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Site-Specific Ser/Thr/Tyr Phosphoproteome of Sinorhizobium meliloti at Stationary Phase.

Liu T, Tian CF, Chen WX - PLoS ONE (2015)

Bottom Line: Phosphoproteins identified in S. meliloti showed a wide distribution pattern regarding to functional categories, such as replication, transcription, translation, posttranslational modification, transport and metabolism of amino acids, carbohydrate, inorganic ion, succinoglycan etc.Moreover, phosphorylation in proteins involved in processes related to rhizobial adaptation was also discussed, such as those identified in SMa0114 and PhaP2 (polyhydroxybutyrate synthesis), ActR (pH stress and microaerobic adaption), SupA (potassium stress), chaperonin GroEL2 (viability and potentially symbiosis), and ExoP (succinoglycan synthesis and secretion).These Ser/Thr/Tyr phosphosites identified herein would be helpful for our further investigation and understanding of the role of phosphorylation in rhizobial physiology.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Agrobiotechnology, and College of Biological Sciences, China Agricultural University, Beijing, China; Key Laboratory of Soil Microbiology, Ministry of Agriculture, China Agricultural University, Beijing, China; Rhizobium Research Center, China Agricultural University, Beijing, China.

ABSTRACT
Sinorhizobium meliloti, a facultative microsymbiont of alfalfa, should fine-tune its cellular processes to live saprophytically in soils characterized with limited nutrients and diverse stresses. In this study, TiO2 enrichment and LC-MS/MS were used to uncover the site-specific Ser/Thr/Tyr phosphoproteome of S. meliloti in minimum medium at stationary phase. There are a total of 96 unique phosphorylated sites, with a Ser/Thr/Tyr distribution of 63:28:5, in 77 proteins. Phosphoproteins identified in S. meliloti showed a wide distribution pattern regarding to functional categories, such as replication, transcription, translation, posttranslational modification, transport and metabolism of amino acids, carbohydrate, inorganic ion, succinoglycan etc. Ser/Thr/Tyr phosphosites identified within the conserved motif in proteins of key cellular function indicate a crucial role of phosphorylation in modulating cellular physiology. Moreover, phosphorylation in proteins involved in processes related to rhizobial adaptation was also discussed, such as those identified in SMa0114 and PhaP2 (polyhydroxybutyrate synthesis), ActR (pH stress and microaerobic adaption), SupA (potassium stress), chaperonin GroEL2 (viability and potentially symbiosis), and ExoP (succinoglycan synthesis and secretion). These Ser/Thr/Tyr phosphosites identified herein would be helpful for our further investigation and understanding of the role of phosphorylation in rhizobial physiology.

No MeSH data available.


Bioinformatics analysis of phosphorylation sites.(a) Relative abundances of amino acids flanking pS/pT or non-phosphorylated Ser/Thr. Phospho-13-mers and non-phospho-13-mers (6 amino acids upstream and downstream of the phosphorylated or non-phosphorylated site) are shown. Amino acids are colored according to their charge scale. (b) Phosphorylated and non-phosphorylated Ser/Thr/Tyr in protein secondary structures. P-values based on T-test are indicated.
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pone.0139143.g002: Bioinformatics analysis of phosphorylation sites.(a) Relative abundances of amino acids flanking pS/pT or non-phosphorylated Ser/Thr. Phospho-13-mers and non-phospho-13-mers (6 amino acids upstream and downstream of the phosphorylated or non-phosphorylated site) are shown. Amino acids are colored according to their charge scale. (b) Phosphorylated and non-phosphorylated Ser/Thr/Tyr in protein secondary structures. P-values based on T-test are indicated.

Mentions: To visualize potential preferred sequence pattern around pS/pT in S. meliloti, relative abundances of amino acids flanking pS/pT or non-phosphorylated Ser/Thr identified in this study were compared. As shown in Fig 2a, neutral amino acids including Ala, Gly, Ser etc. dominate those downstream sites of non-phosphorylated Ser/Thr, whereas the frequency of charged amino acids such as Asp, Glu, and Arg increases in downstream sites of pS and pT. In line with this pattern, the observed phosphorylation sites showed a slightly increased probability of being located on the protein surface than non-phosphorylated sites (Fig 2b, 52.8% versus 49.0%, T-test, P-value = 0.09) as determined by using NetSurfP [38]. Similar trend in solvent accessibility of identified phosphorylation sites was reported in Phaeodactylum tricornutum [49]. Moreover, our results also showed that pS/pT is more frequently present in unstructured coil region as demonstrated earlier in other bacteria [28, 50, 51]. Among the identified phosphoproteins, cellular localization for 60/77 proteins could be predicated by using pSORTb [39]. 45 and 11 phosphoproteins are cytoplasmic and cytoplasmic membrane proteins, respectively, whereas four phosphoproteins were predicted as outer-membrane or periplasmic proteins (Fig 3a). As shown in S3 Table, at a stringency of 5%, 18 phosphopeptides matches the target motifs of eukaryotic kinases such as Casein kinase -1 and -2, GSK3b, PLK1, PKC mu, PKC delta, Calmodulin dependent kinase 2, PIP3-binding PH, Erk1 kinase, SH3 etc. This implies potential diverse sources of protein phosphorylation in S. meliloti as reported in other bacteria [28].


Site-Specific Ser/Thr/Tyr Phosphoproteome of Sinorhizobium meliloti at Stationary Phase.

Liu T, Tian CF, Chen WX - PLoS ONE (2015)

Bioinformatics analysis of phosphorylation sites.(a) Relative abundances of amino acids flanking pS/pT or non-phosphorylated Ser/Thr. Phospho-13-mers and non-phospho-13-mers (6 amino acids upstream and downstream of the phosphorylated or non-phosphorylated site) are shown. Amino acids are colored according to their charge scale. (b) Phosphorylated and non-phosphorylated Ser/Thr/Tyr in protein secondary structures. P-values based on T-test are indicated.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4581636&req=5

pone.0139143.g002: Bioinformatics analysis of phosphorylation sites.(a) Relative abundances of amino acids flanking pS/pT or non-phosphorylated Ser/Thr. Phospho-13-mers and non-phospho-13-mers (6 amino acids upstream and downstream of the phosphorylated or non-phosphorylated site) are shown. Amino acids are colored according to their charge scale. (b) Phosphorylated and non-phosphorylated Ser/Thr/Tyr in protein secondary structures. P-values based on T-test are indicated.
Mentions: To visualize potential preferred sequence pattern around pS/pT in S. meliloti, relative abundances of amino acids flanking pS/pT or non-phosphorylated Ser/Thr identified in this study were compared. As shown in Fig 2a, neutral amino acids including Ala, Gly, Ser etc. dominate those downstream sites of non-phosphorylated Ser/Thr, whereas the frequency of charged amino acids such as Asp, Glu, and Arg increases in downstream sites of pS and pT. In line with this pattern, the observed phosphorylation sites showed a slightly increased probability of being located on the protein surface than non-phosphorylated sites (Fig 2b, 52.8% versus 49.0%, T-test, P-value = 0.09) as determined by using NetSurfP [38]. Similar trend in solvent accessibility of identified phosphorylation sites was reported in Phaeodactylum tricornutum [49]. Moreover, our results also showed that pS/pT is more frequently present in unstructured coil region as demonstrated earlier in other bacteria [28, 50, 51]. Among the identified phosphoproteins, cellular localization for 60/77 proteins could be predicated by using pSORTb [39]. 45 and 11 phosphoproteins are cytoplasmic and cytoplasmic membrane proteins, respectively, whereas four phosphoproteins were predicted as outer-membrane or periplasmic proteins (Fig 3a). As shown in S3 Table, at a stringency of 5%, 18 phosphopeptides matches the target motifs of eukaryotic kinases such as Casein kinase -1 and -2, GSK3b, PLK1, PKC mu, PKC delta, Calmodulin dependent kinase 2, PIP3-binding PH, Erk1 kinase, SH3 etc. This implies potential diverse sources of protein phosphorylation in S. meliloti as reported in other bacteria [28].

Bottom Line: Phosphoproteins identified in S. meliloti showed a wide distribution pattern regarding to functional categories, such as replication, transcription, translation, posttranslational modification, transport and metabolism of amino acids, carbohydrate, inorganic ion, succinoglycan etc.Moreover, phosphorylation in proteins involved in processes related to rhizobial adaptation was also discussed, such as those identified in SMa0114 and PhaP2 (polyhydroxybutyrate synthesis), ActR (pH stress and microaerobic adaption), SupA (potassium stress), chaperonin GroEL2 (viability and potentially symbiosis), and ExoP (succinoglycan synthesis and secretion).These Ser/Thr/Tyr phosphosites identified herein would be helpful for our further investigation and understanding of the role of phosphorylation in rhizobial physiology.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Agrobiotechnology, and College of Biological Sciences, China Agricultural University, Beijing, China; Key Laboratory of Soil Microbiology, Ministry of Agriculture, China Agricultural University, Beijing, China; Rhizobium Research Center, China Agricultural University, Beijing, China.

ABSTRACT
Sinorhizobium meliloti, a facultative microsymbiont of alfalfa, should fine-tune its cellular processes to live saprophytically in soils characterized with limited nutrients and diverse stresses. In this study, TiO2 enrichment and LC-MS/MS were used to uncover the site-specific Ser/Thr/Tyr phosphoproteome of S. meliloti in minimum medium at stationary phase. There are a total of 96 unique phosphorylated sites, with a Ser/Thr/Tyr distribution of 63:28:5, in 77 proteins. Phosphoproteins identified in S. meliloti showed a wide distribution pattern regarding to functional categories, such as replication, transcription, translation, posttranslational modification, transport and metabolism of amino acids, carbohydrate, inorganic ion, succinoglycan etc. Ser/Thr/Tyr phosphosites identified within the conserved motif in proteins of key cellular function indicate a crucial role of phosphorylation in modulating cellular physiology. Moreover, phosphorylation in proteins involved in processes related to rhizobial adaptation was also discussed, such as those identified in SMa0114 and PhaP2 (polyhydroxybutyrate synthesis), ActR (pH stress and microaerobic adaption), SupA (potassium stress), chaperonin GroEL2 (viability and potentially symbiosis), and ExoP (succinoglycan synthesis and secretion). These Ser/Thr/Tyr phosphosites identified herein would be helpful for our further investigation and understanding of the role of phosphorylation in rhizobial physiology.

No MeSH data available.