Role of small proteins in the stress response
Role of small proteins in the stress response of Alphaproteobacteria
For many years little attention was given to small open reading frames in bacterial genomes. Recently, it emerged that many of these ORFs encode small proteins (< 100 amino acids) with important cellular functions. In the frame of the DFG priority program SPP2002: "Small Proteins in Prokaryotes: an Unexplored World" we will elucidate the role of small proteins in the stress response of Alphaproteobacteria.
The project will initially focus on the small DUF1127 (domain of unknown function) proteins, which are found in many Alpha- and Gammaproteobacteria and are structurally related to domains from eukaryotic RNA binding proteins.
The DUF1127 domain comprises 40 amino acids (aa), which either represent almost a complete protein of 45-50 aa or the C-terminal domain of proteins with 60-75 aa, in rare cases up to 100 aa. We will first concentrate on the elucidation of the function of the two DUF1127 proteins RSP_6037 and RSP_0557 in the facultative photosynthetic bacterium Rhodobacter sphaeroides, for which preliminary data are available. RSP_6037 is co-transcribed with 4 sRNAs (CcsR1-4) with a function in singlet oxygen and other stress responses. DUF1127 protein genes often co-localize with genes for CcsR homologs in genomes from Alphaproteobacteria (Billenkamp et al., 2015).
Fig. 1: The RSP_6037 gene for a DUF1127 protein is co-transcribed with the genes for the CcsR 1-4 sRNAs in R. sphaeroides 2.4.1. (modified from Berghoff et al., 2009)
In R. sphaeroides the CcsR sRNAs are generated by RNase E-mediated processing from the primary transcript and the RSP_6037 protein strongly affects their level.
Fig. 2: Effect of RSP_6037 on CcsR1-4 sRNA levels in strains harboring different plasmids. The probe used for Northern blot preferentially detects CcsR1. 5S RNA was probed as a loading control.
There is experimental evidence that the CcsR and CcaF orthologs also function in stress responses in other Alphaproteobacteria. CcsR sRNAs in two R. capsulatus strains (Type 1) and in Sinorhizobium meliloti (Type 3) are also induced by singlet oxygen and heat. However, CcsR sRNAs in S. fredii (Type 3) are only induced by heat and CcsRs from Paracoccus denitrificans (Type 1) do not respond to singlet oxygen or heat (Doktorarbeit Billenkamp, 2015).
Overexpression of CcsR1-4 alone resulted in increased resistance to paraquat (generates superoxide) and tertiary butylhydroperoxide. Co-expression of RSP_6037 together with CcsR1-4 abolished this effect: resistances were similar to that of the control strain (Billenkamp et al., 2015). When the ATG start codon of RSP_6037 was replaced by TGA, overexpression of CcsR1-4 together with the mutated RSP_6037 revealed similar resistance levels as overexpression of CcsR1-4 alone ( Doktorarbeit Fabian Billenkamp, Universität Gießen, 2015). These data hint to an inhibiting effect of RSP_6037 on CcsR levels and consequently stress resistance, which depends on the presence of the protein.
The second DUF1127 protein, RSP_0577, is also induced by several stresses (Müller et al., 2016), plays a role in stress defense and has a moderate effect on the levels of the CcsR sRNAs. While RSP_6037 is essential, RSP_0557 is not essential and not part of an operon. There is evidence that the DUF1127 proteins are RNA binding proteins, which may affect RNA processing.
References:
Grützner J, Billenkamp F, Spanka DT, Rick T, Monzon V, Förstner KU, Klug G. The small DUF1127 protein CcaF1 from Rhodobacter sphaeroides is an RNA-binding protein involved in sRNA maturation and RNA turnover. Nucleic Acids Res. 2021 Mar 12:gkab146. doi: 10.1093/nar/gkab146
Kubatova N, Pyper DJ, Jonker HRA, Saxena K, Remmel L, Richter C, Brantl S, Evguenieva-Hackenberg E, Hess WR, Klug G, Marchfelder A, Soppa J, Streit W, Mayzel M, Orekhov VY, Fuxreiter M, Schmitz RA, Schwalbe H. Rapid Biophysical Characterization and NMR Spectroscopy Structural Analysis of Small Proteins from Bacteria and Archaea. Chembiochem. 2019 Nov 9. doi: 10.1002/cbic.201900677.
Müller KM, Berghoff BA, Eisenhardt BD, Remes B, Klug G. Characteristics of Pos19 - A Small Coding RNA in the Oxidative Stress Response of Rhodobacter sphaeroides. PLoS One. 2016 Sep 26;11(9):e0163425. doi: 10.1371/journal.pone.0163425. eCollection 2016.
Billenkamp, F., Peng, T., Berghoff, B.A., Klug, G. (2015) A cluster of four homologous small RNAs modulates C1 metabolism and the pyruvate dehydrogenase complex in Rhodobacter sphaeroides under various stress conditions. J. Bacteriol. 197: 1839-1852.