A global data-driven census ofSalmonellasmall proteins and their potential functions in bacterial virulence

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 (TraDIS), 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.

The small DUF1127 protein CcaF1 from Rhodobacter sphaeroides is an RNA-binding protein involved in sRNA maturation and RNA turnover

“…In Brucella abortus deletion of the gene for a DUF1127 protein caused a defect in fucose metabolism ( 50 ). Recently a role of DUF1127 proteins in phosphate and carbon metabolism in Agrobacterium tumefaciens was demonstrated ( 51 ), as well as a role of the Salmonella protein YjiS in virulence ( 52 ). YjiS is a DUF1127 protein with 20% identity to CcaF1.…”

RiboReport - Benchmarking tools for ribosome profiling-based identification of open reading frames in bacteria

“…In order to evaluate the ORF detection tools, we used part of a pre-release version of our HRIBO (High-throughput annotation by Ribo-seq) workflow [16], which we have developed to analyse prokaryotic ribosome profiling experiments [51]. The genomes and annotations of E. coli K-12 substr.…”

“…Moreover, the limited information content of small proteins makes them challenging to predict using in silico alignment- and sequence-based tools. Small ORFs are likely under-represented in genome annotations [21,38], despite emerging evidence that they play central roles in diverse physiological processes in bacteria, including those underlying virulence [21, 51].…”

The World of Stable Ribonucleoproteins and Its Mapping With Grad-Seq and Related Approaches

“…This can be achieved either by the identification of the molecule in question (by MS or western blotting) or by demonstrating the involvement of the corresponding mRNA in translation ( Makarewich and Olson, 2017 ). The former approach has been recently used to support the existence of 170 small proteins, including 89 uncharacterized ones, in a S. Typhimurium Grad-seq dataset ( Venturini et al, 2020 ). Their detection has become possible thanks to a higher sensitivity offered by Grad-seq protein profiling which, by fractionating the cell lysate, considerably decreases the complexity of analyzed samples.…”

“…To date, Grad-seq has been used to profile stable RNPs in several model bacteria, such as the γ-proteobacteria S. Typhimurium (Smirnov et al, 2016;Gerovac et al, 2020;Venturini et al, 2020), Escherichia coli (Hör et al, 2020a), and Pseudomonas aeruginosa (Gerovac et al, 2021), the firmicute Streptococcus pneumoniae (Hör et al, 2020b), the cyanobacterium Synechocystis sp. (Riediger et al, 2020), and even in human cells (Aznaourova et al, 2020).…”