Purification and Characterization of WaaP from Escherichia coli, a Lipopolysaccharide Kinase Essential for Outer Membrane Stability

In Escherichia coli, Salmonella enterica, and Pseudomonas aeruginosa, thewaaP (rfaP) gene product is required for the addition of phosphate to O-4 of the first heptose residue of the lipopolysaccharide (LPS) inner core region. This phosphate substitution is particularly important to the biology of these bacteria; it has previously been shown that WaaP is necessary for resistance to hydrophobic and polycationic antimicrobials in E. coli and that it is required for virulence in invasive strains of S. enterica. WaaP function is also known to be essential for the viability of P. aeruginosa. The predicted WaaP protein shows low levels of similarity (10–15% identity) to eukaryotic protein kinases, but its kinase activity has never been tested. Here we report the purification of WaaP and the reconstitution of its enzymatic activity in vitro. The purified enzyme catalyzes the incorporation of 33P from [γ-33P]ATP into acceptor LPS purified from a defined E. coli waaP mutant. Enzymatic activity is dependent upon the presence of Mg2+and is maximal from pH 8.0 to 9.0. The apparent K m(determined at saturating concentrations of the second substrate) is 0.13 mm for ATP and 76 μm for LPS. These data are the first proof that WaaP is indeed an LPS kinase. Further, site-directed mutagenesis of a predicted catalytic residue suggests that WaaP shares a common mechanism of action with eukaryotic protein kinases. In Escherichia coli, Salmonella enterica, and Pseudomonas aeruginosa, thewaaP (rfaP) gene product is required for the addition of phosphate to O-4 of the first heptose residue of the lipopolysaccharide (LPS) inner core region. This phosphate substitution is particularly important to the biology of these bacteria; it has previously been shown that WaaP is necessary for resistance to hydrophobic and polycationic antimicrobials in E. coli and that it is required for virulence in invasive strains of S. enterica. WaaP function is also known to be essential for the viability of P. aeruginosa. The predicted WaaP protein shows low levels of similarity (10–15% identity) to eukaryotic protein kinases, but its kinase activity has never been tested. Here we report the purification of WaaP and the reconstitution of its enzymatic activity in vitro. The purified enzyme catalyzes the incorporation of 33P from [γ-33P]ATP into acceptor LPS purified from a defined E. coli waaP mutant. Enzymatic activity is dependent upon the presence of Mg2+and is maximal from pH 8.0 to 9.0. The apparent K m(determined at saturating concentrations of the second substrate) is 0.13 mm for ATP and 76 μm for LPS. These data are the first proof that WaaP is indeed an LPS kinase. Further, site-directed mutagenesis of a predicted catalytic residue suggests that WaaP shares a common mechanism of action with eukaryotic protein kinases. lipopolysaccharide 2-aminoethyl phosphate 2-aminoethyl disphosphate 3-deoxy-d-manno-oct-2-ulosonic acid l-glycero-d-manno-heptose isopropyl-1-thio-β-d-galactopyranoside nitrilotriacetic acid polyacrylamide gel electro- phoresis polymerase chain reaction The outer membrane of a Gram-negative bacterium is a barrier to many antibiotics and host defense factors (1Nikaido H. Vaara M. Microbiol. Rev. 1985; 49: 1-32Crossref PubMed Google Scholar, 2Vaara M. Microbiol. Rev. 1992; 56: 395-411Crossref PubMed Google Scholar). This barrier function is due in large part to structural features of the lipopolysaccharide (LPS)1 molecules that make up the outer leaflet of the outer membrane bilayer. In Escherichia coli, Salmonella enterica, and a variety of other Gram-negative pathogens including members of the familiesPseudomonadaceae and Vibrionaceae, the LPS molecule is conceptually divided into three distinct regions: 1) a hydrophobic membrane anchor designated lipid A; 2) a short, branched chain of sugar residues with multiple phosphoryl substituents, referred to as the core oligosaccharide; and 3) a structurally diverse polysaccharide composed of repeating oligosaccharide units, termed the O antigen (3Raetz C.R.H. Neidhardt F.C. Curtiss III, R. Ingraham J.L. Lin E.C.C. Low K.B. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella: Cellular and Molecular Biology. 1. American Society for Microbiology Press, Washington, D. C.1996: 1035-1063Google Scholar) (Fig. 1). The presence of phosphoryl substituents on the inner (lipid A proximal) region of the LPS core oligosaccharide is a key structural feature required for the formation of a stable outer membrane in these bacteria (4Yethon J.A. Heinrichs D.E. Monteiro M.A. Perry M.B. Whitfield C. J. Biol. Chem. 1998; 273: 26310-26316Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 5Yethon J.A. Gunn J.S. Ernst R.K. Miller S.I. Laroche L. Malo D. Whitfield C. Infect. Immun. 2000; 68: 4485-4491Crossref PubMed Scopus (77) Google Scholar, 6Walsh A.G. Matewish M.J. Burrows L.L. Monteiro M.A. Perry M.B. Lam J.S. Mol. Microbiol. 2000; 35: 718-727Crossref PubMed Scopus (71) Google Scholar). These phosphoryl substituents are postulated to be critical to outer membrane integrity because their negative charge allows neighboring LPS molecules to be cross-linked by divalent cations (1Nikaido H. Vaara M. Microbiol. Rev. 1985; 49: 1-32Crossref PubMed Google Scholar, 2Vaara M. Microbiol. Rev. 1992; 56: 395-411Crossref PubMed Google Scholar). The genes involved in core phosphorylation have only recently been identified with certainty. In E. coli, waaP was shown to be required for phosphate addition to HepI (4Yethon J.A. Heinrichs D.E. Monteiro M.A. Perry M.B. Whitfield C. J. Biol. Chem. 1998; 273: 26310-26316Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar), and this reaction was found to be a prerequisite for the addition of the HepIII residue by the waaQ gene product, which in turn was required for the waaY-mediated addition of a second phosphate at HepII (see Fig. 1). Core phosphorylation in S. entericaproceeds in similar fashion (5Yethon J.A. Gunn J.S. Ernst R.K. Miller S.I. Laroche L. Malo D. Whitfield C. Infect. Immun. 2000; 68: 4485-4491Crossref PubMed Scopus (77) Google Scholar). Given the sequential action of thewaaP, waaQ, and waaY gene products, mutation of waaP alone is enough to eliminate all phosphate from the heptose region of the LPS inner core, resulting in a strain that is hypersensitive to detergents and hydrophobic antibiotics. Although mutation of waaY does reduce the amount of core phosphate, this reduction is not sufficiently serious to cause hypersensitivity to such compounds (4Yethon J.A. Heinrichs D.E. Monteiro M.A. Perry M.B. Whitfield C. J. Biol. Chem. 1998; 273: 26310-26316Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). Mutants of E. coli and S. enterica with highly truncated core oligosaccharides, such that they lack the inner core heptose residues that serve as the sites for phosphorylation (see Fig.1), exhibit a pleiotropic phenotype called “deep-rough.” Characteristics of the deep-rough phenotype include 1) hypersensitivity to detergents and hydrophobic antibiotics, 2) the appearance of phospholipid bilayer patches in the outer membrane, 3) leakage of periplasmic proteins into the culture medium, and 4) a marked decrease in the protein content of the outer membrane (reviewed in Refs. 7Heinrichs D.E. Yethon J.A. Whitfield C. Mol. Microbiol. 1998; 30: 221-232Crossref PubMed Scopus (284) Google Scholar and8Schnaitman C.A. Klena J.D. Microbiol. Rev. 1993; 57: 655-682Crossref PubMed Google Scholar). It has also been shown that the LPS from deep-rough mutants cannot support the proper folding of some outer membrane proteins (9de Cock H. Brandenburg K. Wiese A. Holst O. Seydel U. J. Biol. Chem. 1999; 274: 5114-5119Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). It was thought originally that all these characteristics could be explained simply by loss of core phosphoryl substituents. However, this was recently shown to be somewhat of an oversimplification, at least in the case of the outer membrane protein defect; a defined waaPmutant was shown to have wild-type levels of outer membrane proteins despite a complete lack of core phosphate (10Yethon J.A. Vinogradov E. Perry M.B. Whitfield C. J. Bacteriol. 2000; 182: 5620-5623Crossref PubMed Scopus (90) Google Scholar). Therefore, while the loss of core phosphate undoubtedly plays an important role in the manifestation of the deep-rough phenotype, clearly other factors must also be involved. The importance of LPS core phosphorylation and of WaaP activity in particular extend beyond the obvious membrane defects. For example, mutation of waaP in S. enterica serovar Typhimurium has been shown to cause a complete loss of virulence in mouse infection models (5Yethon J.A. Gunn J.S. Ernst R.K. Miller S.I. Laroche L. Malo D. Whitfield C. Infect. Immun. 2000; 68: 4485-4491Crossref PubMed Scopus (77) Google Scholar). In addition, WaaP activity is intuitively a prerequisite for the functioning of the currently unidentified enzyme responsible for 2-aminoethyl phosphate (PEtN) modification of theE. coli and S. enterica inner core heptose region (see Fig. 1). PEtN modification of the LPS inner core is correlated with resistance to polymyxin in these bacteria (11Nummila K. Kilpeläinen I. Zähringer U. Vaara M. Helander I.M. Mol. Microbiol. 1995; 16: 271-278Crossref PubMed Scopus (173) Google Scholar, 12Helander I.M. Kilpeläinen I. Vaara M. Mol. Microbiol. 1994; 11: 481-487Crossref PubMed Scopus (146) Google Scholar) and may provide a means by which these pathogens can modulate their surface charge. Inhibition of WaaP would thus hinder the ability of these bacteria to adapt their surfaces to particular microenvironments during pathogenesis. The predicted WaaP protein shares limited similarity with eukaryotic protein kinases (4Yethon J.A. Heinrichs D.E. Monteiro M.A. Perry M.B. Whitfield C. J. Biol. Chem. 1998; 273: 26310-26316Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar), but kinase activity has never been demonstrated. Here, we report the purification of a His6-tagged derivative of WaaP, and the development of an in vitro assay that demonstrates unequivocally that WaaP is an LPS core heptose kinase. We also give the first characterization of the catalytic properties of the WaaP enzyme and provide site-directed mutagenesis data that suggest that the enzyme's mechanism of action is similar to that of eukaryotic protein kinases. Materials and kits were purchased from the following suppliers: PCR primers (Guelph Molecular Supercentre, University of Guelph); restriction enzymes (New England Biolabs and Roche Molecular Biochemicals); QIAprep Spin Miniprep Kit and QIAquick PCR Purification Kit (Qiagen); antibiotics (Sigma); nickel-nitrilotriacetic acid-agarose (Ni2+-NTA-agarose) (Qiagen); PD-10 desalting columns (Amersham Pharmacia Biotech); [γ-33P]ATP (3000 Ci/mmol) (PerkinElmer Life Sciences); other assay reagents (Tris, (Sigma); and LPS acceptor for the WaaP assay was purified from defined waaPmutant strain (4Yethon J.A. Heinrichs D.E. Monteiro M.A. Perry M.B. Whitfield C. J. Biol. Chem. 1998; 273: 26310-26316Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar) by of O. K. Chem. coli strain is from and E. coli strain is from Life primers for the of waaP from the E. coli core B. K. J. PubMed Scopus Google Scholar) were to restriction sites for The of thewaaP and the the waaP in the and PCR was polymerase Molecular as by the The region for as a and the sites of and to an the WaaP protein and from the The and of E. coli were with the to the of WaaP protein on the of B. M. J. P. U. S. A. 1995; PubMed Scopus Google Scholar). The and from the E. coli Neidhardt F.C. Curtiss III, R. Ingraham J.L. Lin E.C.C. Low K.B. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella: Cellular and Molecular Biology. American Society for Microbiology Press, Washington, D. C.1996: Scholar). primers were to restriction sites for the the and the the shown in the and The region was as and and sites of The region was from the derivative as an with the and the and sites of D. M.J. J. J. Bacteriol. 1995; PubMed Scopus Google Scholar). The resulting allows for from the and was from an culture of and to of at with at was to and and were for an at which was to of were by and and at The was on and in a of mm phosphate, pH mm mm mm was to and and A were to reduce the was on with to complete and to of was to of and was to for with at The was into a the was to The was with of mm phosphate, pH mm mm and the His6-tagged WaaP was in of mm phosphate, pH mm mm The second the of The enzyme from the was purified by to the from the was first into a mm phosphate, pH mm PD-10 columns as by the (Amersham Pharmacia The was to a in mm phosphate, pH mm and the was in reaction mm pH mm of to μm with and mm LPS. The enzyme was to the and of the reaction were at for and by the addition of an of phosphate mm LPS was in a by and three in to the LPS was by for the the LPS was in and the was in a LPS was from reaction and in of The acid was at for to the the of and the of lipid A and and lipid A was by The core were to a of with as The and were and were and for by The gene the His6-tagged derivative of WaaP with its was from the WaaP as an and the and sites of D. M.J. J. J. Bacteriol. 1995; PubMed Scopus Google Scholar). The resulting was as the for site-directed It was necessary to of for these to for of WaaP and its in strain (4Yethon J.A. Heinrichs D.E. Monteiro M.A. Perry M.B. Whitfield C. J. Biol. Chem. 1998; 273: 26310-26316Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar), which is not for the primers were the mutation in the of with in the and PCR of the was polymerase Molecular as 1) at for 2) at for for and for The was purified the QIAquick PCR Purification Kit and with for the thus for the the The was into E. coli and on purified from the resulting was to that only the mutation been into thewaaP region. The the and of WaaP and were into by and WaaP was by the addition of the ability of WaaP to the of the in and were as previously (4Yethon J.A. Heinrichs D.E. Monteiro M.A. Perry M.B. Whitfield C. J. Biol. Chem. 1998; 273: 26310-26316Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). The addition of an WaaP was shown to have on its ability to the waaP phenotype not thus the of the for of the His6-tagged WaaP derivative from in a amount of as by the appearance of an at in The predicted of WaaP is and the of His6-tagged WaaP is a that could be by not The of the protein was by with for the not with the also that the of the protein was not with the from the waaP the were and by and the protein was found in were to the of WaaP medium, of of of the from and of these in the of We a that would for the of the E. coli and on the of B. M. J. P. U. S. A. 1995; PubMed Scopus Google Scholar), with the that this would the of the WaaP a resistance and a of we the on which has a resistance and a of from D. M.J. J. J. Bacteriol. 1995; PubMed Scopus Google Scholar). only can these be in a but from is from while from The ability to of WaaP for of to the of this it was to protein to for the purification of the protein the of protein (Fig. also a for the His6-tagged WaaP to with was to the to WaaP and the of The of WaaP with was not the enzymes involved in LPS core are predicted to function as membrane proteins at the of the inner membrane C.A. Klena J.D. Microbiol. Rev. 1993; 57: 655-682Crossref PubMed Google Scholar), they have to their and their lipid acceptor The incorporation of an the WaaP protein for the of as an purification (Fig. Given that the predicted of the WaaP protein is the was to However, the protein to during the required to on the this the enzyme was purified by to the WaaP to be in the (Fig. In this the protein could be in a to the The of the protein in the from the was as by (Fig. this the activity of the WaaP was the with a activity of of His6-tagged WaaP from in a the presence of mm in the (see was found to the activity of the purified protein data not Further, the purified enzyme could be in at loss of activity for The kinase activity of purified WaaP was a of concentrations from to as shown in Fig. A. The enzyme an for that cannot be with not kinase activity was at mm The activity of WaaP was a pH from to as shown in Fig. B. The kinase was shown to be at pH with maximal activity pH 8.0 and 9.0. The activity of WaaP was shown to be dependent on to and protein not the of ATP in the was at mm and the of LPS was the K the LPS acceptor was to be 76 the of LPS acceptor was at mm and the of ATP was the K for ATP was to be 0.13 In the for the reaction was the acceptor LPS purified from is on an gel and with distinct are (see Fig. The of the of these is known to of complete lipid the and on the heptose region of the core (see Fig. 1) (4Yethon J.A. Heinrichs D.E. Monteiro M.A. Perry M.B. Whitfield C. J. Biol. Chem. 1998; 273: 26310-26316Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). The is a truncated of the (see Fig. 1) as by with LPS from a defined on not WaaP was of of LPS from the waaP the LPS reaction were first in acid to the lipid A of the with acid at for the the of and the of lipid A and and and of the was in the as core not The core were on a of (Fig. distinct of were the of the of for that WaaP is indeed of the and from the waaP strain (Fig. The that at the to a amount of 33P from the reaction during the acid of the predicted WaaP proteins from E. coli and S. enterica shows while WaaP is to of E. coli and S. enterica not particular residues and coli residue are all of the WaaP the other of the E. coli WaaP protein with eukaryotic protein kinases shows only (Fig. It is that the of in Fig. with residues that are known in eukaryotic protein kinases to be important for on similarity S. PubMed Scopus Google Scholar) and data J. PubMed Scopus Google Scholar, J.A. 1994; PubMed Scopus Google Scholar). the of these we a acid into the waaP in that in the of We because it is all of the known WaaP the and because similarity that it as the WaaP catalytic In this the WaaP residue would a from the of thus an of at the of data have the role of this residue in eukaryotic protein kinases P. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar), but is that it is essential for The of with would thus be to WaaP WaaP was into defined waaPmutant it was to the and not Further, purified WaaP kinase activity in in vitro assay not clearly that is an essential of the LPS core region is essential for outer membrane in E. coli and S. enterica and has been the of many (reviewed in Refs. 7Heinrichs D.E. Yethon J.A. Whitfield C. Mol. Microbiol. 1998; 30: 221-232Crossref PubMed Scopus (284) Google Scholar, C.A. Klena J.D. Microbiol. Rev. 1993; 57: 655-682Crossref PubMed Google Scholar, and of I. Scholar). the activity of an LPS core enzyme was as as acceptor LPS from an of S. enterica and from wild-type bacteria as the enzyme P. J. 11: PubMed Scopus Google Scholar). the kinase by these was never was it that ATP was the of a gene called was in all of the phosphoryl of the E. coli LPS core C.A. M.A. S. J. Bacteriol. 1992; PubMed Google Scholar). However, the from these were limited by their on strains with and also in by the sequential action of the core kinases. The of the waaP gene product in the phosphorylation of the E. coli LPS core at HepI was only recently with on the LPS core resulting from defined and waaY (4Yethon J.A. Heinrichs D.E. Monteiro M.A. Perry M.B. Whitfield C. J. Biol. Chem. 1998; 273: 26310-26316Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). mutation of waaP in S. enterica was shown to in the LPS (5Yethon J.A. Gunn J.S. Ernst R.K. Miller S.I. Laroche L. Malo D. Whitfield C. Infect. Immun. 2000; 68: 4485-4491Crossref PubMed Scopus (77) Google Scholar), and the of WaaP in P. was shown to a waaP enterica A.G. Matewish M.J. Burrows L.L. Monteiro M.A. Perry M.B. Lam J.S. Mol. Microbiol. 2000; 35: 718-727Crossref PubMed Scopus (71) Google Scholar). The data provide the first characterization of WaaP activity and unequivocally that WaaP is indeed an LPS core heptose kinase. protein was to the assay and catalytic properties of activity was pH 8.0 and and kinases, the enzyme an for The for the reaction were to be 0.13 mm for and 76 LPS at saturating concentrations of the second In this the of WaaP phosphorylation was only to the core oligosaccharide of the LPS that was by acid However, the known of the E. coli core K. S. H. Holst O. J. 1999; PubMed Scopus Google Scholar, O. H. J.L. I. Press, Scholar) and the core resulting from defined waaY and of phosphate on HepII and loss of phosphate on HepII and (4Yethon J.A. Heinrichs D.E. Monteiro M.A. Perry M.B. Whitfield C. J. Biol. Chem. 1998; 273: 26310-26316Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar), it is to that WaaP is in a LPS core and is responsible for phosphorylation at Further, the WaaP enzyme is known to the addition of phosphate to only HepI not LPS purified from a defined waaY strain could not serve as an acceptor for the WaaP enzyme not These data thus to the of in core that in the of phosphoryl on HepI (see Fig. 1). It was previously that all LPS molecules be by the addition of to with a of these molecules to PEtN and phosphate C.A. Klena J.D. Microbiol. Rev. 1993; 57: 655-682Crossref PubMed Google Scholar). with the of WaaP as the HepI the is clearly that WaaP catalyzes the of phosphate to HepI with a of molecules at this phosphate residue with PEtN by a currently unidentified enzyme (see Fig. 1). The of the gene responsible for this PEtN modification is of particular the role of PEtN core modification in resistance to polymyxin and of surface charge (11Nummila K. Kilpeläinen I. Zähringer U. Vaara M. Helander I.M. Mol. Microbiol. 1995; 16: 271-278Crossref PubMed Scopus (173) Google Scholar, 12Helander I.M. Kilpeläinen I. Vaara M. Mol. Microbiol. 1994; 11: 481-487Crossref PubMed Scopus (146) Google Scholar). Given the importance of WaaP to membrane (4Yethon J.A. Heinrichs D.E. Monteiro M.A. Perry M.B. Whitfield C. J. Biol. Chem. 1998; 273: 26310-26316Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar) and its for virulence in (5Yethon J.A. Gunn J.S. Ernst R.K. Miller S.I. Laroche L. Malo D. Whitfield C. Infect. Immun. 2000; 68: 4485-4491Crossref PubMed Scopus (77) Google Scholar), to the reaction could function as a for the of it would be to the that is of by The that WaaP is of the and low LPS from a waaP strain (Fig. to this the outer core to are not required (see Fig. 1). This is by the report that LPS from a defined strain the phosphate modification on at only of wild-type levels (10Yethon J.A. Vinogradov E. Perry M.B. Whitfield C. J. Bacteriol. 2000; 182: 5620-5623Crossref PubMed Scopus (90) Google Scholar). the HepII phosphate is in the LPS (see Fig. that the kinase by waaY the presence of as part of its acceptor (10Yethon J.A. Vinogradov E. Perry M.B. Whitfield C. J. Bacteriol. 2000; 182: 5620-5623Crossref PubMed Scopus (90) Google Scholar). it is that WaaP was never kinase function on the of theE. coli waaP gene has been known for some E. C.A. J. Bacteriol. 1992; PubMed Google Scholar), and have been identified in S. enterica J.D. E. C.A. J. Bacteriol. 1993; PubMed Google Scholar) A.G. Matewish M.J. Burrows L.L. Monteiro M.A. Perry M.B. Lam J.S. Mol. Microbiol. 2000; 35: 718-727Crossref PubMed Scopus (71) Google Scholar). The has been that the of these predicted proteins such limited to kinases that cannot be with Miller J. Mol. Biol. PubMed Scopus Google Scholar). However, with the of the J. Miller PubMed Scopus Google Scholar) it was that WaaP to kinases only key residues the of the protein (4Yethon J.A. Heinrichs D.E. Monteiro M.A. Perry M.B. Whitfield C. J. Biol. Chem. 1998; 273: 26310-26316Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). We the of of these key by the WaaP predicted by to function as the enzyme's catalytic for mutation of a residue essential to the WaaP protein in vitro. It is this of catalytic that WaaP may have a similar mechanism of action to other eukaryotic protein kinases. We are currently structural to in support of this the of the kinase from which catalyzes the phosphorylation of a of antibiotics, was recently Full Text Full Text PDF PubMed Scopus Google Scholar). WaaP, the protein similarity to eukaryotic kinases at key such as the in this despite this lack of the is similar to that of eukaryotic protein kinases Full Text Full Text PDF PubMed Scopus Google Scholar).

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