ABSTRACT: Background: Xylella fastidiosa, a Gram-negative fastidious bacterium, grows exclusively in the xylem of several plants, causing diseases such as citrus variegated chlorosis. As the xylem sap contains low concentrations of amino acids and other compounds, X. fastidiosa needs to cope with nitrogen limitation in its natural habitat. Results: In this work, we performed a whole-genome microarray analysis of the X. fastidiosa nitrogen starvation response. A time-course experiment (2, 8 and 12 hours) revealed many differentially expressed genes under nitrogen starvation, such as genes related to transport, nitrogen assimilation, amino acid biosynthesis, transcriptional regulation, and many genes encoding hypothetical proteins. In addition, a decrease in the expression levels of many genes involved in carbon metabolism and energy generation pathways was also observed. Comparison of gene expression profiles between the wild type strain and the rpoN null mutant allowed the identification of genes induced by nitrogen starvation in a ?54-dependent manner. A more complete picture of the ?54 regulon was achieved by combining the transcriptome data with an in silico search for potential ?54-dependent promoters, using a position weight matrix approach. One of these ?54-predicted binding sites, located upstream of the glnA gene (encoding a glutamine synthetase), was validated by primer extension assays, confirming that this gene has a ?54-dependent promoter and contains a predicted NtrC binding site. Conclusions: Together, these results show that nitrogen starvation causes intense changes in the X. fastidiosa transcriptome and some of these differentially expressed genes belong to the ?54 regulon. For time-course studies, cells cultivated at late-exponential phase in PWG medium were used to inoculate a culture in 100 ml XDM2 medium to an optical density at 600 nm (OD600 nm) of 0.1. Cells were grown during 12 days in the XDM2 medium (mid-log phase) and harvested by centrifugation. Then, the culture was divided into two 6 portions and cells were washed with XDM2 medium (zero time) or XDM2 medium lacking all nitrogen sources (XDM0), respectively. The cultivation was continued for 2h, 8h and 12h in XDM0 to establish nitrogen starvation conditions. For each time point, cells in a 25-ml culture were collected by centrifugation and rapidly frozen in dry ice, until RNA isolation. Three RNA samples isolated from independently grown cultures of the cells at each starvation period (2h, 8h and 12h) were examined, and each preparation was subjected to microarray analysis. As the genes were spotted at least in duplicate, we obtained six replicates for each gene from three independent data sets per gene per starvation period. Comparison of gene expression profiles between the wild type strain and the rpoN null mutant allowed the identification of genes induced by nitrogen starvation in a ?54-dependent manner. To determine the effect of rpoN inactivation on gene expression after nitrogen starvation, the transcriptomes of the wild type and the rpoN strains were compared using DNA microarrays, with both strains grown on XDM2 medium and submitted to nitrogen starvation (XDM0) during 2 hours. Three RNA samples isolated from independently grown cultures of the cells were examined. Due to the platform design, each microarray slide was divided into "LEFT" and "RIGHT", allowing the probing of two technical replicates per slide.
