Identification of novel non-coding small RNAs from Streptococcus pneumoniae TIGR4 using high-resolution genome tiling arrays

Abstract

Background: The identification of non-coding transcripts in human, mouse, and Escherichia coli has revealed their widespread occurrence and functional importance in both eukaryotic and prokaryotic life. In prokaryotes, studies have shown that non-coding transcripts participate in a broad range of cellular functions like gene regulation, stress and virulence. However, very little is known about non-coding transcripts in Streptococcus pneumoniae (pneumococcus), an obligate human respiratory pathogen responsible for significant worldwide morbidity and mortality. Tiling microarrays enable genome wide mRNA profiling as well as identification of novel transcripts at a high-resolution.

Results: Here, we describe a high-resolution transcription map of the S. pneumoniae clinical isolate TIGR4 using genomic tiling arrays. Our results indicate that approximately 66% of the genome is expressed under our experimental conditions. We identified a total of 50 non-coding small RNAs (sRNAs) from the intergenic regions, of which 36 had no predicted function. Half of the identified sRNA sequences were found to be unique to S. pneumoniae genome. We identified eight overrepresented sequence motifs among sRNA sequences that correspond to sRNAs in different functional categories. Tiling arrays also identified approximately 202 operon structures in the genome.

Conclusions: In summary, the pneumococcal operon structures and novel sRNAs identified in this study enhance our understanding of the complexity and extent of the pneumococcal 'expressed' genome. Furthermore, the results of this study open up new avenues of research for understanding the complex RNA regulatory network governing S. pneumoniae physiology and virulence.

Figure 1

Tiling array data analysis workflow. Analysis workflow includes sRNA identification, compilation of sRNA additional features as well as operon identification in S. pneumoniae TIGR4.

Figure 2

A S. pneumoniae TIGR4 sRNA SN1 visualized in the genome browser. The sRNA and the additional features are shown as different tracks in the genome browser. sRNA track (in blue) shows the presence of small RNA SN1. Tiling array expression track indicates the higher level of expression in the sRNA SN1 region (located in-between genes SP0019 and SP0020) relative to the intensity threshold cutoff (11.0). Rho-independent terminator track shows a predicted terminator near the 3' end of sRNA. B. Circular representation of S. pneumoniae TIGR4 genome depicting open reading frames and sRNAs. The outermost track (solid black circle, track one) is TIGR4 genome. With reference to track one, moving inward, tracks two and three represent sRNAs in the forward and reverse strand respectively. Tracks four and five (gray) shows the presence of genes on the forward and reverse strand respectively. Track six is the GC plot and the seventh (innermost track) shows the GC skew of the genome.

Figure 3

Sequence motifs identified in sRNAs by MEME. Overrepresented sequence motifs among non-aligned sRNA sequences were identified by MEME. Rfam annotation for sRNAs are shown where available.

Figure 4

A S. pneumoniae TIGR4 genes expressed in different TIGR protein families (TIGRFAMs). The gene expression is shown as a percentage of the total number of genes present in TIGR4 genome in a particular TIGRFAM category. B. Genome browser visualization of S10, a 15 gene operon (SP0208 - SP0222). Track two shows the DNA sequence translation in six frames and track one shows the genes. The color of the expressed genes is in accordance with the six frame translation. S10 operonic genes SP0208 - SP0222 are present in the forward strand. The "tiling array expression" track clearly demonstrates that all genes predicted in S10 operon are expressed at similar level and this expression is higher than the intensity threshold for expression (11.0).