Molecular Tools to Study Azospirillum sp. and Other Related Plant Growth Promoting Rhizobacteria

Abstract

Molecular methods have been used in the study of Azospirillum and other related PGPRs to carry out gene functional analysis, create gene knockouts, generate genetically engineered strains, and carry out gene expression studies. Genetic transformation has routinely been carried out using conjugation, while chromosomal modifications have been performed using unstable, suicide plasmids, or more stable, broad host-range vectors. Gene expression studies are often carried out using promoter-bound reporter genes; however, quantitative methods such as reverse transcribed polymerase chain reaction can now be used to directly study gene expression. In this chapter we describe the common types of vectors used in Azospirillum, as well as methods for transformation and mutagenesis. We also describe the use of promoter-bound reporter genes and the applications of quantitative RT-PCR for Azospirillum gene expression studies. Methods for the isolation of DNA and RNA from Azospirillum for use in molecular and gene expression studies are also described.