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. 2013 Dec;12(12):3908-23.
doi: 10.1074/mcp.M113.027375. Epub 2013 Sep 27.

Archaeal signal transduction: impact of protein phosphatase deletions on cell size, motility, and energy metabolism in Sulfolobus acidocaldarius

Julia Reimann  1 , Dominik Esser, Alvaro Orell, Fabian Amman, Trong Khoa Pham, Josselin Noirel, Ann-Christin Lindås, Rolf Bernander, Phillip C Wright, Bettina Siebers, Sonja-Verena Albers
Affiliations

Archaeal signal transduction: impact of protein phosphatase deletions on cell size, motility, and energy metabolism in Sulfolobus acidocaldarius

Julia Reimann et al. Mol Cell Proteomics. 2013 Dec.

Abstract

In this study, the in vitro and in vivo functions of the only two identified protein phosphatases, Saci-PTP and Saci-PP2A, in the crenarchaeal model organism Sulfolobus acidocaldarius were investigated. Biochemical characterization revealed that Saci-PTP is a dual-specific phosphatase (against pSer/pThr and pTyr), whereas Saci-PP2A exhibited specific pSer/pThr activity and inhibition by okadaic acid. Deletion of saci_pp2a resulted in pronounced alterations in growth, cell shape and cell size, which could be partially complemented. Transcriptome analysis of the three strains (Δsaci_ptp, Δsaci_pp2a and the MW001 parental strain) revealed 155 genes that were differentially expressed in the deletion mutants, and showed significant changes in expression of genes encoding the archaella (archaeal motility structure), components of the respiratory chain and transcriptional regulators. Phosphoproteome studies revealed 801 unique phosphoproteins in total, with an increase in identified phosphopeptides in the deletion mutants. Proteins from most functional categories were affected by phosphorylation, including components of the motility system, the respiratory chain, and regulatory proteins. In the saci_pp2a deletion mutant the up-regulation at the transcript level, as well as the observed phosphorylation pattern, resembled starvation stress responses. Hypermotility was also observed in the saci_pp2a deletion mutant. The results highlight the importance of protein phosphorylation in regulating essential cellular processes in the crenarchaeon S. acidocaldarius.

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Figures

Fig. 1.
Fig. 1.
Biochemical characterization of the protein phosphatases Saci-PTP. The activity of Saci-PTP was analyzed in the presence of pNPP (A), of the phospho-Tyr peptide TEVGKRI(pY)RLV (B), the phospho-Ser peptide NIDAIRA(pS)LNIMSR (C), and the phospho-Thr peptide ETTYERW(pT)TITQRER (D). All experiments were performed at 70 °C in triplicate. Saci_PTP showed general phosphatase activity with pNPP as substrate (A). Furthermore, Saci-PTP displayed a dual-specific phosphatase activity using p-Tyr peptides (B) as well as p-Ser (C) and p-Thr (D) peptides as substrate.
Fig. 2.
Fig. 2.
Biochemical characterization of the protein phosphatases Saci-PP2A. The activity of Saci-PP2A was determined in the presence of pNPP (A), the phospho-Thr peptide RRA(pT)VA (B) as well as in the presence of okadaic acid (p-Thr peptide RRA(pT)VA)(C). All experiments were performed at 70 °C in triplicate. Saci_PP2A revealed phosphatase activity with pNPP as well as the p-Thr peptide, whereas no activity was observed with the p-Tyr peptide. Furthermore, Saci_PP2A was significantly inhibited in presence of okadaic acid, a typical inhibitor of PPPs.
Fig. 3.
Fig. 3.
Transcriptionally regulated genes in Δsaci_ptp and Δsaci_pp2a as compared with the strain MW001. A, The number of differentially regulated gene products in Δsaci_ptp and Δsaci_pp2a are depicted in a Venn diagram. Up and down arrows represent the number of genes which were found to be either up- or down-regulated for each deletion strain. In Δsaci_ptp, most genes were down-regulated, whereas in Δsaci_pp2a most were up-regulated. Eight genes were differentially regulated in both phosphatase deletion strains. B, Heat-map representation of all eight commonly regulated genes in Δsaci_ptp (X) and Δsaci_pp2a (Y). The graph is based on the differential gene expression analysis by RNA-seq, showing all genes that are significantly altered in their expression in both deletion strains. The right side panel shows the functional classification for each gene product.
Fig. 4.
Fig. 4.
Bar plot representation of the transcriptomic changes in Δsaci_ptp and Δsaci_pp2a. A differential gene expression analysis was performed for each deletion strain, Δsaci_ptp (A) and Δsaci_pp2a (B), in comparison to the wild-type strain MW001. All genes of whose expression was significantly altered (≥twofold) are shown. Red and green bars represent down- and up-regulated genes, respectively. Genes with a p value below 0.054 were considered to be significantly differentially regulated. The annotation of selected genes is indicated to the right of each column.
Fig. 5.
Fig. 5.
Proposed model of S. acidocaldarius branched aerobic respiratory chain. The respiratory chain model is based on separate biochemical characterization studies. Electrons from NADH or succinate oxidation presumably enter electron transport chain via NADH:quinone oxidoreductases or succinate:quinone oxidoreductases (not shown). Reduced quinones (CQH2) then deliver electrons to cytochrome oxidase bc1 complex SoxLN-CbsAB-OdsN (42) and subsequently to one of the three terminal oxidase complexes. These terminal complexes are SoxABCD-SoxL (45, 54, 55), the bb3 terminal oxidase complex SoxEFGHIM (–58), and the DoxBCE (59). Components whose transcript levels were altered in each deletion strain are depicted either in red or green for down- and up-regulation, respectively. Gene identifiers for each component are shown below.
Fig. 6.
Fig. 6.
Differential expression levels of archaella operon genes and respiratory chain genes in Δsaci_pp2a and Δsaci_ptp. Total RNA isolated from S. acidocaldarius MW001, Δsaci_pp2a and Δsaci_ptp cultures were used for cDNAs synthesis. qRT-PCR analysis was performed using specific primers for each archaella component (A) and components of the terminal oxidase complexes (B). Relative transcript levels of each gene were normalized to an internal control gene secY. The values reflect the fold change compared with cDNA prepared from MW001. The means and standard deviations of three biological replicates are shown. Up-regulation of all archaella and the terminal oxidase genes in the saci_pp2a deletion strain reflect the RNA-seq results. * significant (p value ≤ 0.05), ** highly significant (p value ≤ 0.01).
Fig. 7.
Fig. 7.
Impact of phosphatase deletion on archaella expression and motility. A, For the nutrient limitation assay MW001, Δsaci_ptp and Δsaci_pp2a were grown to an OD600 of about 0.4 in Brock medium supplemented with 0.1% NZ-amine, 0.2% sucrose and 10 μg/ml uracil and then transferred to medium lacking the nutrient source. After 6 h samples were collected and immunoblotting was performed with specific antibodies against the archaellum proteins FlaB and FlaX. The amount of protein in Δsaci_ptp is comparable to the strain MW001, whereas for Δsaci_pp2a highly elevated expression of FlaB and FlaX was observed after the starvation stress. Interestingly, already before stress induction the FlaB levels were increased in the Saci-PP2A deletion mutant. B, The wild type strain MW001, the two phosphatase deletion strains Δsaci_ptp and Δsaci_pp2a, the hypermotile positive control strain ΔaapF and the negative nonmotile strain ΔaapFΔflaH were spotted on semi-solid Gelrite dishes and incubated for 5 days at 76 °C. Only a small halo is visible for MW001 and Δsaci_ptp, whereas Δsaci_pp2a was as hypermotile as the positive control strain.
Fig. 8.
Fig. 8.
Model of the involvement of phosphorylation in the regulation of motility in S. acidocaldarius. During exponential growth (A) archaellum synthesis from the flaB-flaJ (saci1178–1172) operon is repressed. Part of the repression network are ArnA (Saci1210) and ArnB (Saci1211) that are phosphorylated and dephosphorylated by ePK (Saci1193) and PP2A, respectively. ArnA and B closely interact and repress the flaX promoter (35). Probably another, yet unknown, factor is repressing the flaB promoter. When S. acidocaldarius cells enter stationary phase and undergo starvation, or in the Δsaci_pp2a deletion mutant (B), the ArnaA/B complex exists only in its phosphorylated form and repression is released. Moreover, the expression of ArnR (Saci1180) and ArnR1 (Saci1171) is induced and these factors then drive high expression of FlaB which leads to the assembly of archaella on the cell surface and therefore to increased motility.
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