A family of small intrinsically disordered proteins involved in flagella-dependent motility in Salmonella .

By screening a collection of Salmonella mutants deleted for genes encoding small proteins ≤60 amino acids, we identified three paralogous small genes ( ymdF , STM14_1829 , and yciG ) required for wild-type flagella-dependent swimming and swarming motility. The ymdF , STM14_1829, and yciG genes encode small proteins of 55, 60, and 60 amino-acid residues, respectively. Bioinformatics analysis predicted that these small proteins are intrinsically disordered proteins, and circular dichroism analysis of purified recombinant proteins confirmed that all three proteins are unstructured in solution. A mutant deleted for STM14_1829 showed the most severe motility defect, indicating that among the three paralogs, STM14_1829 is a key protein required for wild-type motility. We determined that relative to the wild type, the expression of the flagellin protein FliC is lower in the Δ STM14_1829 mutant due to downregulation of the flhDC operon encoding the FlhDC master regulator. By comparing the gene expression profiles between the wild-type and Δ STM14_1829 strains using RNA sequencing, we found that the gene encoding the response regulator PhoP is upregulated in the Δ STM14_1829 mutant, suggesting the indirect repression of the flhDC operon by the activated PhoP. Homologs of STM14_1829 are conserved in a wide range of bacteria including E. coli and P. aeruginosa We showed that inactivation of STM14_1829 homologs in E. coli and P. aeruginosa also alters motility, suggesting that this family of small intrinsically disordered proteins may play a role in the cellular pathway(s) that affect motility. IMPORTANCE This study reports the identification of a novel family of small intrinsically disordered proteins that are conserved in a wide range of flagellated and non-flagellated bacteria. Although this study identifies the role of these small proteins in the scope of flagella-dependent motility in Salmonella , they likely play larger roles in more conserved cellular pathway(s) that indirectly affect flagella expression in the case of motile bacteria. Small intrinsically disordered proteins have not been well characterized in prokaryotes, and the results of our study provide a basis for their detailed functional characterization.