Abstract:In Once considered as a phytopathogen, Burkholderia cepacia, a multi-drug-resistant bacteria, is now recognized as an important pathogen in the lung of patients with cystic fibrosis (CF) and can lead to severe pneumonia and death (15). B. cepacia strain KF1 isolated from a non-CF patient with pneumonia, produces extracellular metalloprotease in large quantities (37), and causes lung infections in mice on intratracheal inoculation (42). We have been analyzing the mechanism of protease production to explore its involvement in the pathogenesis.In a previous study, we showed that the protease-negative, lipase-positive mutant KFT1007, a Tn5-Tp insertion mutant of B. cepacia KF1, was impaired in the dsbB gene which encodes a membranebound disulfide bond oxidoreductase, DsbB, resulting in secretion of a premature and catalytically inactive form of protease (1). In Escherichia coli, DsbB couples with DsbA, a periplasmic disulfide bond oxidoreductase that directly makes S-S bonds on target molecules (26). Thus, the reduced-DsbA is reoxidized by oxidized-DsbB that is recycled with the aid of a respiratory electron transfer chain (14). The DsbA-DsbB circuit in E. coli is involved in flagellar basal body assembly (9), type IV pilin biogenesis (44), and the maturation of heat-stable enterotoxin (22) and alkaline phosphatase (10). In some Gram-negative bacteria, homologs of E. coli DsbA and their target proteins are found in Vibrio cholerae TcpG for enterotoxin (23)

Thiaminase I (EC 2.5.1.2) catalyzes the replacement of the thiazole moiety of thiamin with a wide variety of nucleophiles. Here we report the sequencing of a thiaminase I clone from Bacillus thiaminolyticus, the overexpression of the cloned gene in Escherichia coli, and the purification and characterization of the enzyme. Recombinant thiaminase I functions as a monomer with a Km for thiamin of 3.7 +/- 0.6 microM and a kcat of 34 s-1. Electrospray ionization Fourier-transform mass spectrometry identified a single sequencing error and demonstrated heterogeneity, finding molecular weights of 42,127, 42,198, and 42,255 due to added Ala and Gly-Ala at the amino terminus. Similar analysis of the 4-amino-2-methyl-6-chloropyrimidine inactivated enzyme indicated that the active site nucleophile involved in catalysis of the substitution reaction is located between Pro79 and Thr177. Subsequent cysteine-specific labeling and site-directed mutagenesis identified Cys113 as the active site nucleophile.

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Hemolytic and antifungal substances, cepalycin I and cepalycin II, have been isolated from Pseudomonas cepacia JN106. A large amount of cepalycins were produced by growing the cells on 1% glycerin‐nutrient agar medium covered with a cellophane membrane. The cell‐washed supernatant was applied to an Amberlite XAD2 column, and cepalycins were eluted with 70% ethanol containing 1mM HCl. Cepalycins were separated by reverse phase HPLC in two fractions which were designated as cepalycin I and cepalycin II. The two cepalycins have indistinguishable UV absorption spectra but have different levels of hemolytic activity relative to the UV absorption. From the inhibition of hemolytic activity of cepalycin by sterols, both cepalycins were suggested to interact with cholesterol in erythrocyte membrane. Such an interaction may contribute to their hemolytic and antifungal activities.