A Transcriptome Map of <i>Actinobacillus pleuropneumoniae</i> at Single-Nucleotide Resolution Using Deep RNA-Seq

<div><p><i>Actinobacillus pleuropneumoniae</i> is the pathogen of porcine contagious pleuropneumoniae, a highly contagious respiratory disease of swine. Although the genome of <i>A</i>. <i>pleuropneumoniae</i> was sequenced several years ago, limited information is available on the genome-wide transcriptional analysis to accurately annotate the gene structures and regulatory elements. High-throughput RNA sequencing (RNA-seq) has been applied to study the transcriptional landscape of bacteria, which can efficiently and accurately identify gene expression regions and unknown transcriptional units, especially small non-coding RNAs (sRNAs), UTRs and regulatory regions. The aim of this study is to comprehensively analyze the transcriptome of <i>A</i>. <i>pleuropneumoniae</i> by RNA-seq in order to improve the existing genome annotation and promote our understanding of <i>A</i>. <i>pleuropneumoniae</i> gene structures and RNA-based regulation. In this study, we utilized RNA-seq to construct a single nucleotide resolution transcriptome map of <i>A</i>. <i>pleuropneumoniae</i>. More than 3.8 million high-quality reads (average length ~90 bp) from a cDNA library were generated and aligned to the reference genome. We identified 32 open reading frames encoding novel proteins that were mis-annotated in the previous genome annotations. The start sites for 35 genes based on the current genome annotation were corrected. Furthermore, 51 sRNAs in the <i>A</i>. <i>pleuropneumoniae</i> genome were discovered, of which 40 sRNAs were never reported in previous studies. The transcriptome map also enabled visualization of 5'- and 3'-UTR regions, in which contained 11 sRNAs. In addition, 351 operons covering 1230 genes throughout the whole genome were identified. The RNA-Seq based transcriptome map validated annotated genes and corrected annotations of open reading frames in the genome, and led to the identification of many functional elements (e.g. regions encoding novel proteins, non-coding sRNAs and operon structures). The transcriptional units described in this study provide a foundation for future studies concerning the gene functions and the transcriptional regulatory architectures of this pathogen.</p></div

sRNAs identified in <i>A</i>. <i>pleuropneumoniae</i> JL03.

<p>sRNAs identified in <i>A</i>. <i>pleuropneumoniae</i> JL03.</p

Corrections made to the existing genome annotation.

<p>Corrections made to the existing genome annotation.</p

Novel protein coding genes identified in the <i>A</i>. <i>pleuropneumoniae</i> JL03 genome.

<p>Novel protein coding genes identified in the <i>A</i>. <i>pleuropneumoniae</i> JL03 genome.</p

Single-nucleotide resolution transcriptome map revealing the operon structures of <i>A</i>. <i>pleuropneumoniae</i> JL03.

<p>The operons predicted by RNA-seq and the operons predicted by DOOR were compared. (A) An operon common to both DOOR and RNA-Seq. (B) An operon identified based on RNA-seq data but not previously predicted by DOOR. (C) An operon identified based on RNA-seq data having different sizes with the one predicted by DOOR.</p

Correction made to the start site of a gene.

<p>Visualization of the single-nucleotide resolution transcriptome map shows the transcription of upstream of the gene ‘‘APJL_1947”. The predicted and actual start codons within the ORF are marked.</p

Clusters of orthologous groups (COG) classification of the expressed <i>A</i>. <i>pleuropneumoniae</i> genes.

<p>Bars in dark gray indicate numbers of expressed genes (n = 1845); while bars in white indicate numbers of all protein-coding genes (n = 2,036) in <i>A</i>. <i>pleuropneumoniae</i> JL03. COG categories: J, translation; A, RNA processing and modification; K, transcription; L, DNA replication, recombination and repair; D, cell division and chromosome partitioning; V, defense mechanisms; T, signal transduction; M, cell wall/membrane biogenesis; U, intracellular trafficking, secretion and vesicular transport; O, posttranslational modification, protein turnover and chaperones; C, energy production and conversion; G, carbohydrate transport and metabolism; E, amino acid transport and metabolism; F, nucleotide transport and metabolism; H, coenzyme metabolism; I, lipid metabolism; P, inorganic ion transport and metabolism; Q, secondary metabolites biosynthesis, transport and catabolism; R, general functional prediction only; S, function-unassigned; -, unknown proteins not in the COG categories.</p

Identification of a novel protein coding gene within the single-nucleotide resolution transcriptome map of <i>A</i>. <i>pleuropneumoniae</i>.

<p>Genomic loci and transcriptional profiles were visualized in the Artemis genome browser. The figure shows identification of a novel protein coding gene ‘‘APP-P04” using RNA-seq data. A BLASTX search of the ORF shows the homology (similarity 100%, sequence coverage 100%) to a hypothetical protein in <i>A</i>. <i>pleuropneumoniae</i>.</p

The effect of transferrin on <i>A</i>. <i>pleuropneumoniae</i> growth in CDM).

<p><i>A</i>. <i>pleuropneumoniae</i> was cultured in TSB medium overnight and then sub-cultured into CDM at an inoculation dose of 10⁴ CFU/ml. Optical densities of bacterial cultures (OD_600nm) were recorded at selected time points. Various concentrations of apo-transferrin (ATF) <b>(A)</b> or holo-transferrin (HTF) <b>(B)</b> were added into CDM. <b>(C)</b>. ATF at 400μg/ml was added into CDM (ATF) and FeCl₃ at different concentrations were supplemented into the medium containing ATF (ATF + Fe). <i>A</i>. <i>pleuropneumoniae</i> cultured in CDM without any supplementation was used as a control (CDM). Data are shown as means ± SD from four independent replications.</p

The effects of catecholamines on growth of <i>A</i>. <i>pleuropneumoniae ΔtonB1</i> and <i>ΔtonB2</i> mutants.

<p><i>A</i>. <i>pleuropneumoniae</i> was cultured in TSB medium overnight and then sub-cultured into CDM using an inoculation dose of 10⁴ CFU/ml. <i>A</i>. <i>pleuropneumoniae</i> 4074 and the mutants were sub-cultured in CDM containing 50μM of Epi and 1/40 of serum (S in figure A) or 400μg/ml ATF (figure B). Optical densities of bacterial cultures (OD_600nm) were recorded at selected time points. Data are shown as means ± SD from four independent replications.</p