Periplasmic Protein-Protein Contacts in the Inner Membrane Protein Wzc Form a Tetrameric Complex Required for the Assembly of Escherichia coli Group 1 Capsules

The K antigenic capsular polysaccharide forms a structural layer, the capsule, on the surfaces of Escherichia coli cells. The capsule provides an important protective covering that helps protect encapsulated bacteria from host immune defenses. The assembly and translocation of the capsule requires proteins in the inner and outer membranes. The inner membrane protein Wzc is a tyrosine autokinase that plays an essential role in what is believed to be a coordinated biosynthesis and secretion process. Mutants lacking Wzc can form K antigen oligosaccharides but are unable to polymerize high molecular weight capsular polymers. Wzc homologs have been identified in exopolymer biosynthesis systems in many different Gram-negative and -positive bacteria. Using single particle averaging on cryo-negatively stained samples, we have produced the first three-dimensional structure of this type of membrane protein in its phosphorylated state at ∼14 Å resolution. Perfluoro-octanoate-PAGE analysis of detergent-solubilized oligomeric Wzc and symmetry analysis of the transmission electron microscopy data clearly demonstrated that Wzc forms a tetrameric complex with C4 rotational symmetry. Viewed from the top of the complex, the oligomer is square with a diameter of ∼100 Å and can be divided into four separate densities. From the side, Wzc is ∼110 Å high and has a distinctive appearance similar to an extracted molar tooth. The upper “crown” region is ∼55 Å high and forms a continuous ring of density. Four unconnected “roots” (∼65 Å high) emerge from the underside of the crown. We propose that the crown is formed by protein-protein contacts from the four Wzc periplasmic domains, while each root represents an individual cytoplasmic tyrosine autokinase domain. The K antigenic capsular polysaccharide forms a structural layer, the capsule, on the surfaces of Escherichia coli cells. The capsule provides an important protective covering that helps protect encapsulated bacteria from host immune defenses. The assembly and translocation of the capsule requires proteins in the inner and outer membranes. The inner membrane protein Wzc is a tyrosine autokinase that plays an essential role in what is believed to be a coordinated biosynthesis and secretion process. Mutants lacking Wzc can form K antigen oligosaccharides but are unable to polymerize high molecular weight capsular polymers. Wzc homologs have been identified in exopolymer biosynthesis systems in many different Gram-negative and -positive bacteria. Using single particle averaging on cryo-negatively stained samples, we have produced the first three-dimensional structure of this type of membrane protein in its phosphorylated state at ∼14 Å resolution. Perfluoro-octanoate-PAGE analysis of detergent-solubilized oligomeric Wzc and symmetry analysis of the transmission electron microscopy data clearly demonstrated that Wzc forms a tetrameric complex with C4 rotational symmetry. Viewed from the top of the complex, the oligomer is square with a diameter of ∼100 Å and can be divided into four separate densities. From the side, Wzc is ∼110 Å high and has a distinctive appearance similar to an extracted molar tooth. The upper “crown” region is ∼55 Å high and forms a continuous ring of density. Four unconnected “roots” (∼65 Å high) emerge from the underside of the crown. We propose that the crown is formed by protein-protein contacts from the four Wzc periplasmic domains, while each root represents an individual cytoplasmic tyrosine autokinase domain. Escherichia coli produces ∼80 structurally and immunochemically distinct capsular polysaccharides (1Jann K. Jann B. Sussman M. Escherichia coli: Mechanisms of Virulence. Cambridge University Press, Cambridge, UK1997: 113-143Google Scholar). These major surface structures define the K antigen in serotyping analyses, and they form a surface structure known as the capsule. The capsule is a continuous and coherent surface layer, and its primary role is to provide a protective barrier and maintain the cell in a hydrated state. It is also an important virulence determinant that enables pathogenic bacteria to evade or counteract the nonspecific host defense during the early (preimmune) phase of infection by interfering with the action of complement and phagocytes (1Jann K. Jann B. Sussman M. Escherichia coli: Mechanisms of Virulence. Cambridge University Press, Cambridge, UK1997: 113-143Google Scholar). Capsular polysaccharides vary in monosaccharide composition, glycosidic linkages, and substitution with side branch glycoses and non-carbohydrate residues. Based on structural, genetic, and biochemical criteria, the capsules of E. coli have been classified into four groups (2Whitfield C. Roberts I.S. Mol. Microbiol. 1999; 31: 1307-1319Crossref PubMed Scopus (417) Google Scholar), and in terms of biosynthesis, there are two distinct pathways. Groups 1 and 4 capsules use a Wzy-dependent system, whereas assembly of groups 2 and 3 follows an ATP-binding cassette transporter-dependent process. Both biosynthesis mechanisms are widely represented in capsules and other glycoconjugates in a variety of bacterial species. The E. coli system therefore serves as a paradigm for this process in a broad range of bacteria, including significant pathogens of plants, livestock, and humans (2Whitfield C. Roberts I.S. Mol. Microbiol. 1999; 31: 1307-1319Crossref PubMed Scopus (417) Google Scholar, 3Raetz C.R.H. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3423) Google Scholar, 4Whitfield C. Paiment A. Carbohydr. Res. 2003; 338: 2491-2502Crossref PubMed Scopus (106) Google Scholar). The current working model for the Wzy-dependent assembly pathway is based on dissection of the system by biochemical and genetic means with some interpretation of corresponding reactions in lipopolysaccharide O antigen synthesis (3Raetz C.R.H. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3423) Google Scholar, 4Whitfield C. Paiment A. Carbohydr. Res. 2003; 338: 2491-2502Crossref PubMed Scopus (106) Google Scholar). In this model, individual repeat unit oligosaccharides are assembled on undecaprenol diphosphate lipid carriers at the cytoplasmic face of the inner membrane, which provide a pool of subunits for assembly. These intermediates are then transported across the inner membrane in what is proposed to be a “flippase”-type reaction by a process involving the Wzx protein. Once the oligosaccharide moiety is transferred to the periplasm, it forms the substrate for the polymerization machinery. The characteristic component of this process is the putative polymerase Wzy, an integral inner membrane protein. These initial steps in the Wzy-dependent pathway are conserved in groups 1 and 4 E. coli capsules and in lipopolysaccharide O antigens, capsules, and exopolysaccharides from a variety of bacteria. Notably, these early reactions are found in some Gram-positive bacteria, including staphylococci and streptococci (4Whitfield C. Paiment A. Carbohydr. Res. 2003; 338: 2491-2502Crossref PubMed Scopus (106) Google Scholar). The terminal stages in assembly of these polymers on bacterial cell surfaces differ according to the nature of the glycoconjugate involved and the structure of the cell envelope (i.e. Gram-negative versus Gram-positive). The terminal stages in group 1 capsule assembly require the outer membrane protein Wza, a member of the outer membrane auxiliary family (5Paulsen I.T. Beness A.M. Saier Jr., M.H. Microbiology. 1997; 143: 2685-2699Crossref PubMed Scopus (132) Google Scholar). The presence of Wza is essential for the assembly of the prototype group 1 capsule structure on the surface of E. coli serotype K30 (6Drummelsmith J. Whitfield C. Mol. Microbiol. 1999; 31: 1321-1332Crossref PubMed Scopus (123) Google Scholar, 7Drummelsmith J. Whitfield C. EMBO J. 2000; 19: 57-66Crossref PubMed Scopus (129) Google Scholar). Wza forms stable oligomeric “donut”-shaped complexes with a molecular mass of ∼300 kDa, and a preliminary study of Wza, based on small two-dimensional crystalline areas, revealed ring-like complexes with an average outer diameter of ∼90–110 Å and a central stained region of ∼20–35 Å in diameter (20Nesper J. Hill C.M.D. Paiment A. Harauz G. Beis K. Naismith J.H. Whitfield C. J. Biol. Chem. 2003; 278: 49763-49772Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). A three-dimensional structure for Wza, calculated using single particle analysis of cryo-negatively stained data (9Beis K. Collins R.F. Ford R.C. Kamis A.B. Whitfield C. Naismith J.H. J. Biol. Chem. 2004; 279: 28227-28232Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar), revealed that Wza forms an octamer with an unusual rotational organization. Oligomeric Wza appears to be formed by a novel tetrameric ring of dimerically associated Wza (i.e. Wza is a tetramer of dimers). Viewed from the side, the oligomer has two distinctive layers with a cavity in the center of the complex (9Beis K. Collins R.F. Ford R.C. Kamis A.B. Whitfield C. Naismith J.H. J. Biol. Chem. 2004; 279: 28227-28232Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Wza is thought to provide a capsule translocon for export of capsular polysaccharide across the outer membrane, and it certainly has structural features in common with members of the secretin superfamily of proteins (e.g. PilQ, pIV, PulD, XcpQ, and YscC (10Bitter W. Arch. Microbiol. 2003; 179: 307-314Crossref PubMed Scopus (50) Google Scholar, 11Bitter W. Koster M. Latjinhouwers M. de Cock H. Tommassen J. Mol. Microbiol. 1998; 27: 209-219Crossref PubMed Scopus (194) Google Scholar, 12Collins R.F. Ford R.C. Kitmitto A. Olsen R. Tonjum T. Derrick J.P. J. Bacteriol. 2003; 183: 3825-3832Crossref Scopus (108) Google Scholar, 13Collins R.F. Frye S.A. Kitmitto A. Ford R.C. Tonjum T. Derrick J.P. J. Biol. Chem. 2004; 279: 39751-39756Google Scholar, 14Nouwen N. Stahleberg H. Pugsley A.P. Engel A. EMBO J. 2000; 19: 2229-2236Crossref PubMed Scopus (108) Google Scholar)). These secretins translocate a variety of proteins and pili through the outer membrane. However, Wza shares no sequence homology with them (6Drummelsmith J. Whitfield C. Mol. Microbiol. 1999; 31: 1321-1332Crossref PubMed Scopus (123) Google Scholar, 7Drummelsmith J. Whitfield C. EMBO J. 2000; 19: 57-66Crossref PubMed Scopus (129) Google Scholar). The remaining outer membrane component Wzi is required for surface association of the capsule (15Rahn A. Beis K. Naismith J.H. Whitfield C. J. Bacteriol. 2003; 185: 5882-5890Crossref PubMed Scopus (67) Google Scholar), and the means of capsule attachment is currently unclear. The polymerization and assembly of group 1 capsular polymer appears to be a coupled process in that mutants defective in late steps do not accumulate intracellular or periplasmic polymer (6Drummelsmith J. Whitfield C. Mol. Microbiol. 1999; 31: 1321-1332Crossref PubMed Scopus (123) Google Scholar, 16Wugeditsch T. Paiment A. Hocking J. Drummelsmith J. Forrester C. Whitfield C. J. Biol. Chem. 2001; 276: 2361-2371Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 20Nesper J. Hill C.M.D. Paiment A. Harauz G. Beis K. Naismith J.H. Whitfield C. J. Biol. Chem. 2003; 278: 49763-49772Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). This would suggest a connection between the inner and outer membranes, and Wza can, in fact, be cross-linked to an integral inner membrane component, Wzc in vivo (20Nesper J. Hill C.M.D. Paiment A. Harauz G. Beis K. Naismith J.H. Whitfield C. J. Biol. Chem. 2003; 278: 49763-49772Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Analysis of the primary sequence of the highly conserved Wzc homolog from E. coli K12 predicts a bitopic protein with large cytoplasmic and periplasmic domains (17Doublet P. Grangeasse C. Obadia B. Vaganay E. Cozzone A.J. J. Biol. Chem. 2002; 277: 37339-37348Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), and no structural studies have been performed in detail on any member of the Wzc family. In Gram-positive bacteria, functional homologs of Wzc are composed of two polypeptides, one representing the periplasmic component and the other the cytoplasmic C-terminal domain (4Whitfield C. Paiment A. Carbohydr. Res. 2003; 338: 2491-2502Crossref PubMed Scopus (106) Google Scholar, 18Cozzone A.J. Grangeasse C. Doublet P. Duclos B. Arch. Microbiol. 2004; 181: 170-181Crossref Scopus (81) Google Scholar). The C-terminal cytoplasmic domain of Wzc from E. coli K30 contains tyrosine autokinase activity with seven tyrosine residues in the last 17 residues (ASSYYRYGHNHYGYSYYDKK721) (16Wugeditsch T. Paiment A. Hocking J. Drummelsmith J. Forrester C. Whitfield C. J. Biol. Chem. 2001; 276: 2361-2371Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 19Paiment A. Hocking J. Whitfield C. J. Bacteriol. 2002; 184: 6437-6447Crossref PubMed Scopus (97) Google Scholar). In the prototype system from E. coli K30, capsule assembly is dependent on the activity of Wzc and its cognate cytoplasmic phosphotyrosine phosphatase, (6Drummelsmith J. Whitfield C. Mol. Microbiol. 1999; 31: 1321-1332Crossref PubMed Scopus (123) Google Scholar, 16Wugeditsch T. Paiment A. Hocking J. Drummelsmith J. Forrester C. Whitfield C. J. Biol. Chem. 2001; 276: 2361-2371Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 19Paiment A. Hocking J. Whitfield C. J. Bacteriol. 2002; 184: 6437-6447Crossref PubMed Scopus (97) Google Scholar). Analysis of different mutants with in the or in the C-terminal domain that are essential for capsule assembly and that the process the of Wzc (16Wugeditsch T. Paiment A. Hocking J. Drummelsmith J. Forrester C. Whitfield C. J. Biol. Chem. 2001; 276: 2361-2371Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). single tyrosine is essential or required for in capsule it appears that the of tyrosine is important for A. Hocking J. Whitfield C. J. Bacteriol. 2002; 184: 6437-6447Crossref PubMed Scopus (97) Google Scholar). The for the is with a of during export (16Wugeditsch T. Paiment A. Hocking J. Drummelsmith J. Forrester C. Whitfield C. J. Biol. Chem. 2001; 276: 2361-2371Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). The of Wzc not the cell from K30 antigen but high molecular weight capsular polymers are (6Drummelsmith J. Whitfield C. Mol. Microbiol. 1999; 31: 1321-1332Crossref PubMed Scopus (123) Google Scholar, 16Wugeditsch T. Paiment A. Hocking J. Drummelsmith J. Forrester C. Whitfield C. J. Biol. Chem. 2001; 276: 2361-2371Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 19Paiment A. Hocking J. Whitfield C. J. Bacteriol. 2002; 184: 6437-6447Crossref PubMed Scopus (97) Google Scholar, 20Nesper J. Hill C.M.D. Paiment A. Harauz G. Beis K. Naismith J.H. Whitfield C. J. Biol. Chem. 2003; 278: 49763-49772Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Wzc provide a role in capsule and Wza to the inner membrane. studies of the protein in export in these molecular are important in the of capsular polysaccharide biosynthesis and the of this essential in many this complex system, we have the three-dimensional structure of the inner membrane protein Wzc to a of This is the first structural data on this important of integral inner membrane protein. Wzc of the E. coli Wzc with an in the (20Nesper J. Hill C.M.D. Paiment A. Harauz G. Beis K. Naismith J.H. Whitfield C. J. Biol. Chem. 2003; 278: 49763-49772Abstract Full Text Full Text PDF PubMed Scopus (75) Google J. J. Bacteriol. PubMed Scopus Google and into E. coli (16Wugeditsch T. Paiment A. Hocking J. Drummelsmith J. Forrester C. Whitfield C. J. Biol. Chem. 2001; 276: 2361-2371Abstract Full Text Full Text PDF PubMed Scopus (157) Google cells. of to bacteria of Wzc at with and the for a 4 at The then by at for and the cell in and 1 using a and the by in and transmission electron and at 4 by at for 1 at 4 and the a with and with a using the 1 The then and and a The then using of the with one major at The in and that the Wzc protein in a phosphorylated state with C-terminal residues not of Oligomeric performed using a on E. Biochem. J. 1999; PubMed Scopus Google Scholar). Wzc with and and at 4 for then and in a and for 2 at 4 and to 4 for of and side on the surface of a of for 2 and then on a of for then and data in system at ∼100 K. for each oligomeric complex and as in microscopy and analysis at range to to of at in a and from using and The for each using the W. J. Biol. 1999; PubMed Scopus Google Scholar), and for and for in each using a structure to by for A of then with the complex in particle R.F. Frye S.A. Kitmitto A. Ford R.C. Tonjum T. Derrick J.P. J. Biol. Chem. 2004; 279: 39751-39756Google Scholar), a preliminary three-dimensional model from that represented distinct of the Wzc The of the characteristic using a common and the to the preliminary three-dimensional The three-dimensional structure using C4 using of with each by by of the of the three-dimensional from each The three-dimensional of and by analysis as R.F. Frye S.A. Kitmitto A. Ford R.C. Tonjum T. Derrick J.P. J. Biol. Chem. 2004; 279: 39751-39756Google Scholar). of of with of for at 4 then at in a for as structure of A of the protein with of the for to the Wzc structure using residues of of the a region that shares and with a continuous region of the Wzc sequence and which to of its cytoplasmic domain. The for the and that many of the residues are involved at the not of Wzc an of a of cryo-negatively can be the of complexes and in The in a and of for analysis in this of high be from the of the of particle between and Å in with a square appearance and a large stained The other of and a appearance with a the center of the Analysis of the using two-dimensional identified many of the Wzc some of which are in The the particle top which an rotational symmetry The a symmetry and the side of the Wzc oligomer The other represented between these two central The that the the in and for using W. J. Biol. 1999; PubMed Scopus Google Scholar). of C4 in Wzc to the of on other demonstrated that Wzc has a tetrameric organization. of Wzc using produced one which at The nature of these Wzc using is a that high in many membrane protein complexes E. Biochem. J. 1999; PubMed Scopus Google Scholar). of the Wzc clearly identified two that at and that the Wzc is kDa, the are as and tetrameric Wzc species. The of the tetramer is not an of the protein and is across a range of protein with molecular in of no oligomer complexes by These therefore contacts in or to the molecular mass we the symmetry in the two-dimensional of rotational analysis to the single particle data This the of a C4 for and C4 rotational with the from the C4 structure on the Wzc data therefore performed using C4 symmetry. of the Wzc three-dimensional structure calculated from the Wzc single particle data with C4 symmetry A molecular envelope of Wzc at 1 the would a molecular mass of kDa, a protein of M. C. Full Text Full Text PDF PubMed Scopus Google Scholar). This data is with a protein complex of this Wzc tetramer of with associated The of the to be Å using the for J. of Press, Google The structure and analysis of the revealed no significant in data through three-dimensional a surface of the Wzc oligomer Viewed from the at a of 2 the the complex is with small the The top of the complex is with a square of Å and a of The side of Wzc it is Å high) and has a distinctive appearance similar to an extracted molar with distinctive “crown” and separate The upper crown is ∼55 Å high and forms a ring of Å the of the From the underside of the four unconnected Å high the of the Wzc structure in an in vivo the of the E. coli inner membrane by in hydrated is Å A. J. J. Bacteriol. 2003; 185: Scopus Google Scholar). of the Wzc in the is a bitopic inner membrane and the periplasmic of the domain by the two has been by (17Doublet P. Grangeasse C. Obadia B. Vaganay E. Cozzone A.J. J. Biol. Chem. 2002; 277: 37339-37348Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). The be cytoplasmic to tyrosine and as a the also be in the the membrane of the Wzc three-dimensional structure in a the for this is a highly that is and Using to oligomeric complexes also single particle the electron as a for two-dimensional a of cryo-negatively stained Wzc which have been with These Wzc complexes in appearance to but can be to the a data we to a three-dimensional structure at Å with the in found in two on the upper of the root and at the of the cavity formed by the the crown. this is not to the protein it also nonspecific or However, the of in that the the and are in the whereas the crown is in the the of the on a the is with of the protein We therefore that the root contains the tyrosine domain and is the structure of ATP-binding from P. into an individual root and is a between the model and molecular envelope of and this is with of the two It be at Å the model can as an and of the model the root domain is not The appearance of the structure this the are and as would be for a of the tetramer in the cytoplasmic membrane. The of into the root of the Wzc three-dimensional The is at 1 and the and the is in The on the the of Wzc with the corresponding domain the three-dimensional E. coli K12 and K30 Wzc homologs sequence and and they to similar Whitfield C. J. Bacteriol. PubMed Scopus Google Scholar). The K12 homolog has been to form molecular weight as and with (17Doublet P. Grangeasse C. Obadia B. Vaganay E. Cozzone A.J. J. Biol. Chem. 2002; 277: 37339-37348Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). However, the that Wzc can be cross-linked to Wza (20Nesper J. Hill C.M.D. Paiment A. Harauz G. Beis K. Naismith J.H. Whitfield C. J. Biol. Chem. 2003; 278: 49763-49772Abstract Full Text Full Text PDF PubMed Scopus (75) Google these The data of the of Wzc of Wzc is by contacts with the periplasmic of the protein. this domain contains with a high of structures R. C. 2000; PubMed Scopus Google Scholar), which are in protein-protein in a variety of different systems R. J. Biol. 2001; Google Scholar). we no between the an is from the of the protein to (16Wugeditsch T. Paiment A. Hocking J. Drummelsmith J. Forrester C. Whitfield C. J. Biol. Chem. 2001; 276: 2361-2371Abstract Full Text Full Text PDF PubMed Scopus (157) Google Wzc complexes be involved in this but The role of proteins and in that would between the and are the of this complexes have been identified in other assembly and some of these intracellular proteins with the type pathway in requires the cytoplasmic for of a with membrane proteins and M. Mol. Microbiol. 2001; PubMed Scopus Google Scholar). This to to protein export through the type complex M. J. Bacteriol. PubMed Scopus Google Scholar, M. J. Mol. Biol. 2003; PubMed Scopus Google Scholar). is proposed to form a ring-like and it represents a family involved in protein secretion in M. J. Bacteriol. PubMed Scopus Google Scholar). In the type secretion system of is one of at associated with the inner membrane M. J. Bacteriol. PubMed Scopus Google Scholar). In a Wzc be involved in the assembly of a functional capsule assembly complex or in for capsule Wzc is not in sequence or domain to these and the mutants provide no into the structure is from a phosphorylated Wzc is not essential for oligomer not and its role be to in a complex or the of Wzc with data clearly that Wza and Wzc (20Nesper J. Hill C.M.D. Paiment A. Harauz G. Beis K. Naismith J.H. Whitfield C. J. Biol. Chem. 2003; 278: 49763-49772Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). the periplasmic domain in these and there is an of sequence in the of Wza homologs by cognate Wzc proteins Whitfield C. J. Bacteriol. PubMed Scopus Google Scholar). These are essential for in capsule assembly. The oligomeric structure of Wza is an octamer (9Beis K. Collins R.F. Ford R.C. Kamis A.B. Whitfield C. Naismith J.H. J. Biol. Chem. 2004; 279: 28227-28232Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 20Nesper J. Hill C.M.D. Paiment A. Harauz G. Beis K. Naismith J.H. Whitfield C. J. Biol. Chem. 2003; 278: 49763-49772Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), whereas Wzc is a the in it is that Wza and Wzc rotational symmetry and have similar This is that a complex forms between the two during a complex it would the periplasmic complexes are and for systems (e.g. the type export system requires the assembly of the complex T. E. C. EMBO J. 1998; PubMed Scopus Google and the from E. coli H. J. Mol. Microbiol. 2001; Google Scholar)). In these structural are a has been performed on the complex of the type system T. A. 2004; PubMed Scopus Google Scholar). However, the in the process and proteins involved in type secretion and involved in capsular polysaccharide export any from the type protein structural structural studies be required to the putative microscopy provides a to the structure and of the capsule export system the of this it is that the of be to study in the A structural of the mechanisms of capsule export would not provide a for the of but also and Gram-negative secretion in We and for microscopy and and Derrick for

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