Formation of nitrogen-fixing nodules on legume roots by Rhizobium sp. NGR234 requires an array of bacterial factors, including nodulation outer proteins (Nops) secreted through a type III secretion system (TTSS). Secretion of Nops is abolished upon inactivation of ttsI (formerly y4xI), a protein with characteristics of two-component response regulators that was predicted to activate transcription of TTSS-related genes. During the symbiotic interaction, the phenotype of NGR omega ttsI differs from that of a mutant with a nonfunctional secretion machine, however. This indicated that TtsI regulates the synthesis of other symbiotic factors as well. Conserved sequences, called tts boxes, proposed to act as binding sites for TtsI, were identified not only within the TTSS cluster but also in the promoter regions of i) genes predicted to encode homologs of virulence factors secreted by pathogenic bacteria, ii) loci involved in the synthesis of a rhamnose-rich component (rhamnan) of the lipopolysaccharides (LPS), and iii) open reading frames that play roles in plasmid partitioning. Transcription studies showed that TtsI and tts boxes are required for the activation of TTSS-related genes and those involved in rhamnose synthesis. Furthermore, extraction of polysaccharides revealed that inactivation of ttsI abolishes the synthesis of the rhamnan component of the LPS. The phenotypes of mutants impaired in TTSS-dependent protein secretion, rhamnan synthesis, or in both functions were compared to assess the roles of some of the TtsI-controlled factors during symbiosis.

The plant pathogenic bacterium Xanthomonas campestris pv. vesicatoria expresses a type III secretion system that is necessary for both pathogenicity in susceptible hosts and the induction of the hypersensitive response in resistant plants. This specialized protein transport system is encoded by a 23-kb hrp (hypersensitive response and pathogenicity) gene cluster. Here we show that X. campestris pv. vesicatoria produces filamentous structures, the Hrp pili, at the cell surface under hrp-inducing conditions. Analysis of purified Hrp pili and immunoelectron microscopy revealed that the major component of the Hrp pilus is the HrpE protein which is encoded in the hrp gene cluster. Sequence homologues of hrpE are only found in other xanthomonads. However, hrpE is syntenic to the hrpY gene from another plant pathogen, Ralstonia solanacearum. Pathogenic bacteria exploit different strategies to successfully colonize their eukaryotic hosts. One of the key bacterial pathogenicity mechanisms is the translocation of proteins into eukaryotic host cells by a type III secretion (TTS) system consisting of a trans-envelope multiprotein complex. Components of TTS systems are generally encoded by gene clusters which often reside in pathogenicity islands (25). Several TTS systems have been studied, but our knowledge on the mechanism of substrate recognition and translocation is still very limited. The injectisome of Yersinia is the prototype example of a TTS system (13). There are three hallmarks of type III protein secretion. First, upon secretion, there is no processing of the protein substrate (42). Second, targeting to the TTS system involves sequence information at the N terminus of the protein and/or the corresponding region of the mRNA (14).Third, secretion across the bacterial cell envelope appears to occur in one step without a periplasmic intermediate (11).Xanthomonas campestris pv. vesicatoria is the causal agent of bacterial spot disease in pepper and tomato (8). The X. campestris pv. vesicatoria TTS system is encoded by a 23-kb chromosomal hrp (hypersensitive response and pathogenicity) gene cluster which contains six operons, hrpA to hrpF (Fig. 1) (5, 20, 21, 30, 51; U. Bonas, unpublished data). Loss of hrp gene function results in a pleiotropic phenotype: hrp mutants are unable to grow in the plant, no longer cause disease symptoms, and fail to induce the hypersensitive reaction in resistant host and nonhost plants (5). The hypersensitive reaction is a rapid, local, programmed cell death that is induced upon recognition of the pathogen and is concomitant with the inhibition of pathogen growth within the infected plant tissue (35).hrp gene expression is induced in planta (55) and is controlled by the regulatory genes hrpG and hrpX, which are located outside the hrp gene cluster. The HrpG protein belongs to the OmpR family of two-component regulatory systems (64) and controls the expression of a large gene regulon including hrpX. The AraC-type transcriptional activator HrpX regulates the expression of the operons hrpB t...

Molecular signals, including Nod factors and succinoglycan, are necessary for the establishment of nitrogenfixing nodules (Fix ؉ ) in Medicago truncatula-Sinorhizobium meliloti symbiosis. This report shows that M. truncatula-S. meliloti interactions involve ecotype-strain specificity, as S. meliloti Rm41 and NRG247 are Fix ؉ (compatible) on M. truncatula A20 and Fix ؊ (incompatible) on M. truncatula A17, the Fix phenotypes are reversed with S. meliloti NRG185 and NRG34, and there is a correlation between the host specificity and succinoglycan oligosaccharide structure. S. meliloti NRG185 produces oligosaccharides that are almost fully succinylated, with two succinate groups per subunit, whereas the oligosaccharides produced by S. meliloti Rm41 include many nonsuccinylated subunits, as well as subunits with a single succinate group and others with malate. The results of this study demonstrated the following: (i) incompatibility is not a consequence of an avirulence factor or lack of Nod factor activity; (ii) the Fix ؉ phenotypes are succinoglycan dependent; (iii) there is structural variability in the succinoglycan oligosaccharide populations between S. meliloti strains; (iv) the structural nature of the succinoglycan oligosaccharides is correlated to compatibility; most importantly, (v) an S. meliloti Rm41 derivative, carrying exo genes from an M. truncatula A17-compatible strain, produced a modified population of succinoglycan oligosaccharides (similar to the donor strain) and was Fix ؉ on A17.Sinorhizobium meliloti and Medicago sativa (alfalfa) enter into a nitrogen-fixing symbiosis, and the initiation of a functional symbiosis involves an exchange of signal molecules. Plant-derived flavonoids activate the transcription of bacterial nodulation (nod) genes, resulting in the biosynthesis of Nacylated chito-oligosaccharides (Nod factors). The Nod factors then elicit root hair curling and nodule initiation in the plant (11,14). The subsequent infection of the nodule by the microsymbiont results in the development of nitrogen-fixing nodules (Fix ϩ ) containing physiologically distinct bacteroids. Sinorhizobium spp. lipopolysaccharides, capsular polysaccharides (K antigens), and exopolysaccharides (succinoglycan or galactoglucan) are involved in the infection process, and specific polysaccharide mutants yield nonfunctional "pseudonodules" and a Fix Ϫ phenotype (9,26,29,36). Succinoglycan (exo) mutants of S. meliloti Rm1021 elicit Fix Ϫ nodules due to a lack of infection thread development (10,31,38). Succinoglycan is composed of octasaccharide repeat units (one galactosyl and seven glucosyl residues) substituted with pyruvyl (1-carboxyethylidene), acetyl, and succinyl groups (2, 23, 24, 43). The functional oligosaccharin, an oligosaccharide with signal activity (1), consists of three repeats (5, 53), and the presence of the succinyl groups is essential for activity (30). S. meliloti also produces K antigens (46), and the K R 5 antigen of S. meliloti Rm41 exoB functionally replaces succinoglycan in the infection of a...

The fimA gene of Xanthomonas campestris pv. vesicatoria was identified and characterized. A 20-mer degenerate oligonucleotide complementary to the N-terminal amino acid sequence of the purified 15.5-kDa fimbrillin was used to locate fimA on a 2.6-kb SalI fragment of the X. campestris pv. vesicatoria 3240 genome. The nucleotide sequence of a 1.4-kb fragment containing the fimA region revealed two open reading frames predicting highly homologous proteins FimA and FimB. FimA, which was composed of 136 amino acids and had a calculated molecular weight of 14,302, showed high sequence identity to the type IV fimbrillin precursors. fimB predicted a protein product of 135 amino acids and a molecular weight of 13,854. The open reading frame for fimB contained near the 5 end a palindromic sequence with a terminator loop potential, and the expression level of fimB in vitro and in Xanthomonas was considerably lower than that of fimA. We detected an efficiently transcribed fimA-specific mRNA of 600 bases as well as two weakly expressed, longer mRNA species that reacted with both fimA and fimB. A homolog of fimA but not of fimB was detected by Southern hybridization in strains of X. campestris pv. vesicatoria, campestris, begoniae, translucens, and graminis. A fimA::⍀ mutant of strain 3240 was not significantly reduced in virulence or adhesiveness to tomato leaves. However, the fimA mutant was dramatically reduced in cell aggregation in laboratory cultures and on infected tomato leaves. The fimA mutant strain also exhibited decreased tolerance to UV light.Xanthomonas campestris is a plant pathogen divided into more than 140 pathovars on the basis of the host plants of X. campestris isolates (48). Commonly, the pathovars exhibit a high degree of host specificity in causing the disease, which makes X. campestris infections an interesting target for studies on bacterium-plant interactions and pathogenetic mechanisms of the infections. The ability of X. campestris isolates to cause disease is controlled by hrp (hypersensitive reaction and pathogenicity) genes, whose pathogenetic functions have not yet been characterized. Some of the identified hrp gene products of X. campestris exhibit sequence homology to proteins functioning in secretion of virulence factors of bacteria causing infections in animals (reviewed in reference 5), suggesting that secreted proteins play a role in pathogenesis of X. campestris infections. Isolates of X. campestris secrete plant cell walldegrading enzymes whose function in the pathogenetic processes of X. campestris infections, however, has remained unclear (reviewed in reference 13).In contrast to bacterial infections in mammals, the importance of fimbriae and bacterial adhesion to plant tissue in the pathogenetic processes of plant pathogens has remained controversial (reviewed in references 4 and 34). Fimbriae have been indicated to mediate adhesion of Pseudomonas syringae to bean leaves and to affect the virulence of P. syringae in bean (35). Recently, van Doorn et al. (47) isolated fimbriae from ...

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