Identification of Salmonella enterica genes with a role in persistence on lettuce leaves during cold storage by recombinase-based in vivo expression technology.

Recurrent outbreaks of enteric illness linked to lettuce and a lack of efficacious strategies to decontaminate produce underscores the need for a better understanding of the molecular interactions of foodborne pathogens with plants. This study aimed at identifying Salmonella enterica genes involved in the persistence of this organism on post-harvest lettuce during cold storage using recombinase-based in vivo expression technology (RIVET). In total, 37 potentially induced loci were identified in four distinct screenings. Knockout mutations in eight upregulated genes revealed that four of them have a role in persistence of the pathogen in this system. These genes included stfC, bcsA, misL, and yidR, encoding a fimbrial outer membrane usher, a cellulose synthase catalytic subunit, an adhesin of the autotransporter family expressed from the Salmonella pathogenicity island-3, and a putative ATP/GTP-binding protein, respectively. bcsA, misL, and yidR but not stfC mutants were impaired also in attachment and biofilm formation, suggesting that these functions are required for survival of S. enterica on post-harvest lettuce. This is the first report that MisL, which has a role in Salmonella binding to fibronectin in animal hosts, is involved also in adhesion to plant tissue. Hence, our study uncovered a new plant attachment factor in Salmonella and demonstrates that RIVET is an effective approach for investigating human pathogen-plant interactions in a post-harvest leafy vegetable.

The bitter side of the nightshades: Genomics drives discovery in Solanaceae steroidal alkaloid metabolism.

Steroidal alkaloids (SAs) and their glycosylated forms (SGAs) are toxic compounds largely produced by members of the Solanaceae and Liliaceae plant families. This class of specialized metabolites serves as a chemical barrier against a broad range of pest and pathogens. In humans and animals, SAs are considered anti-nutritional factors because they affect the digestion and absorption of nutrients from food and might even cause poisoning. In spite of the first report on SAs nearly 200 years ago, much of the molecular basis of their biosynthesis and regulation remains unknown. Aspects concerning chemical structures and biological activities of SAs have been reviewed extensively elsewhere; therefore, in this review the latest insights to the elucidation of the SAs biosynthetic pathway are highlighted. Recently, co-expression analysis combined with metabolic profiling revealed metabolic gene clusters in tomato and potato that contain core genes required for production of the prominent SGAs in these two species. Elaborating the knowledge regarding the SAs biosynthetic pathway, the subcellular transport of these molecules, as well as the identification of regulatory and signaling factors associated with SA metabolism will likely advance understanding of chemical defense mechanisms in Solanaceae and Liliaceae plants. It will also provide the means to develop, through classical breeding or genetic engineering, crops with modified levels of anti-nutritional SAs.

Purification of the major outer membrane protein of Azospirillum brasilense, its affinity to plant roots, and its involvement in cell aggregation.

The major outer membrane protein (MOMP) of the nitrogen-fixing rhizobacterium Azospirillum brasilense strain Cd was purified and isolated by gel filtration, and antiserum against this protein was obtained. A screening of the binding of outer membrane proteins (OMPs) of A. brasilense to membrane-immobilized root extracts of various plant species revealed different affinities for the MOMP, with a stronger adhesion to extracts of cereals in comparison with legumes and tomatoes. Moreover, this protein was shown to bind to roots of different cereal seedlings in an in vitro adhesion assay. Incubation of A. brasilense cells with MOMP-antiserum led to fast agglutination, indicating that the MOMP is a surface-exposed protein. Cells incubated with Fab fragments obtained from purified MOMP-antiserum immunoglobulin G exhibited significant inhibition of bacterial aggregation as compared with controls. Bacteria preincubated with Fab fragments showed weaker adhesion to corn roots in comparison to controls without Fab fragments. These findings suggest that the A. brasilense MOMP acts as an adhesin involved in root adsorption and cell aggregation of this bacterium.

Extracellular polysaccharide composition of Azospirillum brasilense and its relation with cell aggregation.

The exopolysaccharide (EPS) and capsular polysaccharide (CPS) composition of four Azospirillum brasilense strains differing in their aggregation capacity was analyzed by high performance anion exchange chromatography. When growing the different strains in an aggregation inducing medium containing a high carbon:nitrogen (C:N) ratio, both EPS and CPS showed a positive correlation between aggregation and the relative amount of arabinose. Arabinose was not detected in polysaccharides from Sp72002, a pleiotrophic Tn5 mutant strain impaired in aggregation. Arabinose was also not detected in extracellular polysaccharides of bacteria grown in a low C:N ratio, non-inducing aggregation medium, with exception for a relatively small amount found in the CPS of FAJ0204, a super-aggregating mutant strain. The only monosaccharides able to significantly inhibit aggregation at low sugar concentration when tested in a bioassay were arabinose (at a higher extent) and galactose. The possibility that residues of arabinose present in the extracellular polysaccharides are involved in the aggregation of A. brasilense is discussed.

Identification and characterization of the omaA gene encoding the major outer membrane protein of Azospirillum brasilense.

The major outer membrane protein (MOMP) of Azospirillum brasilense was purified and degenerate oligonucleotides were constructed on the basis of partial internal amino acid sequences. PCR products were obtained using total DNA of A. brasilense as template. One of these, a 766-bp fragment, was DIG-labelled and used in Southern hybridization against A. brasilense DNA and a genomic library of A. brasilense in Escherichia coli. A clone containing a 20-kb EcoRI insert in pLAFR3 was identified by PCR screening. From this insert, an EcoRI-SalI fragment of approximately 3.5-kb was subcloned in pUC19. The gene encoding the A. brasilense MOMP was sequenced and analyzed. The deduced amino acid sequence contains a putative signal peptide of 23 residues, followed by 367 amino acids of the mature protein with a molecular mass of 38,753 Da. The deduced amino acid sequence shows similarity to certain bacterial porins.

Antagonistic effects of the endophytic fungus Meira geulakonigii on the citrus rust mite Phyllocoptruta oleivora.

AIMS: The fungus Meira geulakonigii has been shown to reduce populations of citrus rust mite (CRM; Phyllocoptruta oleivora) on citrus leaves and fruits, in both the field and laboratory. However, attempts to isolate the fungus from leaves and fruits have been unsuccessful. The aims of this study were therefore to determine whether M. geulakonigii is a citrus endophyte, and to assess possible mechanisms involved in its mite-antagonist activity. METHODS AND RESULTS: A quantitative real-time PCR and regular PCR approaches were developed to detect M. geulakonigii in both the field and laboratory. The fungus was detected throughout. Different methods revealed that M. geulakonigii is an endophyte, which colonizes both the peel of grapefruits. Applications of conidia protected the grapefruits against CRM, and fungal secretions extracted from growth media caused 100% CRM mortality. CONCLUSIONS: Meira geulakonigii is a beneficial endophyte of grapefruits that colonizes the fruit's peel, and protects it from CRM. SIGNIFICANCE AND IMPACT OF THE STUDY: Findings from this study demonstrate the endophytic nature of M. geulakonigii in its interaction with grapefruits. In addition, a molecular approach was developed to specifically detect the fungus inside the grapefruit peel. This approach can be used to assess the natural occurrence of M. geulakonigii in grapefruit.

Surface characteristics of Azospirillum brasilense in relation to cell aggregation and attachment to plant roots.

The free-living bacteria of the genus Azospirillum live in close association with plant roots and represent one of the best-characterized plant growth promoting rhizobacteria (PGPR). The attachment of Azospirillum to the roots is essential for the establishment of an efficient association with the host plant. Azospirillum cells are able to aggregate under certain environmental conditions, leading to the formation of bacterial flocs. The bacterial surface plays an important role in the establishment of the bacteria-plant association as well as in the bacterial aggregation and data suggesting the involvement of extracellular polysaccharides and proteins in these phenomena have been published. This review summarizes the current knowledge on the involvement of surface components in the adhesion processes of Azospirillum. Emphasis is placed on A. brasilense, the species that has been the subject of most studies in the Azospirillum genus.

Promotion of nod Gene Inducers and Nodulation in Common Bean (Phaseolus vulgaris) Roots Inoculated with Azospirillum brasilense Cd.

Inoculation of Phaseolus vulgaris with Azospirillum brasilense Cd promoted root hair formation in seedling roots and significantly increased total and upper nodule numbers at different concentrations of Rhizobium inoculum. In experiments carried out in a hydroponic system, A. brasilense caused an increase in the secretion of nod gene-inducing flavonoids, as was observed by nod gene induction assays of root exudates fractionated by high-performance liquid chromatography. Possible mechanisms involved in the influence of A. brasilense on this symbiotic system are discussed.