens C58 is highly attenuated in virulence. The identity of the mutant was initially determined from its amino acid sequence, which is 68% identical to Escherichia coli and 77% identical to Brucella melitensis. The mutant lost all CS enzymatic activity, and a cloned CS gene complemented a CS mutation in Sinorhizobium. The CS mutation resulted in a 10-fold reduction in vir gene expression, which likely accounts for the attenuated virulence. When a plasmid containing a constitutive virG [virG(Con)] locus was introduced into this mutant, the level of vir gene induction was restored to nearly wild-type level. Further, the virG(Con)-complemented CS mutant strain induced tumors that were similar in size and number to those induced by the parental strain. The CS mutation resulted in only a minor reduction in growth rate in a glucose-salts medium. Both the CS mutant and the virG(Con)-complemented CS strain displayed similar growth deficiencies in a glucose-salts medium, indicating that the reduced growth rate of the CS mutant could not be responsible for the attenuated virulence. A search of the genome of A. tumefaciens C58 revealed four proteins, encoded on different replicons, with conserved CS motifs. However, only the locus that when mutated resulted in an attenuated phenotype has CS activity. Mutations in the other three loci did not result in attenuated virulence and any loss of CS activity, and none were able to complement the CS mutation in Sinorhizobium. The function of these loci remains unknown.     

Genome structure of Ri plasmid (3). Sequencing analysis of the vir region of pRi1724 in Japanese Agrobacterium rhizogenes.

The entire genome of the pRi1724 (217.6-kb) in the mikimopine type Agrobacterium rhizogenes strain MAFF03-01724 has been completely sequenced. The vir region covering 30.2-kb has found to be composed of 21 genes resembling virH1, virA, virB1-11, virG, virC1-2, and virD1-5. The structural organization of the pRi1724 vir operons in this study is exactly the same as that of the previously reported vir operons of other Ri or Ti plasmids, although the size of some ORFs showed little variations among the plasmids. We also found virE3 gene in the pRi1724 (1), but different from Ti plasmids, virE1 and virE2 that are also important for the virulence do not exist in the vir region of pRi1724.