Tyrosine phosphorylation of CpsD negatively regulates capsular polysaccharide biosynthesis in Streptococcus pneumoniae

Abstract

SummaryIn Streptococcus pneumoniae, the first four genes of the capsule locus (cpsA to cpsD) are common to most serotypes. By analysis of various in-frame deletion and site-directed mutants, the function of their gene products in capsular polysaccharide (CPS) biosynthesis was investigated. We found that while CpsB, C and D are essential for encapsulation, CpsA is not. CpsC and CpsD have similarity to the aminoterminal and carboxy-terminal regions, respectively, of the autophosphorylating protein-tyrosine kinase Wzc from Escherichia coli. Alignment of CpsD with Wzc and other related proteins identified conserved Walker A and B sequence motifs and a tyrosine rich domain close to the carboxy-terminus. We have shown that CpsD is also an autophosphorylating protein-tyrosine kinase and that point mutations in cpsD affecting either the ATP-binding domain (Walker A motif) or the carboxy-terminal [YGX] 4 repeat domain eliminated tyrosine phosphorylation of CpsD. We describe, for the first time, the phenotypic impact of these two mutations on polysaccharide production and show that they affect CPS production differently. Whereas a mutation in the Walker A motif resulted in loss of encapsulation, mutation of the tyrosines in the [YGX] 4 repeat domain resulted in an apparent increase in encapsulation and a mucoid phenotype. These data suggest that autophosphorylation of CpsD at tyrosine attenuates its activity and reduces the level of encapsulation. Additionally, we demonstrated that CpsC is required for CpsD tyrosine phosphorylation and that CpsB influences dephosphorylation of CpsD. These results are consistent with CpsD tyrosine phosphorylation acting to negatively regulate CPS production. This has implications for the function of CpsC/CpsD homologues in both Gram-positive and Gram-negative bacteria and provides a mechanism to explain regulation of CPS production during pathogenesis.

Autophosphorylation of the Escherichia coli Protein Kinase Wzc Regulates Tyrosine Phosphorylation of Ugd, a UDP-glucose Dehydrogenase

“…Thus, when Wzc is phosphorylated, no colanic acid is synthesized by bacteria, whereas when Wzc is dephosphorylated by Wzb, colanic acid is produced. The same observation has been made in Streptococcus pneumoniae where autophosphorylation of the kinase CpsD decreases its activity and, consequently, lowers the production of capsular polysaccharides (12).…”

“…Indeed, although intermolecular autophosphorylation of Wzc at the C-terminal tyrosine cluster (five tyrosines from position 708 to position 715) is stimulated by intramolecular autophosphorylation at Tyr 569 (24), autophosphorylation of the S. pneumoniae kinase CpsD only occurs on the tyrosine cluster when the membrane protein modulator CpsC is also present (Fig. 1) (11,12). Several studies have been performed to assess the biological role of phosphorylation of the tyrosine cluster located at the C-terminal end of PTKs.…”

Coiled-coil regions play a role in the function of the Shigella flexneri O-antigen chain length regulator WzzpHS2

“…We have unpublished data showing that fusion of GFP to the C-terminus of Wzz SF results in an 85 % reduction in Wzz activity (Daniels, 1999) and furthermore, studies of hybrid Wzz proteins also support the idea that the C-terminus is important for function (Daniels & Morona, 1999). While post-translational regulation of Wzz activity has not been demonstrated as yet, activity of the related PCP2 proteins, Wzc and CpsC/D, is affected by phosphorylation that is mediated via cytoplasmic domains or subunits of these proteins (Morona et al, 2000a;Vincent et al, 1999;Wugeditsch et al, 2001). It may be possible that a similar mechanism for regulating the activity of Wzz pHS2 and other Wzz proteins exists.…”

PhpA, a tyrosine phosphatase of Myxococcus xanthus, is involved in the production of exopolysaccharide

“…BLASTP analysis showed that PhpA is a member of the PHP superfamily. PhpA shared 28, 24, 23, 28, (Morona et al, 2002) and EpsB from Streptococcus thermophilus (Minic et al, 2007), respectively (Fig. S1, available with the online version of this paper).…”

“…PHPs are divalent metal ion-dependent enzymes, and the phosphatase activities of PHPs are stimulated by various metal ions (Morona et al, 2002;Hagelueken et al, 2009). The protein phosphatase activity of PhpA required Mn 2+ or Co 2+ as an essential cofactor (Table 1).…”