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ABSTRACT: Cultured cells of Sinorhizobium sp. NGR234 produce an abundance of capsular polysaccharides, or K antigens; however, cells that are cultured in the presence of apigenin, a nod gene inducer, exhibited a significant reduction in K-antigen production. The flavonoid-induced modulation in capsule production appeared to be related to the phase-shift changes associated with bacteroid differentiation. Therefore, the polysaccharides were extracted from Sinorhizobium sp. NGR234 bacteroids recovered from Vigna unguiculata cv Red Caloona root nodules, and subsequent analyses showed that the bacteroid extracts were virtually devoid of K-antigen. Polysaccharide extracts from two nodulation mutants cultured in the presence of apigenin were then analyzed, and the results showed that the flavonoid-inducible decrease in K-antigen production is y4gM- and nodD1-dependent.
Carbohydrate research 08/2009; 344(15):1947-50. · 2.03 Impact Factor
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ABSTRACT: Molecular signals, including Nod factors and succinoglycan, are necessary for the establishment of nitrogen-fixing 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.
Journal of Bacteriology 12/2007; 189(21):7733-40. · 3.83 Impact Factor
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ABSTRACT: Crude phenol-phase extracts containing bacterial lipopolysaccharides (LPS) from 5 strains of Escherichia coli were investigated to differentiate the strains using Fourier transform infrared (FTIR) spectroscopy and multivariate statistical analysis. The strains used were E. coli K12, E. coli DH5α, E. coli O157:H7, E. coli O157:H12, and E. coli O157:H19. LPS-containing extracts were isolated from each E. coli strain using a hot phenol-water extraction procedure. The extracts were 1st analyzed by deoxycholic acid-polyacrylamide gel electrophoresis and visualized by silver-staining. Analysis of the extracts from E. coli K12 and E. coli DH5α showed rough-type LPS on the lower half of the gel, whereas E. coli O157:H7, E. coli O157:H12, and E. coli O157:H19 yielded abundant smooth LPS (high-molecular-weight LPS that include the O-polysaccharides). Spectra (4000 cm-1 to 700 cm-1) of crude E. coli LPS extracts and intact cells were collected using a FTIR spectrometer. Spectral data were compressed by principle component analysis and analyzed using canonical variate analysis (CVA) of 4000 cm-1 to 700 cm-1 or 1200 cm-1 to 900 cm-1 spectral regions. CVA showed better separation between strains using LPS extracts than intact cells in the 1200 cm-1 to 900 cm-1 spectral region. The same separation trend was found using Mahalanobis distances that quantified spectral differences between the E. coli strains, providing 80% and >95% correct classifications of intact cells and LPS extracts, respectively. This article is the first to report the successful differentiation of E. coli strains at a serotype level using FTIR spectra of bacterial phenol-phase extracts (crude LPS preparations).
Journal of Food Science 02/2006; 71(2):M57 - M61. · 1.66 Impact Factor
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ABSTRACT: 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 (NGRomegafixF); 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)-alpha-L-Rhap-(1,3)-alpha-L-Rhap-(1,2)-alpha-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.
Journal of Bacteriology 10/2005; 187(18):6479-87. · 3.83 Impact Factor
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ABSTRACT: 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.
Journal of Bacteriology 05/2005; 187(7):2458-68. · 3.83 Impact Factor
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ABSTRACT: 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.
Molecular Plant-Microbe Interactions 10/2004; 17(9):958-66. · 4.43 Impact Factor
