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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.
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.