Evaluation of Reference Genes for Gene Expression Analysis Using Quantitative RT-PCR in 'Azospirillum brasilense'
'Azospirillum brasilense' is a nitrogen fixing bacterium that has been shown to have various beneficial effects on plant growth and yield. Under normal conditions 'A. brasilense' exists in a motile flagellated form, which, under starvation or stress conditions, can undergo differentiation into an encapsulated, cyst-like form. Quantitative RT-PCR can be used to analyse changes in gene expression during this differentiation process. The accuracy of quantification of mRNA levels by qRT-PCR relies on the normalisation of data against stably expressed reference genes. No suitable set of reference genes has yet been described for A. brasilense. Here we evaluated the expression of ten candidate reference genes ('16S' 'rRNA', 'gapB', 'glyA', 'gyrA', 'proC', 'pykA', 'recA', 'recF', 'rpoD', and 'tpiA') in wild-type and mutant 'A. brasilense' strains under different culture conditions, including conditions that induce differentiation. Analysis with the software programs BestKeeper, NormFinder and GeNorm indicated that 'gyrA', 'glyA' and 'recA' are the most stably expressed reference genes in 'A. brasilense'. The results also suggested that the use of two reference genes ('gyrA and glyA') is sufficient for effective normalisation of qRT-PCR data.
Cellular Responses during Morphological Transformation in 'Azospirillum brasilense' and Its flcA Knockout Mutant
FlcA is a response regulator controlling flocculation and the morphological transformation of 'Azospirillum' cells from vegetative to cyst-like forms. To understand the cellular responses of 'Azospirillum' to conditions that cause morphological transformation, proteins differentially expressed under flocculation conditions in 'A. brasilense' Sp7 and its flcA knockout mutant were investigated. Comparison of 2-DE protein profiles of wild-type (Sp7) and a flcA deletion mutant (Sp7-flcAΔ) revealed a total of 33 differentially expressed 2-DE gel spots, with 22 of these spots confidently separated to allow protein identification. Analysis of these spots by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and MASCOT database searching identified 48 proteins (≥10% emPAI in each spot). The functional characteristics of these proteins included carbon metabolism (beta-ketothiolase and citrate synthase), nitrogen metabolism (Glutamine synthetase and nitric oxide synthase), stress tolerance (superoxide dismutase, Alkyl hydroperoxidase and ATP-dependent Clp protease proteolytic subunit) and morphological transformation (transducer coupling protein). The observed differences between Sp7 wild-type and flcA⁻ strains enhance our understanding of the morphological transformation process and help to explain previous phenotypical observations. This work is a step forward in connecting the Azospirillum phenome and genome.
Molecular Tools to Study 'Azospirillum' sp. and Other Related Plant Growth Promoting Rhizobacteria
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 modifi cations 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.