Construction and validation of a first-generation Bordetella bronchiseptica long-oligonucleotide microarray by transcriptional profiling the Bvg regulon

Abstract

Background: Bordetella bronchiseptica is a bacterial respiratory pathogen that infects a broad range of mammals, causing chronic and often subclinical infections. Gene expression in Bordetella is regulated by a two-component sensory transduction system, BvgAS, which controls the expression of a spectrum of phenotypic phases transitioning between a virulent (Bvg+) phase and a non-virulent (Bvg-) phase.

Results: Based on the genomic sequence and using the freely available software ArrayOligoSelector, a long oligonucleotide B. bronchiseptica microarray was designed and assembled. This long-oligonucleotide microarray was subsequently tested and validated by comparing changes in the global expression profiles between B. bronchiseptica RB50 and its Bvg- phase-locked derivative, RB54. Data from this microarray analysis revealed 1,668 Bvg-regulated genes, which greatly expands the BvgAS regulon defined in previous reports. For previously reported Bvg-regulated transcripts, the gene expression data presented here is congruent with prior findings. Additionally, quantitative real-time PCR data provided an independent verification of the microarray expression values.

Conclusion: The results presented here provide a comprehensive, genome-wide portrait of transcripts encompassing the BvgAS regulon, while also providing data validating the long-oligonucleotide microarray described here for studying gene expression in Bordetella bronchiseptica.

Figure 1

Expression of demonstrated Bvg-regulated genes. Hierarchical clustering of known B. bronchiseptica Bvg-regulated genes performed using MeV [32]. Expression profiles of genes are in rows. Data are mean centered for each array element and averaged from biological replicates. Red, indicates increased expression in RB50; green, decreased gene expression in RB50 (and increased expression in RB54); black, no significant change in gene expression.

Figure 2

Comparison of gene expression measurements by microarray hybridization and quantitative real-time PCR. Changes in gene expression were log transformed (in base 2), and the real-time qRT-PCR log₂ values (y axis) were plotted against the microarray data log₂ values (x axis). The coefficient of determination (R²) is given.