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High-resolution Mapping of Plasmid Transcriptomes in Different Host Bacteria

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High-resolution Mapping of Plasmid Transcriptomes in Different Host Bacteria

Masatoshi Miyakoshi et al. BMC Genomics.

Abstract

Background: Plasmids are extrachromosomal elements that replicate autonomously, and many can be transmitted between bacterial cells through conjugation. Although the transcription pattern of genes on a plasmid can be altered by a change in host background, the expression range of plasmid genes that will result in phenotypic variation has not been quantitatively investigated.

Results: Using a microarray with evenly tiled probes at a density of 9 bp, we mapped and quantified the transcripts of the carbazole catabolic plasmid pCAR1 in its original host Pseudomonas resinovorans CA10 and the transconjugant P. putida KT2440(pCAR1) during growth on either carbazole or succinate as the sole carbon source. We identified the operons in pCAR1, which consisted of nearly identical transcription units despite the difference in host background during growth on the same carbon source. In accordance with previous studies, the catabolic operons for carbazole degradation were upregulated during growth on carbazole in both hosts. However, our tiling array results also showed that several operons flanking the transfer gene cluster were transcribed at significantly higher levels in the transconjugant than in the original host. The number of transcripts and the positions of the transcription start sites agreed with our quantitative RT-PCR and primer extension results.

Conclusion: Our tiling array results indicate that the levels of transcription for the operons on a plasmid can vary by host background. High-resolution mapping using an unbiased tiling array is a valuable tool for the simultaneous identification and quantification of prokaryotic transcriptomes including polycistronic operons and non-coding RNAs.

Figures

Figure 1
Figure 1
High-resolution mapping of the pCAR1 transcriptome. Single-stranded cDNA from KT2440(pCAR1) grown on succinate (cyan) or carbazole (blue) and from CA10 grown on succinate (magenta) or carbazole (red) was mapped on both strands of pCAR1, which are transcribed from left to right (above) and from right to left (below). The identified transcripts and annotated ORFs are indicated by orange arrows and green rectangles, respectively. The scale bars indicate the signal intensities from 0 to 10,000 on a linear scale.
Figure 2
Figure 2
Scatterplots of the median signal intensities (log2) within a sense region and its antisense region. (A) KT2440(pCAR1) grown on succinate; r = 0.82 (n = 190). (B) KT2440(pCAR1) grown on carbazole; r = 0.79 (n = 190). (C) CA10 grown on succinate; r = 0.83 (n = 190). (D) CA10 grown on carbazole; r = 0.84 (n = 190). Red lines, y = x-2.
Figure 3
Figure 3
Histograms showing the log2 ratio of the signal intensities for the sense and antisense strands. The frequency of the signal intensity ratios for a total of 21,875 probe pairs was plotted. All four histograms have two peaks (at 2 and -2), indicating that most of the ratios were 4 or 0.25. (A) KT2440(pCAR1) grown on succinate. (B) KT2440(pCAR1) grown on carbazole. (C) CA10 grown on succinate. (D) CA10 grown on carbazole.
Figure 4
Figure 4
Comparison of the dynamic range between the tiling array and the expression array platforms. The plots represent the median signal intensities (log2) of 190 pCAR1 genes calculated from the tiling and the expression arrays. The correlation coefficients are for the signal intensities of the overall genes, highly transcribed genes (> 29 in the tiling array), and weakly transcribed genes (< 29).
Figure 5
Figure 5
High-resolution mapping of the catabolic operons induced during growth on carbazole, the ant operon and antR (A), and the car operon (B). Single-stranded cDNA from KT2440(pCAR1) grown on succinate (cyan) or carbazole (blue) and from CA10 grown on succinate (magenta) or carbazole (red) was mapped on both strands of pCAR1, which are transcribed from left to right (above) and from right to left (below). Pentagons represent the pCAR1 genes and their transcriptional directions; the gene names or ORF numbers are indicated therein. The scale bars indicate the signal intensities from 0 to 10,000 on a linear scale. pCAR1 carries duplicate carAa genes [9]; several probes within the first copy of carAa were deleted by the Affymetrix Custom Array Design Program.
Figure 6
Figure 6
Quantification of antA (A), antR (B), ORF9 (C), carF (D), ORF100 (E), and ORF145 (F) transcription. Quantitative RT-PCR was performed using single-stranded cDNA from KT2440(pCAR1) grown on succinate (cyan) or carbazole (blue) and from CA10 grown on succinate (magenta) or carbazole (red). The relative amount of each pCAR1 transcript to 16S rRNA (%) is indicated.
Figure 7
Figure 7
High-resolution mapping of the IncP-7 plasmid backbone, rep and par (A) and tra (B). Single-stranded cDNA from KT2440(pCAR1) grown on succinate (cyan) or carbazole (blue) and from CA10 grown on succinate (magenta) or carbazole (red) was mapped on both strands of pCAR1, which are transcribed from left to right (above) and from right to left (below). Pentagons represent the pCAR1 genes and their transcriptional directions; the gene names or ORF numbers are indicated therein. The scale bars indicate the signal intensities from 0 to 6,000 on a linear scale.
Figure 8
Figure 8
High-resolution mapping of ORF100-108 (A) and ORF145-146 (B), differentially transcribed operons between KT2440(pCAR1) and CA10. Single-stranded cDNA from KT2440(pCAR1) grown on succinate (cyan) or carbazole (blue) and from CA10 grown on succinate (magenta) or carbazole (red) was mapped on both strands of pCAR1, which are transcribed from left to right (above) and from right to left (below). Pentagons represent the pCAR1 genes and their transcriptional directions; the gene names or ORF numbers are indicated therein. The scale bars indicate the signal intensities from 0 to 10,000 on a linear scale.
Figure 9
Figure 9
Mapping of the transcription start sites for ORF100 (A) and ORF145 (B). Primer extension was performed using equal amounts of total RNA from KT2440(pCAR1) grown on succinate (lane 1) or carbazole (lane 2) and from CA10 grown on succinate (lane 3) or carbazole (lane 4). Lanes G, A, T, and C indicate the sequence ladder obtained using the same primer.

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