Cycling of Etk and Etp Phosphorylation States Is Involved in Formation of Group 4 Capsule by Escherichia coli

Capsules frequently play a key role in bacterial interactions with their environment. Escherichia coli capsules were categorized as groups 1 through 4, each produced by a distinct mechanism. Etk and Etp are members of protein families required for the production of group 1 and group 4 capsules. These members function as a protein tyrosine kinase and protein tyrosine phosphatase, respectively. We show that Etp dephosphorylates Etk in vivo, and mutations rendering Etk or Etp catalytically inactive result in loss of group 4 capsule production, supporting the notion that cyclic phosphorylation and dephosphorylation of Etk is required for capsule formation. Notably, Etp also becomes tyrosine phosphorylated in vivo and catalyzes rapid auto-dephosphorylation. Further analysis identified Tyr121 as the phosphorylated residue of Etp. Etp containing Phe, Glu or Ala in place of Tyr121 retained phosphatase activity and catalyzed dephosphorylation of Etp and Etk. Although EtpY121E and EtpY121A still supported capsule formation, EtpY121F failed to do so. These results suggest that cycles of phosphorylation and dephosphorylation of Etp, as well as Etk, are involved in the formation of group 4 capsule, providing an additional regulatory layer to the complex control of capsule production.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/92200/1/journal_pone_0037984.pd

Identification of an Escherichia coli operon required for formation of the O-antigen capsule

Escherichia coli produces polysaccharide capsules that, based on their mechanisms of synthesis and assembly, have been classified into four groups. The group 4 capsule (G4C) polysaccharide is frequently identical to that of the cognate lipopolysaccharide O side chain and has, therefore, also been termed the O-antigen capsule. The genes involved in the assembly of the group 1, 2, and 3 capsules have been described, but those required for G4C assembly remained obscure. We found that enteropathogenic E. coli (EPEC) produces G4C, and we identified an operon containing seven genes, ymcD, ymcC, ymcB, ymcA, yccZ, etp, and etk, which are required for formation of the capsule. The encoded proteins appear to constitute a polysaccharide secretion system. The G4C operon is absent from the genomes of enteroaggregative E. coli and uropathogenic E. coli. E. coli K-12 contains the G4C operon but does not express it, because of the presence of IS1 at its promoter region. In contrast, EPEC, enterohemorrhagic E. coli, and Shigella species possess an intact G4C operon.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/92199/1/174504.pd

Enteropathogenic <i>E. coli</i> strains.

<p>Enteropathogenic <i>E. coli</i> strains.</p

Etk and Wzc are not essential for Etp phosphorylation.

<p>EtpD119A was expressed in different EPEC strains with deletions in the <i>etp</i> gene and the kinase genes <i>etk</i> and <i>wzc</i>. Proteins were extracted and the amount and phosphorylation state of Etp were tested by Western blot using anti-6His and anti-PY antibodies, respectively. The presence of intact genes is indicated above the lanes.</p

Etk K545M mutant failed to support capsule production.

<p>EPEC strains, including wild type, <i>etk::kan</i> mutant and the mutant complemented with plasmids pOI277 (pEtk) or pMS3239 (pEtk K545M), were grown under conditions that allowed capsule production. After harvesting, proteins and capsular polysaccharide were extracted. (A) The amount of Etk and phosphorylated Etk were assessed by Western blot using anti-Etk and anti-phosphotyrosine (anti-PY) antibodies. Each lane is labeled with the strain (above blot) and antibody (on left). (B) Purified capsular polysaccharide extracted from bacteria was serially diluted to generate a dot blot that was developed with anti-O127 antibody. Wild-type EPEC and <i>etk</i> mutant were used as positive and negative controls, respectively. The strains are indicated above each serial dilution.</p

Oligonucleotide primers used in this study.

*<p>Underlined sequences represent restriction enzyme sites, and lower case sequences are regions complementary to the template plasmid containing the antibiotic cassette.</p

A model for the role of Etp and Etk in capsule formation.

<p>Cycling of Etk phosphorylation and dephosphorylation are required for capsule production (arrow <b>1)</b>. This cycling is mediated by the autokinase and autophosphatase activities of Etk and Etp, respectively <b>(</b>arrows <b>2</b> and <b>3</b>). Etp also cycles between phosphorylated and unphosphorylated forms and this cycling is catalyzed by Etp autodephosphorylation (arrow <b>4</b>) and a yet to be defined kinase (arrow <b>5</b>). Etp cycling <i>per se</i> is not required for capsule formation. However, based on our results we hypothesize that the unphosphorylated Etp has an inhibitory effect on capsule formation (arrow <b>6</b>), which can be removed by Etp phosphorylation.</p

Plasmids.

<p>Plasmids.</p

ESI tandem mass spectrometry confirms the presence and location of the phosphorylated tyrosine.

<p>The ESI-MS/MS fragmentation spectrum of Etp peptide GKTMLFGQWLEQKEIPAP(pY)RK (residues 113–123, pY is the phosphorylated tyrosine). The parent peptide was a +4 H ion with mass 2599.29 amu (−0.0134 amu from predicted size). The schematic at the top of the figure shows the identified <b>y</b> fragments referred to in the spectra. All of the <b>y</b> fragments include the peptide's C-terminus and have masses consistent with the phosphorylated Y121 (pY) residue. For example, the m/z for fragments <b>y3</b> and <b>y2</b> are 546.2 and 303.2, respectively. The difference 243 is the exact mass of a phosphotyrosyl residue. The labeled <b>b</b> fragments originate at the N-terminus of the peptide but do not include the pY residue.</p