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SUMMARYA transcription/translation system for generating poliovirus proteins in vitro has been used to assess the proteolytic activity of various polypeptides containing the viruscoded 3C region towards the poliovirus precursor protein P1. Plasmids containing a phage T7 promoter followed by either the complete poliovirus Pl sequence or various sequences containing the 3C region were used for this purpose. We showed that all except one of the 3C-containing polypeptides had a very restricted activity towards P 1, generating only a small amount of VP1 and no VP0 or VP3. The only polypeptide capable of fully processing P1 into VP0, VP3 and VP1 in vitro was protein 3CD, consisting of the complete 3C and 3D sequences.

Two constructs derived from the alpha-amylase gene (amyA) of Lactobacillus amylovorus were expressed in Lactobacillus plantarum, and their expression products were purified, characterized, and compared. These products correspond to the complete (AmyA) and truncated (AmyADelta) forms of alpha-amylase; AmyADelta lacks the 66-kDa carboxyl-terminal direct-repeating-unit region. AmyA and AmyADelta exhibit similar amylase activities towards a range of soluble substrates (amylose, amylopectin and alpha-cyclodextrin, and soluble starch). The specific activities of the enzymes towards soluble starch are similar, but the K(M) and V(max) values of AmyADelta were slightly higher than those of AmyA, whereas the thermal stability of AmyADelta was lower than that of AmyA. In contrast to AmyA, AmyADelta is unable to bind to beta-cyclodextrin and is only weakly active towards glycogen. More striking is the fact that AmyADelta cannot bind or hydrolyze raw starch, demonstrating that the carboxyl-terminal repeating-unit domain of AmyA is required for raw-starch binding activity.

A 3.6-kb endogenous plasmid was isolated from a Propionibacterium freudenreichii strain and sequenced completely. Based on homologies with plasmids from other bacteria, notably a plasmid from Mycobacterium, a region harboring putative replicative functions was defined. Outside this region two restriction enzyme recognition sites were used for insertion of an Escherichia coli-specific replicon and an erythromycin resistance gene for selection in Propionibacterium. Hybrid vectors obtained in this way replicated in both E. coli and P. freudenreichii. Whereas electroporation of P. freudenreichii with vector DNA isolated from an E. coli transformant yielded 10 to 30 colonies per g of DNA, use of vector DNA reisolated from a Propionibacterium transformant dramatically increased the efficiency of transformation (>10 8 colonies per g of DNA). It could be shown that restriction-modification was responsible for this effect. The high efficiency of the system described here permitted successful transformation of Propionibacterium with DNA ligation mixtures.The genus Propionibacterium can be divided into two groups, a group containing the classical (or dairy) propionibacteria and a group containing the cutaneous propionibacteria (6). Members of the first group, especially Propionibacterium freudenreichii, play an essential role in the manufacture of Swiss and related types of cheeses (12). Other industrial applications are found in the production of propionic acid and vitamin B 12 (5, 28). Of growing interest, but less well documented, are the probiotic properties ascribed to some propionibacterial strains (16,21).Strain improvement and, in general, study of this economically important group of bacteria would be greatly facilitated by the availability of a system for genetic modification. This report describes isolation and characterization of a 3.6-kb plasmid and successful use of this plasmid in the construction of a set of Escherichia coli-Propionibacterium shuttle vectors. Reproducible transformation of P. freudenreichii strains with these shuttle vectors was achieved by means of electroporation. MATERIALS AND METHODSBacterial strains and plasmids. Propionibacterium strains were obtained from the Belgian Coordinated Collections of Microorganisms/LMG (Ghent, Belgium), from the American Type Culture Collection (Rockville, Md.), and from the Deutsche Sammlung von Mikroorganismen (Braunschweig, Germany). P. freudenreichii subsp. freudenreichii VTB1 was obtained from DSM Food Specialties' industrial collection. For amplification of newly constructed shuttle vector DNA E. coli DH5␣ was used. pBluescript SKIIϩ was obtained from Stratagene (La Jolla, Calif.).Media and growth conditions. E. coli DH5␣ was cultivated at 37°C in L medium (24) supplemented with 50 g of ampicillin per ml if necessary. Propionibacteria were cultivated anaerobically at 30°C in MRS (7) or SLB medium (8) supplemented with an appropriate antibiotic when plasmid isolation was to be performed or in SLB medium when electroporation was to be performed.Isolatio...

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