Biochemical characterization of avirulent Agrobacterium tumefaciens chvA mutants: synthesis and excretion of beta-(1-2)glucan.

The chvA gene product of Agrobacterium tumefaciens is required for virulence and attachment of bacteria to plant cells. Three chvA mutants were studied. In vivo, they were defective in the synthesis, accumulation, and secretion of beta-(1-2)glucan; however, the 235-kilodalton (kDa) protein known to be involved in the synthesis of beta-(1-2)glucan (A. Zorreguieta and R. Ugalde, J. Bacteriol. 167:947-951, 1986) was present and active in vitro. was present and active in vitro. Two molecular forms of cyclic beta-(1-2)glucan, designated types I and II, were resolved by gel chromatography. Type I beta-(1-2)glucan was substituted with nonglycosidic residues, and type II beta-(1-2)glucan was nonsubstituted. Wild-type cells accumulated type I beta-(1-2)glucan, and chvA mutant cells accumulated mainly type II beta-(1-2)glucan and a small amount of type I beta-(1-2)glucan. Inner membranes of wild-type and chvA mutants formed in vitro type II nonsubstituted beta-(1-2)glucan. A 75-kDa inner membrane protein is proposed to be the chvA gene product. chvA mutant inner membranes had increased levels of 235-kDa protein; partial trypsin digestion patterns suggested that the 235-kDa protein (the gene product of the chvB region) and the gene product of the chvA region form a complex in the inner membrane that is involved in the synthesis, secretion, and modification of beta-(1-2)glucan. All of the defects assigned to the chvA mutation were restored after complementation with plasmid pCD522 containing the entire chvA region.     

chvA locus may be involved in export of neutral cyclic beta-1,2-linked D-glucan from Agrobacterium tumefaciens

Extracellular and intracellular neutral beta-1,2-linked D-glucan content was determined in a virulent, attachment-deficient mutants of Agrobacterium tumefaciens that map in the chvA locus. chvA mutants contained approximately the same amount of intracellular glucan as cells of the virulent control strain A759, but released into the culture medium only 2% of the glucan released by strain A759. Introduction of a cosmid carrying the wild-type chv region restored attachment and virulence and restored extracellular glucan production to chvA mutant A2505. Exogenous glucan did not enhance or inhibit attachment or tumorigenesis of the virulent control strain or the chvA or chvB mutants. Our results suggest that the chvA locus is involved in the export of glucan from the cell and that export may be required for tumorigenesis.     

Formation in Rhizobium and Agrobacterium spp. of a 235-kilodalton protein intermediate in beta-D(1-2) glucan synthesis.

beta-D(1-2) Glucan was synthesized by Agrobacterium and Rhizobium spp. in vitro with enzymes from the internal membranes upon the addition of UDF glucose and Mg2+ or Mn2+. An intermediate containing protein and beta-D(1-2) glucan was formed during the reaction. It could be precipitated with trichloroacetic acid or separated by polyacrylamide gel electrophoresis under denaturing conditions. After detection with Coomassie blue or a radioactive substrate, the intermediate appeared as a 235-kilodalton protein. The radioactivity could be chased with a nonradioactive substrate. All strains that formed beta-D(1-2) glucan in vitro formed the 235-kilodalton protein, whereas avirulent, beta-D(1-2) glucan-negative mutants did not synthesize it. Transposon insertions in the chvB locus of strains ME2 and ME116 did not alter the virulence of the strains. These strains were able to form beta-D(1-2) glucan in vitro and synthesize the 235-kilodalton protein.     

A Rhizobium meliloti mutant that forms ineffective pseudonodules in alfalfa produces exopolysaccharide but fails to form beta-(1----2) glucan.

A mutant of Rhizobium meliloti that elicited the formation of inactive nodules in alfalfa was found not to form beta-(1----2) glucan in vivo or in vitro. It was nonmotile because it lacks flagella. The 235-kilodalton protein which acts as an intermediate in beta-(1----2) glucan synthesis was undetectable in the mutant. These properties of the mutant are common to those of chvB mutants of Agrobacterium tumefaciens. Exopolysaccharide formation by the R. meliloti mutant was about double that by the wild type.     

Role for [corrected] Agrobacterium tumefaciens ChvA protein in export of beta-1,2-glucan.

Functional chvA and chvB genes are required for attachment of Agrobacterium tumefaciens to plant cells, an early step in crown gall tumor formation. Strains defective in these loci do not secrete normal amounts of cyclic beta-1,2-glucan. Whereas chvB is required for beta-1,2-glucan synthesis, the role of chvA in glucan synthesis or export has not been clearly defined. We found that cultures of chvA mutants contained as much neutral beta-1,2-glucan in the cell pellets as did the wild type, with no detectable accumulation of glucan in the culture supernatant. The cytoplasm of chvA mutant cells contained over three times more soluble beta-1,2-glucan than did the cytoplasm of the wild-type parent. Unlike the wild type, chvA mutants contained no detectable periplasmic glucan. The amino acid sequence of chvA is highly homologous to the sequences of bacterial and eucaryotic export proteins, as observed previously in the case of ndvA, a rhizobial homolog of chvA. Strong sequence homology within this family of export proteins is concentrated in the carboxy-terminal portions of the proteins, but placement of consensus ATP-binding sites, internal signal sequences, and hydrophobic domains are conserved over their entire lengths. These data suggest a model for beta-1,2-glucan synthesis in A. tumefaciens in which glucan is synthesized inside the inner membrane with the participation of ChvB and transported across the inner membrane with the participation of ChvA.     

Osmosensitivity phenotypes of Agrobacterium tumefaciens mutants that lack periplasmic beta-1,2-glucan.

The periplasmic cyclic beta-1,2-glucan of Agrobacterium tumefaciens is believed to maintain high osmolarity in the periplasm during growth of the bacteria on low-osmotic-strength media. Strains with mutations in the chvA or chvB gene do not accumulate beta-1,2-glucan in their periplasm and exhibit pleiotropic phenotypes, including inability to form crown gall tumors on plants. We examined the effects of medium osmolarity to determine whether some or all of these phenotypes result from suboptimal periplasmic osmolarity. The mutants grew more slowly than wild-type cells and exhibited altered periplasmic and cytoplasmic protein content when cultured in low-osmotic-strength media, but not when cultured in high-osmotic-strength media. These observations support a role for periplasmic glucan in osmoadaptation. However, the mutants were avirulent and exhibited reduced motility regardless of the osmolarity of the medium. Therefore, beta-1,2-glucan may play roles in virulence and motility that are unrelated to its role in osmoadaptation.     

The ndvA gene product of Rhizobium meliloti is required for beta-(1----2)glucan production and has homology to the ATP-binding export protein HlyB.

The ndvA locus of Rhizobium meliloti is homologous to and can substitute for the chvA locus of Agrobacterium tumefaciens. We have previously shown that an ndvA mutant exhibited reduced motility and formed small, white, empty nodules on alfalfa roots. Here we show that this ndvA mutant is defective in the production of the cyclic extracellular polysaccharide beta-(1----2)glucan, even though a 235,000-dalton protein intermediate, known to be involved in the synthesis of this molecule, is present and active in vitro. The DNA sequence of the ndvA locus revealed a single large open reading frame encoding a 67,100-dalton protein that was homologous to a number of bacterial ATP-binding transport proteins. The greatest degree of relatedness was seen with Escherichia coli HlyB, a protein involved in the export of hemolysin, and with the mdr gene product of mammalian cells, which is also homologous to HlyB and thought to be involved in export. Based on the overall symbiotic phenotype of ndvA mutants, the extensive homology between NdvA and HlyB, the fact that ndvA mutants retained an active 235,000-dalton membrane intermediate, and the absence of extracellular beta-(1----2)glucan, we propose that NdvA is involved in export of beta-(1----2)glucan from the cell and that this process is fundamentally important for normal alfalfa nodule development.     

Mechanism of action of cyclic beta-1,2-glucan synthetase from Agrobacterium tumefaciens: competition between cyclization and elongation reactions.

We have examined some aspects of the mechanism of cyclic beta-1,2-glucan synthetase from Agrobacterium tumefaciens (235-kDa protein, gene product of the chvB region). The enzyme produces cyclic beta-1,2-glucans containing 17 to 23 glucose residues from UDP-glucose. In the presence of added cyclic beta-1,2-glucans (> 0.5 mg/ml) (containing 17 to 23 glucose residues), the enzyme instead synthesizes larger cyclic beta-1,2-glucans containing 24 to 30 glucose residues. This is achieved by de novo synthesis and not by disproportion reactions with the added product. This is interpreted as inhibition of the specific cyclization reaction for the synthesis of cyclic beta-1,2-glucans containing 17 to 23 glucose residues but with no concomitant effect on the elongation (polymerization) reaction. Temperature and detergents both affect the distribution of sizes of cyclic beta-1,2-glucans, but glucans containing 24 to 30 glucose residues are not produced. We suggest that the size distribution of cyclic beta-1,2-glucan products depends on competing elongation and cyclization reactions.     

Cyclic beta-(1,2)-glucan synthesis in Rhizobiaceae: roles of the 319-kilodalton protein intermediate.

Cyclic beta-(1,2)-glucans are synthesized by members of the Rhizobiaceae family through protein-linked oligosaccharides as intermediates. The protein moiety is a large inner membrane molecule of about 319 kDa. In Agrobacterium tumefaciens and in Rhizobium meliloti the protein is termed ChvB and NdvB, respectively. Inner membranes of R. meliloti 102F34 and A. tumefaciens A348 were first incubated with UDP-[14C]Glc and then solubilized with Triton X-100 and analyzed by polyacrylamide gel electrophoresis under native conditions. A radioactive band corresponding to the 319-kDa protein was detected in both bacteria. Triton-solubilized inner membranes of A. tumefaciens were submitted to native electrophoresis and then assayed for oligosaccharide-protein intermediate formation in situ by incubating the gel with UDP-[14C]Glc. A [14C]glucose-labeled protein with an electrophoretic mobility identical to that corresponding to the 319-kDa [14C]glucan protein intermediate was detected. In addition, protein-linked radioactivity was partially chased when the gel was incubated with unlabeled UDP-Glc. A heterogeneous family of cyclic beta-(1,2)-glucans was formed upon incubation of the gel portion containing the 319-kDa protein intermediate with UDP-[14C]Glc. A protein with an electrophoretic behavior similar to the 319-kDa protein intermediate was "in gel" labeled by using Triton-solubilized inner membranes of an A. tumefaciens exoC mutant, which contains a protein intermediate without nascent glucan. These results indicate that initiation (protein glucosylation), elongation, and cyclization were catalyzed in situ. Therefore, the three enzymatic activities detected in situ reside in a unique protein component (i.e., cyclic beta-(1,2)-glucan synthase). It is suggested that the protein component is the 319-kDa protein intermediate, which might catalyze the overall cyclic beta-(1,2)-glucan synthesis.     

Biochemical characterization of avirulent exoC mutants of Agrobacterium tumefaciens.

The synthesis of periplasmic beta(1-2)glucan is required for crown gall tumor formation by Agrobacterium tumefaciens and for effective nodulation of alfalfa by Rhizobium meliloti. The exoC (pscA) gene is required for this synthesis by both bacteria as well as for the synthesis of capsular polysaccharide and normal lipopolysaccharide. We tested the possibility that the pleiotropic ExoC phenotype is due to a defect in the synthesis of an intermediate common to several polysaccharide biosynthetic pathways. Cytoplasmic extracts from wild-type A. tumefaciens and from exoC mutants of A. tumefaciens containing a cloned wild-type exoC gene synthesized in vitro UDP-glucose from glucose, glucose 1-phosphate, and glucose 6-phosphate. Extracts from exoC mutants synthesized UDP-glucose from glucose 1-phosphate but not from glucose or glucose 6-phosphate. Membranes from exoC mutant cells synthesized beta(1-2)glucan in vitro when exogenous UDP-glucose was added and contained the 235-kilodalton protein, which has been shown to carry out this synthesis in wild-type cells. We conclude that the inability of exoC mutants to synthesize beta(1-2)glucan is due to a deficiency in the activity of the enzyme phosphoglucomutase (EC 2.7.5.1), which in wild-type bacteria converts glucose 6-phosphate to glucose 1-phosphate, an intermediate in the synthesis of UDP-glucose. This interpretation can account for all of the deficiencies in polysaccharide synthesis which have been observed in these mutants.     

Attachment to roots and virulence of a chvB mutant of Agrobacterium tumefaciens are temperature sensitive

Agrobacterium tumefaciens chvB mutants are unable to produce beta-1,2 glucan. They are nonattaching and avirulent and show reduced motility at room temperature. At lower temperatures (16 degrees C), chvB mutants became virulent on Bryophyllum daigremontiana and Lycopersicon esculentum and were able to attach to L. esculentum, Arabidopsis thaliana, Daucus carota, and Tagetes erecta roots. The mutant bacteria also recovered wild-type motility at lower temperatures. Two other nonattaching mutants of A. tumefaciens, AttR and AtrA, were unaffected by the lowered temperature, remaining nonattaching and avirulent.     

The Agrobacterium tumefaciens virE2 gene product is a single-stranded-DNA-binding protein that associates with T-DNA.

Agrobacterium tumefaciens transfers T-DNA into the plant genome by a process mediated by Ti plasmid-encoded vir genes. Cleavage at T-DNA border sequences by the VirD endonuclease generates linear, single-stranded T-DNA molecules. In the work described in this report, we used electrophoretic mobility shift assays to show that the purified virE2 gene product binds to single-stranded DNA. VirE2 protein associates with T-DNA as shown by immunoprecipitation studies with VirE2-specific antiserum. The VirE2 protein was detected primarily in the cytoplasm, but also in the inner and outer membrane and periplasmic fractions. Virulence of a virE2 mutant was restored by mixed infection with strains carrying an intact vir region, but not with virA, virB, virD, virE, or virG mutants or chvA, chvB, or exoC mutants. We propose that the VirE2 protein is involved in the processing of T-DNA and in T-strand protection during transfer to the plant cell.     

Identification of a new virulence locus in Agrobacterium tumefaciens that affects polysaccharide composition and plant cell attachment.

We have identified a new virulence locus in Agrobacterium tumefaciens. Strains carrying Tn5 inserts at this locus could not incite tumors on Kalanchoe daigremontiana, Nicotiana rustica, tobacco, or sunflower and had severely attenuated virulence on carrot disks. We termed the locus pscA, because the mutants that defined the locus were initially isolated as having an altered polysaccharide composition; they were nonfluorescent on media containing Leucophor or Calcofluor, indicating a defect in the production of cellulose fibrils. Further analysis showed that the pscA mutants produced little, if any, of the four species of exopolysaccharide synthesized by the wild-type strain. DNA hybridization analysis and genetic complementation experiments indicated that the pscA locus is not encoded by the Ti plasmid and that it is distinct from the previously described chromosomal virulence loci chvA and chvB. However, like chvA and chvB mutants, the inability of the pscA mutants to form tumors is apparently due to a defect in plant cell attachment. Whereas we could demonstrate binding of the wild-type strain to tobacco suspension cells, attachment of the pscA mutants was drastically reduced or completely absent.     

Rhicadhesin-mediated attachment and virulence of an Agrobacterium tumefaciens chvB mutant can be restored by growth in a highly osmotic medium.

Cyclic beta-1,2-glucan is considered to play a role in osmoadaptation of members of the family Rhizobiaceae in hypotonic media. Agrobacterium tumefaciens chvB mutants, lacking beta-1,2-glucan, exhibit a pleiotropic phenotype, including nonmotility, attachment deficiency, and avirulence. Here we report that by growth of chvB mutant cells in tryptone-yeast extract medium supplemented with 7 mM CaCl2 and 100 mM NaCl, the mutant cells become motile, attach to pea root hair tips, and are virulent on Kalanchoë leaves. Moreover, whereas chvB mutants grown in tryptone-yeast extract medium containing 7 mM CaCl2 do not produce active rhicadhesin, addition of 100 mM NaCl to this medium resulted in restoration of rhicadhesin activity. The presence of CaCl2 appeared to be required for attachment, virulence, and activity of rhicadhesin. The results support a role for cyclic beta-1,2-glucan in osmoadaptation and strengthen the notion that rhicadhesin is required for attachment and virulence of A. tumefaciens.     

Agrobacterium tumefaciens virulence locus pscA is related to the Rhizobium meliloti exoC locus.

Agrobacterium tumefaciens and Rhizobium meliloti carry related genetic loci which have important roles in virulence and symbiosis. Previously, it was shown that two virulence loci of A. tumefaciens, chvA and chvB, are related to two R. meliloti symbiosis loci, ndvA and ndvB, respectively (T. Dylan, L. Ielpi, S. Stanfield, L. Kashyap, C. Douglas, M. Yanofsky, E. Nester, D. R. Helinski, and G. Ditta, Proc. Natl. Acad. Sci. USA 83:4403-4407, 1986). Here we show that these two phytobacteria possess additional related virulence/symbiosis genes. Results of genetic complementation and DNA hybridization experiments indicate that the pscA virulence locus of A. tumefaciens is structurally and functionally related to the exoC symbiosis locus of R. meliloti. Thus, A. tumefaciens and R. meliloti bear at least three related genetic loci that have crucial roles in establishing the interactions that each bacterium has with its respective host plants.     

The effect of cellulose overproduction on binding and biofilm formation on roots by Agrobacterium tumefaciens

Agrobacterium tumefaciens growing in liquid attaches to the surface of tomato and Arabidopsis thaliana roots, forming a biofilm. The bacteria also colonize roots grown in sterile quartz sand. Attachment, root colonization, and biofilm formation all were markedly reduced in celA and chvB mutants, deficient in production of cellulose and cyclic beta-(1,2)-D-glucans, respectively. We have identified two genes (celG and cell) in which mutations result in the overproduction of cellulose as judged by chemical fractionation and methylation analysis. Wild-type and chvB mutant strains carrying a cDNA clone of a cellulose synthase gene from the marine urochordate Ciona savignyi also overproduced cellulose. The overproduction in a wild-type strain resulted in increased biofilm formation on roots, as evaluated by light microscopy, and levels of root colonization intermediate between those of cellulose-minus mutants and the wild type. Overproduction of cellulose by a nonattaching chvB mutant restored biofilm formation and bacterial attachment in microscopic and viable cell count assays and partially restored root colonization. Although attachment to plant surfaces was restored, overproduction of cellulose did not restore virulence in the chvB mutant strain, suggesting that simple bacterial binding to plant surfaces is not sufficient for pathogenesis.     

Identification of the product of an Agrobacterium tumefaciens chromosomal virulence gene

The chvB operon of Agrobacterium tumefaciens is required for bacterial attachment to plant cells and for efficient crown gall tumor formation. As defined by the virulence phenotypes of mutants with transposon insertions mapping in the region, the operon was previously mapped to a 5-kilobase (kb) stretch of chromosomal DNA. We report here that the operon is actually about 8.5 kb long and that it contains a 7-kb gene coding for a large membrane protein involved in the synthesis of cyclic beta-1,2-glucan. Mutants with transposon insertions within the 5-kb phenotypically defined operon do not synthesize this functional protein, do not synthesize beta-1,2-glucan, and do not form tumors. However, mutants with insertions that map up to 3.5 kb downstream of the phenotypically defined operon synthesize truncated proteins that are active in beta-1,2-glucan synthesis. These mutants form tumors. The truncated proteins correspond closely in size with the map positions of the insertions, suggesting that the insertions truncate the proteins by translational termination. A plasmid that contains only the phenotypically defined chvB operon also codes for a truncated protein. A fusion product between the protein and beta-galactosidase carried on a Tn3-HoHo1 insertion was observed in one mutant. Partial trypsin digestion of wild-type inner membranes generated truncated proteins that were active in beta-1,2-glucan synthesis, demonstrating that a large portion of the protein is not required for beta-1,2-glucan synthesis. The correlation between beta-1,2-glucan synthesis by the truncated proteins and tumorigenesis strongly implicates the polysaccharide product of this protein in tumor formation.     

Synthesis of β(1–2)glucan in Rhizobium loti

Fast growing strains of Rhizobium loti isolated from nodules of Lotus tenuis of the flooding Pampas of Argentina produced cellular (1–2)glucans having a higher degree of polymerization and more anionic substituents than (1–2)glucans accumulated by Agrobacterium tumefaciens cells. Inner membranes of R. loti contained a 235 kDa (1–2)glucan intermediate protein indistinguishable by polyacrylamide gel electrophoresis from the intermediate protein present in A. tumefaciens inner membranes. Incubation of inner membrannes of R. loti with UDP-Gle led to the formation of neutral (1–2)glucans with a higher degree of polymerization than glucans formed by A. tumefaciens inner membranes.Introduction in R. loti strains of plasmid pCD523 containing A. tumefaciens chvA and chvB virulence regions yielded strains that accumulated 4 times more cellular (1–2)glucans than wild type cells. This glucan was, regarding anionic substitution and degree of polymerization, indistinguishable from A. tumefaciens (1–2)glucans. Furthermore inner membranes of these R. loti exoconjugant cells contained higher levels of the 235 kDa (1–2)glucan intermediate protein and formed in vitro 8 times more neutral (1–2)glucan with a degree of polymerization corresponding to A. tumefaciens (1–2)glucan than inner membranes isolated from wild type cells.It was concluded that A. tumefaciens chvB gene is expressed in R. loti and determined the degree of polymerization of (1–2)glucan.Abbreviations Nod⁺ effective nodulation - Vir⁺ virulent - Vir^- avirulent - Trp^r trimethoprim resistence - Tc^r tetracycline resistence - TCA trichloroacetic acid - PMSF phenyl methyl sulfonyl fluoride     

Isolation and characterization of an ndvB locus from Rhizobium fredii

A gene (ndvB) in Rhizobium meliloti that is essential for nodule development in Medicago sativa (alfalfa), specifies synthesis of a large membrane protein. This protein appears to be an intermediate in beta-1,2-glucan synthesis by the microsymbiont. Southern hybridization analysis showed strong homology between an ndvB (chvB) probe and genomic DNA of R. fredii but not from Bradyrhizobium japonicum. A cosmid clone containing the putative ndvB locus was isolated from a Rhizobium fredii gene library. The cosmid clone which complemented R. meliloti ndvB mutants for synthesis of beta-1,2-glucans and effective nodulation of alfalfa was mapped and subcloned. Fragment-specific Tn5 mutagenesis followed by homologous recombination into the R. fredii genome indicated that the region was essential for beta-1,2-glucan synthesis and for formation of an effective symbiosis with Glycine max (soybean).     

An intermediate in cyclic beta 1-2 glucan biosynthesis

Incubation of UDP-[14C]Glc with the inner membranes of Agrobacterium tumefaciens leads to the formation of cyclic beta 1-2 glucan and trichloroacetic acid-insoluble compounds. The proteolysis products of the latter show a positive charge in acid and a negative charge in alkaline buffers. The cyclic beta 1-2 glucan and the trichloroacetic acid insoluble compounds yield the same products on partial acid hydrolysis. Addition of excess non-radioactive UDP-Glc to the reaction mixture nearly stops the formation of radioactive beta 1-2 glucan and leads to a rapid fall of radioactivity in the trichloroacetic acid precipitate. Alkaline treatment of the insoluble compounds under conditions of beta-elimination leads to the partial release of free saccharides (about 30%). It is concluded that beta 1-2 glucan chains are built up joined to a protein and then released as free cyclic beta 1-2 glucan.