Identification and characterization of the Brucella abortus phosphoglucomutase gene: role of lipopolysaccharide in virulence and intracellular multiplication.

Smooth lipopolysaccharide (LPS) of Brucella abortus has been reported to be an important virulence factor, although its precise role in pathogenesis is not yet clear. While the protective properties of LPS against complement are well accepted, there is still some controversy about the capacity of rough mutants to replicate intracellularly. The B. abortus phosphoglucomutase gene (pgm) was cloned, sequenced, and disrupted. The gene has a high index of identity to Agrobacterium tumefaciens pgm but is not part of the glycogen operon. A B. abortus null mutant lacks LPS O antigen but has an LPS core with an electrophoretic profile undistinguishable from that of the wild-type core, suggesting that glucose, galactose, or a derivative of these sugars may be part of the linkage between the core and the O antigen. This mutant is unable to survive in mice but replicates in HeLa cells, indicating that the complete LPS is not essential either for invasion or for intracellular multiplication. This behavior suggests that the LPS may play a role in extracellular survival in the animal, probably protecting the cell against complement-mediated lysis, but is not involved in intracellular survival.

The activity of a putative polyisoprenol-linked sugar translocase (Wzx) involved in Escherichia coli O antigen assembly is independent of the chemical structure of the O repeat.

During O antigen lipopolysaccharide (LPS) synthesis in bacteria, transmembrane migration of undecaprenylpyrophosphate (Und-P-P)-bound O antigen subunits occurs before their polymerization and ligation to the rest of the LPS molecule. Despite the general nature of the translocation process, putative O-antigen translocases display a low level of amino acid sequence similarity. In this work, we investigated whether complete O antigen subunits are required for translocation. We demonstrate that a single sugar, GlcNAc, can be incorporated to LPS of Escherichia coli K-12. This incorporation required the functions of two O antigen synthesis genes, wecA (UDP-GlcNAc:Und-P GlcNAc-1-P transferase) and wzx (O-antigen translocase). Complementation experiments with putative O-antigen translocases from E. coli O7 and Salmonella enterica indicated that translocation of O antigen subunits is independent of the chemical structure of the saccharide moiety. Furthermore, complementation with putative translocases involved in synthesis of exopolysaccharides demonstrated that these proteins could not participate in O antigen assembly. Our data indicate that recognition of a complete Und-P-P-bound O antigen subunit is not required for translocation and suggest a model for O antigen synthesis involving recognition of Und-P-P-linked sugars by a putative complex made of Wzx translocase and other proteins involved in the processing of O antigen.

The precursor of pea ferredoxin-NADP+ reductase synthesized in Escherichia coli contains bound FAD and is transported into chloroplasts.

The precursor of the chloroplast flavoprotein ferredoxin-NADP+ reductase from pea was expressed in Escherichia coli as a carboxyl-terminal fusion to glutathione S-transferase. The fused protein was soluble, and the precursor could be purified in a few steps involving affinity chromatography on glutathione-agarose, cleavage of the transferase portion by protease Xa, and ion exchange chromatography on DEAE-cellulose. The purified prereductase contained bound FAD but displayed marginally low levels of activity. Removal of the transit peptide by limited proteolysis rendered a functional protease-resistant core exhibiting enzymatic activity. The FAD-containing precursor expressed in E. coli was readily transported into isolated pea chloroplasts and was processed to the mature size, both inside the plastid and by incubation with stromal extracts in a plastid-free reaction. Import was dependent on the presence of ATP and was stimulated severalfold by the addition of plant leaf extracts.

Corneal penetration of rifampin.

We tested the corneal penetration of rifampin in four vehicles: dimethylsulfoxide, polyethylene glycol, an ocular lubricant, and as rifampin ointment. We measured drug concentrations in the aqueous humor in rabbits after topical instillation of 1 and 2.5% rifampin according to two dosage schedules. Drug concentrations in the aqueous humor were bactericidal to Mycobacterium leprae. Since leprosy of the cornea, iris, and ciliary body may develop despite standard systemic bacteriostatic treatment, treatment of leprotic involvement of the anterior eye may be enhanced by intensive topical application of rifampin.