A novel outer membrane protein, Wzi, is involved in surface assembly of the Escherichia coli K30 group 1 capsule.

Escherichia coli group 1 K antigens form a tightly associated capsule structure on the cell surface. Although the general features of the early steps in capsular polysaccharide biosynthesis have been described, little is known about the later stages that culminate in assembly of a capsular structure on the cell surface. Group 1 capsule biosynthesis gene clusters (cps) in E. coli and Klebsiella pneumoniae include a conserved open reading frame, wzi. The wzi gene is the first of a block of four conserved genes (wzi-wza-wzb-wzc) found in all group 1 K-antigen serotypes. Unlike wza, wzb, and wzc homologs that are found in gene clusters responsible for production of exopolysaccharides (i.e., predominantly cell-free polymer) in a range of bacteria, wzi is found only in systems that assemble capsular polysaccharides. The predicted Wzi protein shows no similarity to any other known proteins in the databases, but computer analysis of Wzi predicted a cleavable signal sequence. Wzi was expressed with a C-terminal hexahistidine tag, purified, and used for the production of specific antibodies that facilitated localization of Wzi to the outer membrane. Circular dichroism spectroscopy indicates that Wzi consists primarily of a beta-barrel structure, and dynamic light scattering studies established that the protein behaves as a monomer in solution. A nonpolar wzi chromosomal mutant retained a mucoid phenotype and remained sensitive to lysis by a K30-specific bacteriophage. However, the mutant showed a significant reduction in cell-bound polymer, with a corresponding increase in cell-free material. Furthermore, examination of the mutant by electron microscopy showed that it lacked a coherent capsule structure. It is proposed that the Wzi protein plays a late role in capsule assembly, perhaps in the process that links high-molecular-weight capsule to the cell surface.

Purification, crystallization and preliminary structural studies of dTDP-4-keto-6-deoxy-glucose-5-epimerase (EvaD) from Amycolatopsis orientalis, the fourth enzyme in the dTDP-L-epivancosamine biosynthetic pathway.

The vancomycin class of antibiotics is regarded as the last line of defence against Gram-positive bacteria. The compounds used clinically are very complex organic molecules and are made by fermentation. The biosynthesis of these is complex and fascinating. Its study holds out the prospect of utilizing genetic engineering of the enzymes in the pathway in order to produce novel vancomycin analogues. In part, this requires detailed structural insight into substrate specificity as well as the enzyme mechanism. The crystallization of one of the enzymes in the chloroeremomycin biosynthetic pathway (a member of the vancomycin family), dTDP-3-amino-4-keto 2,3,6-trideoxy-3-C-methyl-glucose-5-epimerase (EvaD) from Amycolatopsis orientalis, is reported here. The protein is fourth in the pathway which makes a carbohydrate essential for the activity of chloroeremomycin. The crystals of EvaD diffract to 1.5 A and have unit-cell parameters a = 98.6, b = 72.0, c = 57.1 A with space group P2(1)2(1)2. Data to this resolution were collected at the European Synchrotron Radiation Facility.