Protein tyrosine kinases in bacterial pathogens are associated with virulence and production of exopolysaccharide

Background: UDP-glucose dehydrogenase (Ugd) from E. coli K-12 is reported to be activated by tyrosine phosphorylation. Results: Ugd displayed NAD substrate inhibition that could be relieved by the addition of a nucleotide triphosphate in the absence of kinase. Conclusion: Nucleotide triphosphates are allosteric activators of Ugd. Significance: Tyrosine phosphorylation is not required for Ugd activation.

Section: Discussionmentioning

confidence: 72%

“…Consequently, possession of colanic acid and group 1 capsule assembly machinery appears to be mutually exclusive. The second BY-kinase, Etk, is encoded at an unlinked locus that is not expressed in all isolates (25) but can contribute to the formation of group 1 CPSs (26). Etk and Wzc are structural homologs (27,28).…”

mentioning

confidence: 99%

The UDP-glucose Dehydrogenase of Escherichia coli K-12 Displays Substrate Inhibition by NAD That Is Relieved by Nucleotide Triphosphates

Mainprize

Bean

Bouwman

et al. 2013

“…One copy (designated wza-wzb-wzc K30 ) resides in the cps (K30 biosynthesis gene cluster), and the other (wza-wzb-wzc 22min ) is in a locus found in many E. coli isolates (18) at a position corresponding to 22 min on the E. coli K-12 map. The wzb-wzc 22min genes have also been called etp-etk (18), and their products have broader physiological activities (19). Complementation experiments were performed in a background in which the 22-min locus has been inactivated by a polar insertion (wza 22min ::aadA; Ref.…”

Section: Methodsmentioning

confidence: 99%

Translocation of Group 1 Capsular Polysaccharide in Escherichia coli Serotype K30

Nesper

Hill

Paiment

et al. 2003

The late steps in assembly of capsular polysaccharides (CPS) and their translocation to the bacterial cell surface are not well understood. The Wza protein was shown previously to be required for the formation of the prototype group 1 capsule structure on the surface of Escherichia coli serotype K30 (Drummelsmith, J., and Whitfield, C. (2000) EMBO J. 19, 57-66). Wza is a conserved outer membrane lipoprotein that forms multimers adopting a ringlike structure, and collective evidence suggests a role for these structures in the export of capsular polymer across the outer membrane. Wza was purified in the native form and with a C-terminal hexahistidine tag. Wza His 6 was acylated and functional in capsule assembly, although its efficiency was slightly reduced in comparison to the native Wza protein. Ordered two-dimensional crystals of Wza His 6 were obtained after reconstitution of purified multimers into lipids. Electron microscopy of negatively stained crystals and Fourier filtering revealed ringlike multimers with an average outer diameter of 8.84 nm and an average central cavity diameter of 2.28 nm. Single particle analysis yielded projection structures at an estimated resolution of 3 nm, favoring a structure for the Wza His 6 containing eight identical subunits. A derivative of Wza (Wza*) in which the original signal sequence was replaced with that from OmpF showed that the native acylated N terminus of Wza is critical for formation of normal multimeric structures and for their competence for CPS assembly, but not for targeting Wza to the outer membrane. In the presence of Wza*, CPS accumulated in the periplasm but was not detected on the cell surface. Chemical cross-linking of intact cells suggested formation of a transmembrane complex minimally containing Wza and the inner membrane tyrosine autokinase Wzc.In Gram-negative bacteria, macromolecules destined for the cell surface or the extracellular environment must cross both the inner (IM) 1 and the outer membrane (OM). In protein export, where the processes are arguably best understood, secretion systems with varying complexity accomplish the translocation steps; all involve multi-enzyme complexes where an outer membrane protein (channel) is linked directly, or via helper proteins, to IM components.Cell-associated capsular polysaccharides (CPS) and their secreted (cell-free) counterparts, exopolysaccharides (EPS), represent another type of macromolecule that must be transported across the cell envelope. The early steps in assembly of these polymers are reasonably well established. However, there is little understanding of the terminal steps, including the mechanism by which they cross the bacterial cell envelope and the machinery involved.In Escherichia coli, more than 80 antigenically distinct capsular (K) polysaccharide structures are recognized. They are divided into four groups based on the organization of their genetic loci, polymerization mechanisms, and regulation (1). Group 1 and 2 capsules have received the most attention, and they involve biosynthet...

“…Then, other protein-tyrosine kinases, homologous to Ptk, have been characterized in Gram-negative bacteria, namely protein Wzc from E. coli K12 (9) and E. coli K30 (10), protein Etk from E. coli K30 (11), protein ExoP from Sinorhizobium meliloti (12), and protein Yco6 from Klebsiella pneumoniae (13).…”

mentioning

confidence: 99%

Tyrosine Phosphorylation of Protein Kinase Wzc fromEscherichia coli K12 Occurs through a Two-step Process

Grangeasse

Doublet

Cozzone

2002

In bacteria, several proteins have been shown to autophosphorylate on tyrosine residues, but little is known on the molecular mechanism of this modification. To get more information on this matter, we have analyzed in detail the phosphorylation of a particular autokinase, protein Wzc, from Escherichia coli K12. The analysis of the hydropathic profile of this protein indicates that it is composed of two main domains: an Nterminal domain, including two transmembrane ␣-helices, and a C-terminal cytoplasmic domain. The C-terminal domain alone can undergo autophosphorylation and thus appears to harbor the protein-tyrosine kinase activity. By contrast, the N-terminal domain is not phosphorylated when incubated either alone or in the presence of the C-domain, and does not influence the extent of phosphorylation of the C-domain. The C-domain contains six different sites of phosphorylation. Among these, five are located at the C-terminal end of the molecule in the form of a tyrosine cluster (Tyr 708 , Tyr 710 , Tyr 711 , Tyr 713 , and Tyr 715 ), and one site is located upstream, at Tyr 569 . The Tyr 569 residue can autophosphorylate through an intramolecular process, whereas the tyrosine cluster cannot. The phosphorylation of Tyr 569 results in an increased protein kinase activity of Wzc, which can, in turn, phosphorylate the five terminal tyrosines through an intermolecular process. It is concluded that protein Wzc autophosphorylates by using a cooperative two-step mechanism that involves both intra-and interphosphorylation. This mechanism may be of biological significance in the signal transduction mediated by Wzc.