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The function and mode of action of small regulatory RNAs is currently still understudied in archaea. In the halophilic archaeon Haloferax volcanii, a plethora of sRNAs have been identified; however, in-depth functional analysis is missing for most of them. We selected a small RNA (s479) from Haloferax volcanii for detailed characterization. The sRNA gene is encoded between a CRISPR RNA locus and the Cas protein gene cluster, and the s479 deletion strain is viable and was characterized in detail. Transcriptome studies of wild-type Haloferax cells and the deletion mutant revealed upregulation of six genes in the deletion strain, showing that this sRNA has a clearly defined function. Three of the six upregulated genes encode potential zinc transporter proteins (ZnuA1, ZnuB1, and ZnuC1) suggesting the involvement of s479 in the regulation of zinc transport. Upregulation of these genes in the deletion strain was confirmed by northern blot and proteome analyses. Furthermore, electrophoretic mobility shift assays demonstrate a direct interaction of s479 with the target znuC1 mRNA. Proteome comparison of wild-type and deletion strains further expanded the regulon of s479 deeply rooting this sRNA within the metabolism of H. volcanii especially the regulation of transporter abundance. Interestingly, s479 is not only encoded next to CRISPR–cas genes, but the mature s479 contains a crRNA-like 5′ handle, and experiments with Cas protein deletion strains indicate maturation by Cas6 and interaction with Cas proteins. Together, this might suggest that the CRISPR–Cas system is involved in s479 function.
Like eukaryotes, different bacterial species express one or more Ser/Thr kinases and phosphatases that operate in various signaling networks by catalyzing phosphorylation and dephosphorylation of proteins that can immediately regulate biochemical pathways by altering protein function. The human pathogen Streptococcus pneumoniae encodes a single Ser/Thr kinase-phosphatase couple known as StkP-PhpP, which has shown to be crucial in the regulation of cell wall synthesis and cell division. In this study, we applied proteomics to further understand the physiological role of pneumococcal PhpP and StkP with an emphasis on phosphorylation events on Ser and Thr residues. Therefore, the proteome of the non-encapsulated D39 strain (WT), a kinase (ΔstkP), and phosphatase mutant (ΔphpP) were compared in a mass spectrometry based label-free quantification experiment. Results show that a loss of function of PhpP causes an increased abundance of proteins in the phosphate uptake system Pst. Quantitative proteomic data demonstrated an effect of StkP and PhpP on the two-component systems ComDE, LiaRS, CiaRH, and VicRK. To obtain further information on the function, targets and target sites of PhpP and StkP we combined the advantages of phosphopeptide enrichment using titanium dioxide and spectral library based data evaluation for sensitive detection of changes in the phosphoproteome of the wild type and the mutant strains. According to the role of StkP in cell division we identified several proteins involved in cell wall synthesis and cell division that are apparently phosphorylated by StkP. Unlike StkP, the physiological function of the co-expressed PhpP is poorly understood. For the first time we were able to provide a list of previously unknown putative targets of PhpP. Under these new putative targets of PhpP are, among others, five proteins with direct involvement in cell division (DivIVA, GpsB) and peptidoglycan biosynthesis (MltG, MreC, MacP).