Unraveling the Transcriptional Landscape within a Minimized Bacterium via Comparative Analysis

Brier, T. A.; Cournoyer, J. E.; Gilbert, B. R.; Glass, S. A.; Gao, Y.-l.; Yu, Y.; Thornburg, Z. R.; Goglin, K.; John, G.; Mamaghani, T.; Shivakumar, S.; Fields, C. J.; Glass, J. I.; Mehta, A. P.; Luthey-Schulten, Z.

doi:10.1101/2025.10.23.681674

bioRxiv · 10.1101/2025.10.23.681674

Unraveling the Transcriptional Landscape within a Minimized Bacterium via Comparative Analysis

Brier, T. A.·Cournoyer, J. E.·Gilbert, B. R.·Glass, S. A.·Gao, Y.-l.·Yu, Y.·Thornburg, Z. R.·Goglin, K.·John, G.·Mamaghani, T.·Shivakumar, S.·Fields, C. J.·Glass, J. I.·Mehta, A. P.·Luthey-Schulten, Z.

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Abstract

Stochastic nature of gene expression leads to the complex formation of the bacterial transcriptome and proteome. In contrast to typical transcriptome studies, we employ a near wild-type, Syn1.0, of the naturally genome-reduced Mycoplasmas, and the dramatically further genome-reduced JCVI-syn3A thus avoiding additional contributions from many non-essential cellular functions. To aid in profiling the transcriptional landscape within these bacteria, we present a bioinformatic analysis of the genetic sequence motifs implicated in modulating the stochastic gene expression events, coupled with genome-wide short-read (Illumina) and long-read (Oxford Nanopore Technologies and Pacfic Biosciences) RNA sequencing. The bioinformatic analysis coupled with information from structural studies assigns strengths of the Shine-Dalgarno signatures and identifies both transcription initiation and termination sites, leading to predictions of RNA isoforms in Syn1.0 (and related organisms). The long-read and short-read RNA sequencing characterized the predicted transcriptional activity, and the long-read methods provide direct insight into the RNA isoform complexity within Syn1.0. Comparison of the RNA sequencing results with that of the bioinformatic analysis highlights the inability of bioinformatics alone to capture the results of bacterial transcription without including effects of RNA degradation. This study emphasizes the need for comparative analysis and potential dangers of genome reduction, exemplified through the discovery of altered gene expression patterns of JCVI-syn1.0 and JCVI-syn3A, achieved via the union of our transcriptome study with their proteomics data. Analysis of the transcriptomics data sets through a Jupyter notebook allows any genomic region to be easily examined. Table of Content Image O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/681674v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@78a9e0org.highwire.dtl.DTLVardef@1d8c9fcorg.highwire.dtl.DTLVardef@1b4e129org.highwire.dtl.DTLVardef@2a8c3f_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Brier, T. A., Cournoyer, J. E., Gilbert, B. R., Glass, S. A., Gao, Y.-l., Yu, Y., Thornburg, Z. R., Goglin, K., John, G., Mamaghani, T., Shivakumar, S., Fields, C. J., Glass, J. I., Mehta, A. P., Luthey-Schulten, Z.. 2025-10-23. Unraveling the Transcriptional Landscape within a Minimized Bacterium via Comparative Analysis. https://doi.org/10.1101/2025.10.23.681674

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