TtsI, a Key Regulator of <i>Rhizobium </i>Species NGR234 Is Required for Type III-Dependent Protein Secretion and Synthesis of Rhamnose-Rich Polysaccharides

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 NGRΩttsI 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.</p

Structural Characterization of a Flavonoid-Inducible Pseudomonas aeruginosa A-Band-Like O Antigen of Rhizobium sp. Strain NGR234, Required for the Formation of Nitrogen-Fixing Nodules

Rhizobium (Sinorhizobium) sp. strain NGR234 contains three replicons, the smallest of which (pNGR234a) carries most symbiotic genes, including those required for nodulation and lipo-chito-oligosaccharide (Nod factor) biosynthesis. Activation of nod gene expression depends on plant-derived flavonoids, NodD transcriptional activators, and nod box promoter elements. Nod boxes NB6 and NB7 delimit six different types of genes, one of which (fixF) is essential for the formation of effective nodules on Vigna unguiculata. In vegetative culture, wild-type NGR234 produces a distinct, flavonoid-inducible lipopolysaccharide (LPS) that is not produced by the mutant (NGRΩfixF); this LPS is also found in nitrogen-fixing bacteroids isolated from V. unguiculata infected with NGR234. Electron microscopy showed that peribacteroid membrane formation is perturbed in nodule cells infected by the fixF mutant. LPSs were purified from free-living NGR234 cultured in the presence of apigenin. Structural analyses showed that the polysaccharide portions of these LPSs are specialized, rhamnose-containing O antigens attached to a modified core-lipid A carrier. The primary sequence of the O antigen is [-3)-α-l-Rhap-(1,3)-α-l-Rhap-(1,2)-α-l-Rhap-(1-](n), and the LPS core region lacks the acidic sugars commonly associated with the antigenic outer core of LPS from noninduced cells. This rhamnan O antigen, which is absent from noninduced cells, has the same primary sequence as the A-band O antigen of Pseudomonas aeruginosa, except that it is composed of l-rhamnose rather than the d-rhamnose characteristic of the latter. It is noteworthy that A-band LPS is selectively maintained on the P. aeruginosa cell surface during chronic cystic fibrosis lung infection, where it is associated with an increased duration of infection

The Type III-Dependent Hrp Pilus Is Required for Productive Interaction of Xanthomonas campestris pv. vesicatoria with Pepper Host Plants

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. Bioinformatic analyses suggest that all major Hrp pilus subunits from gram-negative plant pathogens may share the same structural organization, i.e., a predominant alpha-helical structure. Analysis of nonpolar mutants in hrpE demonstrated that the Hrp pilus is essential for the productive interaction of X. campestris pv. vesicatoria with pepper host plants. Furthermore, a functional Hrp pilus is required for type III-dependent protein secretion. Immunoelectron microscopy revealed that type III-secreted proteins, such as HrpF and AvrBs3, are in close contact with the Hrp pilus during and/or after their secretion. By systematic analysis of nonpolar hrp/hrc (hrp conserved) and hpa (hrp associated) mutants, we found that Hpa proteins as well as the translocon protein HrpF are dispensable for pilus assembly, while all other Hrp and Hrc proteins are required. Hence, there are no other conserved Hrp or Hrc proteins that act downstream of HrpE during type III-dependent protein translocation