Capsular and exopolysaccharides play crucial roles in the biology of many bacteria, acting either as virulence determinants that withstand host-cell defenses, or in establishing symbiotic relationships between bacteria and plants. More than 80 different capsular structures (known as K antigens) are produced by Escherichia coli isolates and these are subdivided into four different groups based on genetic and biochemical criteria (1). Surface polysaccharides with similar features are formed by other bacterial genera. The his-linked cps loci encode enzymes for the assembly of group 1 capsular K-antigens in E. coli and Klebsiella pneumoniae. The cps loci all contain a conserved region comprising the first 4 genes (orfX, wza, wzb, and wzc cps ) (2), indicating a shared role in CPS 1 expression. Following the conserved genes is a serotype-specific region encoding enzymes that participate in synthesis of polysaccharide repeat units and their polymerization via a Wzy-dependent mechanism (3). The Wzy-mediated polymerization reaction is thought to result in formation of an undecaprenyl pyrophosphate-linked glycan at the periplasmic face of the plasma membrane. The nascent polymer is then translocated to the cell surface via a process that requires outer membrane complexes formed by multimers of Wza cps (4). These complexes resemble the "secretins" for secretion of proteins via type II and type III systems.

No abstract

Wzc CPS is a tyrosine autokinase essential for the assembly of a high-molecular-weight (HMW) group 1 capsular polysaccharide (CPS) in Escherichia coli. Homologues of Wzc participate in the formation of CPS and exopolysaccharides in a variety of gram-positive and gram-negative bacteria. Phosphorylation of tyrosine residues in the Wzc CPS C terminus is essential for HMW CPS assembly. Overexpression of Wzb CPS (phosphatase) in a wild-type background caused a 3.7-fold decrease in the amount of cell-associated K30 CPS produced, confirming the importance of Wzc CPS phosphorylation for capsule assembly. In this study, the tyrosine-rich region was dissected in an attempt to identify residues critical for Wzc CPS phosphorylation and/or capsule expression. Site-directed mutagenesis demonstrated that no single tyrosine residue in this region is sufficient for detectable phosphorylation of Wzc CPS in vivo or for HMW CPS expression. Furthermore, no single tyrosine residue is essential for phosphorylation or capsule assembly, since removal of any one tyrosine residue has no detectable effect. Altering combinations of tyrosine residues (from two to five) led to Wzc CPS derivatives that were still competent for phosphorylation but that could not support assembly of HMW CPS, showing that phosphorylation of Wzc per se is not an accurate measure of its ability to function in capsule assembly. One interpretation of these data is that the overall level of phosphorylation in this region, rather than the precise combination of residues accessible to phosphorylation, is important for the activity of Wzc CPS . Tyrosine 569, a residue shown to modulate the in vitro phosphorylation of Wzc CA from E. coli K-12, was also mutated. The derivative with this mutation still functioned in capsule assembly. Quantitation of K30 CPS from this mutant revealed no difference in the amount of polymer produced. Finally, dithiobis(succinimidylpropionate) cross-linking was used to confirm that Wzc CPS forms complexes in vivo, independent of the phosphorylation state of the protein.Capsular polysaccharides (CPS) and exopolysaccharides (EPS) are important virulence determinants in many plant and animal pathogens and are also important for symbiotic interactions in some plant-associated bacteria. More than 80 types of capsular or K antigens have been identified in Escherichia coli, and these have been classified into groups 1 through 4 based on genetic and biosynthetic criteria (41). Group 1 CPS of E. coli resemble the capsules of Klebsiella pneumoniae, and group 1-like EPS are found in E. coli (colanic acid), Erwinia amylovora (amylovoran), and Sinorhizobium meliloti (succinoglycan), to name a few.E. coli group 1 CPS are assembled via a Wzy-dependent pathway. The current model (reviewed in reference 41) is based extensively on evidence gathered from the parallel system for lipopolysaccharide (LPS) O-antigen assembly, in which repeat units are assembled on undecaprenol phosphate at the cytoplasmic face of the inner membrane by the sequential activity of glyco...

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...

The purpose of this investigation was to determine the effect of Thiobacillus acidophilus on the leaching of a low-grade Cu-Ni sul de ore by Thiobacillus ferrooxidans. A sample of low-grade Cu-Ni sul de ore containing 0.36% Cu, 0.48% Ni, and 7.87% Fe was pulverized and initially leached for a 21-day period using two different pure cultures of T: ferrooxidans, an environmental strain (F2) and a strain from the American Type Culture Collection (ATCC 23270). Samples of the ore slurries were drawn and the pH was monitored over the course of the leaching period. The concentrations of Cu and Ni leached by each strain were determined and compared. No signi cant differences were observed in the concentrations of Cu and Ni leached by the two pure cultures of T: ferrooxidans. Subsequently, the ore was leached with mixed cultures of T: ferrooxidans and T: acidophilus to determine the effect of the latter on the concentrations of Cu and Ni leached from the ore. The environmental strain F2 of T: ferrooxidans was used in combination with both a type strain (ATCC 27807) and an environmental strain (64) of T: acidophilus. After 21 days, the mixed cultures of T: ferrooxidans and T: acidophilus leached signi cantly greater amounts of copper than the pure strain alone, but no such difference was observed for the leaching of nickel.