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Vibrio vulnificus, a marine bacterium capable of causing wound infection and septicemia, secretes a 45-kDa metalloprotease (vEP) with many biological activities. The precursor of vEP consists of four regions: a signal peptide, an N-terminal propeptide (nPP), a C-terminal propeptide, and the mature protease. Two forms of vEP--vEP-45, which contains the mature protease plus the C-terminal propeptide, and vEP-34, which contains only the mature protease--were expressed in Escherichia coli and purified. vEP-45 and vEP-34 had similar activities with azocasein as a substrate, but vEP-34 had reduced activity toward insoluble proteins. The nPP of vEP was expressed as a His tag fusion protein, and its effect on vEP activity was investigated. nPP inhibited the activities of both vEP-45 and vEP-34 but not that of thermolysin, a different but related zinc-dependent protease. The inhibition of vEP by nPP was further examined using vEP-34 as a representative enzyme. The inhibition could be completely reversed under conditions of low enzyme and propeptide concentrations and with prolonged incubation, which resulted from the degradation of nPP by vEP. However, even at high nPP and vEP concentrations, inhibition of vEP by nPP at high temperatures was not effective, resulting in the degradation of both nPP and vEP. These results demonstrate that the nPP of vEP could bind to vEP and inhibit its activity, resulting in the degradation of the propeptide.

Vibrio vulnificus is a causative agent of serious food-borne diseases in humans related to the consumption of raw seafood. It secretes a metalloprotease that is associated with skin lesions and serious hemorrhagic complications. In this study, we purified and characterized an extracellular metalloprotease (designated as vEP) having prothrombin activation and fibrinolytic activities from V. vulnificus ATCC 29307. vEP could cleave various blood clotting-associated proteins such as prothrombin, plasminogen, fibrinogen, and factor Xa, and the cleavage could be stimulated by addition of 1 mM [Mn.sup.2+] in the reaction. The cleavage of prothrombin produced active thrombin capable of converting fibrinogen to fibrin. The formation of active thrombin appeared to be transient, with further cleavage resulting in a loss of activity. The cleavage of plasminogen, however, did not produce an active plasmin, vEP could cleave all three major chains of fibrinogen without forming a clot. It could cleave fibrin polymer formed by thrombin as well as the cross-linked fibrin formed by factor XIIIa. In addition, vEP could also cleave plasma proteins such as bovine serum albumin and gamma globulin, and its broad specificity is reflected in the cleavage sites, which include [Asp.sup.207]-[Phe.sup.208] and [Thr.sup.272]-[Ala.sup.273] bonds in prothrombin and a [Tyr.sup.80]-[Leu.sup.81] bond in plasminogen. Taken together, the data suggest that vEP is a broad-specificity protease that could function as a prothrombin activator and a fibrinolytic enzyme to interfere with blood homeostasis as part of the mechanism associated with the pathogenicity of V. vulnificus in humans and thereby facilitate the development of systemic infection.

The side chain of residue 561 and the backbone atoms of residue 560 are critical for the proper function of pyruvate decarboxylase from Zymomonas mobilis. The carboxy-terminus locks in the cofactors and closes the active site in preparation for decarboxylation.

Background: The success of cuckoo parasitism is thought to depend largely on the extent of egg matching between cuckoo and host eggs, since poor-matching cuckoo egg would lead to more frequent egg rejection by the host. In this study, we investigated how egg-spot matching between the Common Cuckoo (Cuculus canorus) and its host, the Oriental Reed Warbler (Acrocephalus orientalis) is affected by the local parasitism rate, nest availability in breeding synchronization and egg rejection. Methods: We used the paired design of parasitized and their nearest non-parasitized nests where breeding occurred simultaneously to compare egg-spot matching. The image analysis was used to compare four eggshell pattern variables, namely spot size, density, coverage on the different areas of egg surface, and the distribution on the whole egg surface. Egg recognition experiments were conducted to test the effect of egg spots on egg rejection by the host. Results: Our results show that much better matching in almost all spot parameters tested on the side of the egg and the spot distribution on the whole egg occurred in parasitized nests than in non-parasitized nests. Matching of spot density between cuckoo and host eggs in parasitized nests increased with the synchronization between temporal availability of nests and the egg-laying period of female cuckoos. Egg recognition experiments in which the warbler eggs were deliberately painted with extra spots led to a significantly higher egg rejection rate (78.3%) than of unpainted eggs. Conclusion: Our data suggest that both the high temporal encounter rate between cuckoo and warbler nests as well as the high egg rejection ability of the host are important factors for egg-spot matching of the cuckoos. [ABSTRACT FROM AUTHOR]