Requirement for genes with homology to ABC transport systems for attachment and virulence of Agrobacterium tumefaciens.

Transposon mutants of Agrobacterium tumefaciens which were avirulent and unable to attach to plant cells were isolated and described previously. A clone from a library of Agrobacterium tumefaciens DNA which was able to complement these chromosomal att mutants was identified. Tn3HoHo1 insertions in this clone were made and used to replace the wild-type genes in the bacterial chromosome by marker exchange. The resulting mutants were avirulent and showed either no or very much reduced attachment to carrot suspension culture cells. We sequenced a 10-kb region of this clone and found a putative operon containing nine open reading frames (ORFs) (attA1A2BCDEFGH). The second and third ORFs (attA2 and attB) showed homology to genes encoding the membrane-spanning proteins (potB and potH; potC and potI) of periplasmic binding protein-dependent (ABC) transport systems from gram-negative bacteria. The homology was strongest to proteins involved in the transport of spermidine and putrescine. The first and fifth ORFs (attA1 and attE) showed homology to the genes encoding ATP-binding proteins of these systems including potA, potG, and cysT from Escherichia coli; occP from A. tumefaciens; cysA from Synechococcus spp.; and ORF-C from an operon involved in the attachment of Campylobacte jejuni. The ability of mutants in these att genes to bind to host cells was restored by addition of conditioned medium during incubation of the bacteria with host cells.     

Agrobacterium rhizogenes mutants that fail to bind to plant cells.

Transposon insertion mutants of Agrobacterium rhizogenes were screened to obtain mutant bacteria that failed to bind to carrot suspension culture cells. A light microscope binding assay was used. The bacterial isolates that were reduced in binding to carrot cells were all avirulent on Bryophyllum diagremontiana leaves and on carrot root disks. The mutants did not appear to be altered in cellulose production. The composition of the medium affected the ability of the parent and mutant bacteria to bind to carrot cells. The parent strain bound to carrot cells in greatest numbers in low-ionic-strength media such as 4% sucrose but still showed significant binding in Murashige-Skoog tissue culture medium. All of the mutants showed reduced binding in 4% sucrose after 2 h of incubation with carrot cells. One mutant was delayed in binding in 4% sucrose. This mutant and one other mutant also showed reduced binding to carrot cells in Murashige-Skoog medium. To determine whether the Tn5 insertion was responsible for the mutant phenotype, DNA containing the Tn5 insertion was cloned from the mutant bacteria and used to introduce Tn5 into the parent strain in the same location as in the original mutant by marker exchange. The resulting transconjugants had the same avirulent, nonattaching phenotype as the original mutants, suggesting that the mutant phenotype was due to the Tn5 insertion. The cloned DNA containing the Tn5 insertion was also tested for homology to DNA of known genes that affect attachment of Agrobacterium tumefaciens to plant cells by DNA hybridization. No homology to chv, att, or pscA clones was observed. In addition, cloned chv, att, and pscA genes from A. tumefaciens were unable to complement the attachment-minus A. rhizogenes mutants. Thus, the A. rhizogenes nonattaching mutants appear to be different from the previously described A. tumefaciens mutants.     

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.     

Role of bacterial cellulose fibrils in Agrobacterium tumefaciens infection.

During the attachment of Agrobacterium tumefaciens to carrot tissue culture cells, the bacteria synthesize cellulose fibrils. We examined the role of these cellulose fibrils in the attachment process by determining the properties of bacterial mutants unable to synthesize cellulose. Such cellulose-minus bacteria attached to the carrot cell surface, but, in contrast to the parent strain, with which larger clusters of bacteria were seen on the plant cell, cellulose-minus mutant bacteria were attached individually to the plant cell surface. The wild-type bacteria became surrounded by fibrils within 2 h after attachment. No fibrils were seen with the cellulose-minus mutants. Prolonged incubation of wild-type A. tumefaciens with carrot cells resulted in the formation of large aggregates of bacteria, bacterial fibrils, and carrot cells. No such aggregates were formed after the incubation of carrot cells with cellulose-minus A. tumefaciens. The absence of cellulose fibrils also caused an alteration in the kinetics of bacterial attachment to carrot cells. Cellulose synthesis was not required for bacterial virulence; the cellulose-minus mutants were all virulent. However, the ability of the parent bacterial strain to produce tumors was unaffected by washing the inoculation site with water, whereas the ability of the cellulose-minus mutants to form tumors was much reduced by washing the inoculation site with water. Thus, a major role of the cellulose fibrils synthesized by A. tumefaciens appears to be anchoring the bacteria to the host cells, thereby aiding the production of tumors.     

Involvement of a vitronectin-like protein in attachment of Agrobacterium tumefaciens to carrot suspension culture cells.

Infections of dicotyledonous plants by Agrobacterium tumefaciens result in the formation of crown gall tumors. Attachment of the bacteria to plant host cells is required for tumor formation. Human vitronectin and antivitronectin antibodies both inhibited the binding of A. tumefaciens to carrot cells. Wild-type bacteria are able to bind radioactive vitronectin; nonattaching mutants showed a reduction in the ability to bind vitronectin. The binding of biotype 1 A. tumefaciens to carrot cells or to radioactive vitronectin was not affected by high ionic strength. Detergent extraction of carrot cells removed the receptor to which the bacteria bind. The extract was found to contain a vitronectin-like protein. These results suggest that A. tumefaciens utilizes a vitronectin-like protein on the plant cell surface as the receptor for its initial attachment to host cells.     

Inhibition by Agrobacterium tumefaciens and Pseudomonas savastanoi of development of the hypersensitive response elicited by Pseudomonas syringae pv. phaseolicola.

Injection into tobacco leaves of biotype 1 Agrobacterium tumefaciens or of Pseudomonas savastanoi inhibited the development of a visible hypersensitive response to the subsequent injection at the same site of Pseudomonas syringae pv. phaseolicola. This interference with the hypersensitive response was not seen with injection of bacterial growth medium or Escherichia coli cells. Live A. tumefaciens cells were required for the inhibitory effect. Various mutants and strains of A. tumefaciens were examined to determine the genes involved. Known chromosomal mutations generally had no effect on the ability of A. tumefaciens to inhibit the hypersensitive response, except for chvB mutants which showed a reduced (but still significant) inhibition of the hypersensitive response. Ti plasmid genes appeared to be required for the inhibition of the hypersensitive response. The bacteria did not need to be virulent in order to inhibit the hypersensitive response. Deletion of the vir region from pTi had no effect on the inhibition. However, the T region of the Ti plasmid was required for inhibition. Studies of transposon mutants suggested that the tms but not tmr or ocs genes were required. These genes were not acting after transfer to plant cells since they were effective in strains lacking vir genes and thus unable to transfer DNA to plant cells. The results suggest that the expression of the tms genes in the bacteria may inhibit the development of the hypersensitive response by the plant. An examination of the genes required in P. savastanoi for the inhibition of the hypersensitive response suggested that bacterial production of auxin was also required for the inhibition of the hypersensitive response by these bacteria.     

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.     

Gene expression patterns associated with in vitro tracheary element formation in isolated single mesophyll cells of Zinnia elegans.

Tracheary element formation from isolated Zinnia leaf mesophyll cells is an excellent system for the dissection of patterned secondary cell wall thickening and lignification. We used mRNAs from cells cultured for 48 h in the induction medium to isolate differentially regulated genes. Thirteen unique cDNA clones were isolated using a subtractive hybridization method. These clones can be divided into three distinct groups according to their characteristic gene expression in different media. The first group includes those genes whose expression is induced in the basal medium without 1-naphthaleneacetic acid (NAA) and benzyladenine; this indicates that the expression of these genes is regulated by chemical and physical factors other than these hormones. Three of these clones, p48h-229, p48h-114, and p48h-102, show significant homology to a pathogenesis-related protein II, a serine proteinase inhibitor, and a sunflower anther-specific proline-rich protein, respectively. The second group includes those genes whose expression is mainly NAA induced. One of these clones, p48h-10, shows high protein sequence homology to a barley aleurone-specific cDNA, B11E. The p48h-10-encoded protein shares some common characteristics of plant nonspecific lipid transfer proteins (low molecular weight, the secretion signal peptide, eight conserved cysteine residues, and a basic protein), although no significant protein sequence homology is found between p48-10 and other plant nonspecific lipid transfer proteins. The third group includes those genes whose expression is induced primarily in the induction medium; this indicates that the expression of these genes is closely associated with the process of tracheary element formation. Two of these clones, p48h-107 and p48h-17, show high homology to adenylate kinase and papaya proteinase I, respectively. The possible roles of these differentiation-specific genes during tracheary element formation are discussed.     

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.     

Lack of expression of bundle-forming pili in some clinical isolates of enteropathogenic Escherichia coli (EPEC) is due to a conserved large deletion in the bfp operon

Enteropathogenic Escherichia coli (EPEC) produces a plasmid-encoded type IV pilus, called the bundle-forming pilus (BFP), involved in the formation of the localized adhesion onto epithelial cells. In this study, we demonstrate that clinical isolates of serotypes O128ab:H2 and O119:H2 contain a ca. 13-kb deletion in the bfp operon, resulting in a lack of expression of these pili. An IS sequence with homology to the IS66 of Agrobacterium tumefaciens replaced the deleted bfp genes. These results suggest that the bfp operon was deleted through a transpositional event and that other adherence factors may mediate attachment of these bacteria to the host cells.     

GFP-aided confocal laser scanning microscopy can monitor Agrobacterium tumefaciens cell morphology and gene expression associated with infection

We tagged Agrobacterium tumefaciens cells with a mini-Tn5 transposon containing a promoterless gene encoding a green fluorescent protein (GFP). Some of the GFP-tagged individual bacterial cells exhibited strong green fluorescence, which reflected the expression levels of the GFP-tagged genes. Those cells could be readily detected with a confocal laser scanning microscope (CLSM). We observed that the fluorescence and morphology of A. tumefaciens cells grown in plant tissues resembled those grown in a minimal medium of low pH, which is required for expression of the virulence genes responsible for tumorigenesis. This suggests that GFP-aided CLSM can be used to determine which growth medium is more representative of the nutritional conditions that a pathogen encounters in plant tissues. We also observed that the fluorescence and morphology of A. tumefaciens cells changed dramatically during the course of infection. Our data suggested that A. tumefaciens cells were probably better fed upon successful colonization. We believe that GFP-aided CLSM can help study the fate of A. tumefaciens cells inside plant tissues by monitoring cell morphology and gene expression associated with the infection process in situ.     

MB78, a virulent bacteriophage of Salmonella typhimurium

The isolation and some properties of a virulent bacteriophage of Salmonella typhimurium, MB78, which is morphologically, serologically, and physiologically unrelated to P22, are reported. The phage has a noncontractile long tail with partite ends. It cannot multiply in minimal medium in the presence of citrate. MB78-infected cells are, however, killed in such medium. This phage cannot grow in rifampin-resistant mutants of the host. The latent period of growth of this phage is much shorter than that of P22. Both sieA and sieB genes of the resident P22 prophage are required to exclude the superinfecting MB78 phage, whereas all temperate phages related to P22 are excluded by either one or both of the genes individually. Restriction endonuclease cleavage patterns of P22 and MB78 are distinctly different. The absence of homology between the two phages P22 and MB78 suggests that MB78 is not related to phage P22.     

Specific attachment of Agrobacterium tumefaciens to bamboo cells in suspension cultures.

Agrobacterium tumefaciens was tested for its ability to attach to tissue culture cells of bamboo, a monocotyledonous plant. Phase-contrast microscopy and kinetic experiments with radiolabeled bacteria showed that attachment to bamboo cells was indistinguishable from attachment to cells of dicotyledonous plants. Bacterial mutants defective in attachment to dicotyledonous plants showed similar behavior with bamboo, and extensive washing of the bamboo cells had no effect on the number of bacteria which attached.