The Herbaspirillum genus belongs to subdivision b of the Proteobacteria. These diazotrophic endophytic microrganisms are known to associate with many important agricultural crops, such as maize (Zea mays), rice (Oryza sativa), sorgum (Sorghum bicolor), and wheat (Triticum aestivum), as well as sugar cane (Saccharum officinarum), palm trees, banana and pineapple plants.
The association of H. seropedicae with graminaceous crops apparently initiates with attachment of the bacteria to root surfaces, followed by colonization of the emergence points of secondary roots and penetration through discontinuities of the epidermis. Rapid colonization and occupation of root intercellular spaces then occurs, together with aerenquima, root xylem and aerial portions.
The Herbaspirillum-plant interaction is not yet fully understood. However, it is presumed that bacterial surface components must be involved in early stages of the colonization process. Lipopolysaccharides (LPS) are the main constituent of the cell envelope of bacteria and rhamnose is a monosaccharide frequently found as an integral part of LPS. The biosynthetic pathway of rhamnose requires the product of four genes: rmlA, which codes for glucose-1-phosphate thymidylyltransferase, rmlB (dTDP-D-glucose 4,6-dehydratase), rmlC (dTDP-4keto-6deoxy-D-glucose 3,5-epimerase) and rmlD (dTDP-4keto-L-rhamnose reductase). The genes rmlB and rmlC were found in the H. seropedicae genome and site-directed mutant strains were constructed to determine their role in LPS synthesis.
Plasmids pRAMEBB and pRAMEBC containing, respectively, the rmlB and rmlC genes were disrupted by a gene cassette that confers resistance to tetracycline (Tc). These constructs were transformed by electroporation into the H. seropedicae wild strain. Tetracycline-resistant recombinants were selected and insertion of the Tc cassette in the genome was confirmed by Southern blot.
Maize root attachment experiments showed significant differences in behavior between wild type and mutant strains. SDS-PAGE analyses showed that the mutant strains contained quantitatively and qualitatively different LPS products from each other and from the wild type H. seropedicae. Whether these changes affect plant colonization remains to be determined.
Supported by CNPq/PIBIC, CAPES and Fundação Araucária.