Abstract
Small proteins are an emerging class of gene products with diverse roles in bacterial physiology. However, a full understanding of their importance has been hampered by insufficient genome annotations and a lack of comprehensive characterization in microbes other than Escherichia coli. We have taken an integrative approach to accelerate the discovery of small proteins and their putative virulence-associated functions in Salmonella Typhimurium. We merged the annotated small proteome of Salmonella with new small proteins predicted with in silico and experimental approaches. We then exploited existing and newly generated global datasets that provide information on small open reading frame expression during infection of epithelial cells (dual RNA-seq), contribution to bacterial fitness inside macrophages (Transposon-directed insertion sequencing), and potential engagement in molecular interactions (Grad-seq). This integrative approach suggested a new role for the small protein MgrB beyond its known function in regulating PhoQ. We demonstrate a virulence and motility defect of a Salmonella ΔmgrB mutant and reveal an effect of MgrB in regulating the Salmonella transcriptome and proteome under infection-relevant conditions. Our study highlights the power of interpreting available 'omics' datasets with a focus on small proteins, and may serve as a blueprint for a data integration-based survey of small proteins in diverse bacteria.
Highlights
Small proteins, loosely defined as shorter than 100 amino acids, are being increasingly implicated in regulating major biological processes in all kingdoms of life (Storz, Wolf and Ramamurthi 2014; Saghatelian and Couso 2015; Makarewich and Olson 2017)
We report a requirement of MgrB for efficient infection of both epithelial cells and macrophages and describe the impact of this conserved small protein on the Salmonella transcriptome and proteome in infection-relevant conditions
To further expand the small open reading frames (sORFs) annotation in Salmonella, we searched for previously overlooked small protein candidates by combining computational sORF predictions with experimental data
Summary
Loosely defined as shorter than 100 amino acids (aa), are being increasingly implicated in regulating major biological processes in all kingdoms of life (Storz, Wolf and Ramamurthi 2014; Saghatelian and Couso 2015; Makarewich and Olson 2017). Several small proteins have long been known to perform both structural and regulatory functions in ribosomal subunits. Another major class is that of small protein members of toxin-antitoxin systems, type-II, where both the toxin and the antitoxin are proteins (Harms et al 2018). Starting with the discovery almost two decades ago of previously overlooked conserved small open reading frames (sORFs) in the Escherichia coli chromosome (Wassarman et al 2001), interest in other potential roles of bacterial small proteins has been increasing (Storz, Wolf and Ramamurthi 2014; Miravet-Verde et al 2019; Sberro et al 2019). A clear picture of how many bona fide, translated sORFs are encoded even by otherwise well-studied model bacteria is currently lacking
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