RNA Expression Analysis Using an Antisense Bacillus subtilis Genome Array

Abstract

We have developed an antisense oligonucleotide microarray for the study of gene expression and regulation in Bacillus subtilis by using Affymetrix technology. Quality control tests of the B. subtilis GeneChip were performed to ascertain the quality of the array. These tests included optimization of the labeling and hybridization conditions, determination of the linear dynamic range of gene expression levels, and assessment of differential gene expression patterns of known vitamin biosynthetic genes. In minimal medium, we detected transcripts for approximately 70% of the known open reading frames (ORFs). In addition, we were able to monitor the transcript level of known biosynthetic genes regulated by riboflavin, biotin, or thiamine. Moreover, novel transcripts were also detected within intergenic regions and on the opposite coding strand of known ORFs. Several of these novel transcripts were subsequently correlated to new coding regions.Gene expression in bacteria has been traditionally analyzed by transcriptional or translational fusions to promoterless "reporter" genes (e.g., lacZ, cat, and gus) or by direct detection of transcripts using Northern blotting or reverse transcription-PCR (RT-PCR). With the completion of many bacterial genomes and the development of large-scale analysis tools such as DNA genomic arrays, however, researchers have increasingly applied genomics tools in their research. Measurements of mRNA levels using genome arrays for Escherichia coli, Bacillus subtilis, Streptococcus pneumoniae, and Haemophilus influenzae (10,11,12,17,23,24,29,32,38,39,41) have been found to offer many advantages to traditional gene-monitoring methods. Since the structure of bacterial genomes is relatively simple, containing ca. 4,000 genes and few repetitive sequences, DNA arrays can monitor transcript levels of an entire genome in a single hybridization with high sensitivity. This can lead to the elucidation of complex interactions among genetic networks, which then can be coupled with results from other newer technologies that analyze global protein synthesis (proteome) and metabolite levels (metabolome) to provide a comprehensive picture of the physiology of the bacterium (13,14,18,34,35,42).Using the public B. subtilis genome sequence (20), we developed an oligonucleotide B. subtilis genome microarray using Affymetrix GeneChip technology (21,40). This technology offers high sensitivity, high specificity, and excellent reproducibility (19). We show that the microarray can monitor gene expression changes in response to transition from the exponential to the stationary growth phases and exposure to three different vitamins that repress expression of biosynthetic genes. Moreover, we also present evidence that the microarray can be used to detect novel transcripts within intergenic regions and on the opposite strand of known genes, leading in some cases to the identification of previous unreported coding regions. MATERIALS AND METHODSMicroarray design. An "antisense" oligonucleotide array complementary to the Baci...

Riboswitches: the oldest mechanism for the regulation of gene expression?

“…However, two candidates for this role, RibC and RibR, which were identified genetically, were later shown to regulate the rib operon indirectly. Both proteins have flavokinase activity and thus decrease the concentration of flavin mononucleotide (FMN), which represses the rib operon [9][10][11]. The repression was shown to involve premature termination of transcription, generating a short transcript corresponding to the 5 0 -untranslated leader of the operon mRNA, and to be abolished by mutations in this region [11].…”

“…This is not an absolutely universal rule because it does not take into account cases of dual regulation (e.g. the ypaA genes from the Bacillus/Clostridium group, which are regulated both on the transcriptional level [35] and on the translational [14] level). This correlation can also be obscured by horizontal transfer, when a gene or an operon together with the regulating riboswitch is transferred between taxa.…”

“…Both proteins have flavokinase activity and thus decrease the concentration of flavin mononucleotide (FMN), which represses the rib operon [9][10][11]. The repression was shown to involve premature termination of transcription, generating a short transcript corresponding to the 5 0 -untranslated leader of the operon mRNA, and to be abolished by mutations in this region [11]. Sequences that could fold into a conserved RNA structure, called the RFN-element, were found upstream of several genes involved in riboflavin biosynthesis in a variety of bacterial genomes, and it was suggested that regulation by the RFN-element involved direct binding of FMN to mRNA [12].…”

Conservation of the Biotin Regulon and the BirA Regulatory Signal in Eubacteria and Archaea

“…4B,C). One of the bioY paralogs from B. subtilis, yhfU, belongs to the yhfUST operon, and transcription of this operon is repressed by BirA (Lee et al 2001). In addition, yhfU and yhfS-yhfT are clustered in the genomes of B. cereus, Lactococcus lactis, Clostridium difficile, and S. meliloti; whereas Streptococcus pyogenes, Streptococcus equi, and Staphylococcus aureus have separate BirAregulated yhfST and yhfU operons.…”

Functional Analysis of Intergenic Regions for Gene Discovery

“…In our previous studies, we found that some intergenic sequences in M. tuberculosis genome exhibited expression signals, as detected by the Affymetrix GeneChip (Fu 2006;Fu and FuLiu 2007;. The same observation has been made for other bacteria, such as Bacillus subtilis (Lee, Zhang et al 2001), and also holds true in the eukaryotic system (Zheng, Zhang et al 2005). At present, it is not clear whether or how intergenic expression represents gene activity.…”