Customized cDNA microarray for expression profiling of environmentally important genes of Pseudomonas stutzeri strain KC.

Abstract

DNA microarray is a powerful tool for parallel detection of multiple target genes in biological systems. In this study, a low-density DNA microarray has been custom designed by using Pseudomonas stutzeri strain KC ORFs that are implicated in carbon tetrachloride degradation. PCR amplified strain KC probes of varying lengths were obtained using ORF-specific primers. Purified short probes (80-120 bp) and full-length amplicons were directly immobilized on gamma-aminosilane coated and superaldehyde trade mark glass substrates without any chemical modification. The full-length amplicons exhibited a much higher signal compared to the shorter probes upon hybridization with the Cy5/Cy3-labeled unfragmented cDNA targets. The meager signal with the shorter probes limits the advantage of using the multiple probes of the same genes for enhancing the specificity of hybridization with environmental samples. Nevertheless, expression analysis of strain KC genome, under controlled laboratory conditions, revealed the constitutive expression of at least 11 putative ORFs of the pdt operon. Comparatively weaker hybridization signals with the cDNA from mutant cells suggested a low abundance of mRNA transcripts in the KC 1896 mutant. Similar expression levels of the pdt ORFs I, J, K, M, N, O, P, and fur gene both under iron-limiting conditions and in presence of iron (20 micro M Fe(3+)) suggested metal ion-independent regulation of the pdt operon. The tailor-made array with strain KC gene-specific probes served as a model for demonstrating the utility of cDNA microarray technology in monitoring the expression of environmentally important genes in bacteria.