Open Access

Hypoxia is common in marine environments and a major stressor for marine organisms inhabiting benthic and intertidal zones. Several studies have explored the responses of these organisms to hypoxic stress at the whole organism level with a focus on energy metabolism and mitochondrial response, but the instrinsic mitochondrial responses that support the organelle’s function under hypoxia and reoxygenation (H/R) stress are not well understood. We studied the effects of acute H/R stress (10 min anoxia followed by 15 min reoxygenation) on mitochondrial respiration, production of reactive oxygen species (ROS) and posttranslational modifications (PTM) of the proteome in a marine facultative anaerobe, the blue mussel Mytilus edulis. The mussels’ mitochondria showed increased OXPHOS respiration and suppressed proton leak resulting in a higher coupling efficiency after H/R stress. ROS production decreased in both the resting (LEAK) and phosphorylating (OXPHOS) state indicating that M. edulis was able to prevent oxidative stress and mitochondrial damage during reoxygenation. Hypoxia did not lead to rearrangement of the mitochondrial supercomplexes but impacted the mitochondrial phosphoproteome including the proteins involved in OXPHOS, amino acid- and fatty acid catabolism, and protein quality control. This study indicates that mussels’ mitochondria possess intrinsic mechanisms (including regulation via reversible protein phosphorylation) that ensure high respiratory flux and mitigate oxidative damage during H/R stress and contribute to the hypoxia-tolerant mitochondrial phenotype of this metabolically plastic species.

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).

Streptococcus pneumoniae is a commensal of the human upper respiratory tract and moreover, the causative agent of several life-threatening diseases including pneumonia, sepsis, otitis media, and meningitis. Due to the worldwide rise of resistance to antibiotics in pneumococci the understanding of its physiology is of increasing importance. In this context, the analysis of the pneumococcal proteome is helpful as comprehensive data on protein abundances in S. pneumoniae may provide an extensive source of information to facilitate the development of new vaccines and drug treatments. It is known that protein phosphorylation on serine, threonine and tyrosine residues is a major regulatory post-translational modification in pathogenic bacteria. This reversible post-translational modification enables the translation of extracellular signals into cellular responses and therewith adaptation to a steadily changing environment. Consequently, it is of particular interest to gather precise information about the phosphoproteome of pneumococci. S. pneumoniae encodes a single Serine/Threonine kinase-phosphatase couple known as StkP-PhpP. To address the global impact and physiological importance of StkP and PhpP which are closely linked to the regulation of cell morphology, growth and cell division in S. pneumoniae, proteomics with an emphasis on phosphorylation and dephosphorylation events on Ser and Thr residues was applied. Thus, the non-encapsulated pneumococcal D39Δcps strain (WT), a kinase (ΔstkP) and phosphatase mutant (ΔphpP) were analyzed in in a mass spectrometry based label-free quantification experiment. The global proteome analysis of the mutants deficient for stkP or phpP already proved the essential role of StkP-PhpP in the protein regulation of the pneumococcus. Proteins with significantly altered abundances were detected in diverse functional groups in both mutants. Noticeable changes in the proteome of the stkP deletion mutant were observed in metabolic processes such as “Amino acid metabolism” and also in pathways regulating genetic and environmental information processing like “Transcription” and “Signal transduction”. Prominent changes in the metabolism of DNA, nucleotides, carbohydrates, cofactors and vitamins as well as in the categories “Transport and binding proteins” and “Glycan biosynthesis and metabolism” have been additionally detected in the proteome of the phosphatase mutant. Still, the quantitative comparison of WT and mutants revealed more significantly altered proteins in ΔphpP than in ΔstkP. Moreover, the results indicated that the loss of function of PhpP causes an increased abundance of proteins in the pneumococcal phosphate uptake system Pst. Furthermore, the obtained quantitative proteomic data revealed an influence of StkP and PhpP on the twocomponent systems ComDE, LiaRS, CiaRH, and VicRK. Recent studies of the pneumococcal StkP/PhpP couple demonstrated that both proteins play an essential role in cell growth, cell division and separation. Growth analyses and the phenotypic characterization of the mutants by electron-microscopy performed within this work pointed out that ΔphpP and ΔstkP had different growth characteristics and abnormal cell division and cell separation. Nevertheless, the morphological effects could not be explained by changes in protein abundances on a global scale. So, the in-depth analysis of the phosphoproteome was mandatory to deliver further information of PhpP and StkP and their influence in cell division and peptidoglycan synthesis by modulating proteins involved in this mechanisms. For more detailed insights into the activity, targets and target sites of PhpP and StkP the advantages of phosphopeptide enrichment using titanium dioxide and spectral library based data evaluation were combined. Indeed, the application of an adapted workflow for phosphoproteome analyses and the use of a recently constructed broad spectral library, including a large number of phosphopeptides (504) highly enhanced the reliable and reproducible identification of phosphorylated proteins in this work. Finally, already known targets and target sites of StkP and PhpP, detected and described in other studies using different experimental procedures, have been identified as a proof of principle applying the mass spectrometry based phosphoproteome approach presented in this work. Referring to the role of StkP in cell division and cell separation a number of proteins participating in cell wall synthesis and cell division that are apparently phosphorylated by StkP was identified. In comparison to StkP, the physiological function and role of the co-expressed phosphatase PhpP is poorly understood. But, especially the list of previously unknown putative target substrates of PhpP has been extended remarkably in this work. Among others, five proteins with direct involvement in cell division (DivIVA, GpsB) and peptidoglycan biosynthesis (MltG, MreC, MacP) can be found under the new putative targets of PhpP. All in all, this work provides a complex and comprehensive protein repository of high proteome coverage of S. pneumoniae D39 including identification of yet unknown serine/threonine/tyrosine phosphorylation, which might contribute to support various research interests within the scientific community and will facilitate further investigations of this important human pathogen.