Although the antibiotic thiostrepton is best known as an inhibitor of protein synthesis, it also, at extremely low concentrations (< 10-9 M), induces the expression of a regulon of unknown function in certain Streptomyces species. Here, we report the purification of a Streptomyces lividans thiostrepton-induced transcriptional activator protein, TipAL, whose N-terminus is similar to a family of eubacterial regulatory proteins represented by MerR.TipAL was first purified from induced cultures of S.lividans as a factor which bound to and activated transcription from its own promoter. The tipAL gene was overexpressed in Escherichia coli and TipAL protein purifi'ed in a single step using a thiostrepton affinity column. Thiostrepton enhanced binding of TipAL to the promoter and catalysed specific transcription in vitro.TipAs, a second gene product of the same open reading frame consisting of the C-terminal domain of TipAL, is apparently translated using its own in-frame initiation site. Since it is produced in large molar excess relative to TipAL after induction and also binds thiostrepton, it may competitively modulate transcriptional activation.

Bacteriophage #393-A2, isolated from an artisanal cheese whey sample, is a temperate phage able to generate stable lysogens through integration of its DNA into the bacterial genome. One-step growth kinetics of its lytic development revealed eclipse and latent periods of 100 and 140 min, respectively, with a burst size of about 200 p.f.u. per infected cell. #393-A2 virions have an isometric head and a long, non-contractile tail terminating in a baseplate. The capsid is composed of two major and at least nine minor structural polypeptides. The phage genome consists of a double-stranded DNA molecule of 44 kbp bearing 3'-protruding cohesive ends. A physical map of the phage DNA has been constructed for six restriction enzymes. The whole 4393-A2 genome has been cloned in Escherichia coli using plasmid-and phagederived cloning vectors.

Aims:The partial characterization of a bacteriocin produced by a human Lactobacillus delbrueckii isolate with probiotic potential. Methods and Results: A bacterocin, UO004, was partially puri®ed by cation exchange followed by a hydrophobic interaction column, biochemically characterized and the N-terminal region sequenced. Bacteriocin UO004 was found to be a hydrophobic, heat-stable polypeptide with an apparent molecular mass of 6 kDa. It was also stable and active over a wide pH range. Conclusions: The active compound was proteinaceous, heat-stable, and had a bactericidal (and bacteriolytic) mode of action on a limited number of micro-organisms. Such a narrow spectrum of activity is typical for bacteriocins produced by intestinal Lactobacillus. Signi®cance and Impact of the Study: Bacteriocin UO004 from a probiotic strain is a new compound that does not share any homology with any other known lactic acid bacteria bacteriocin. Furthermore, Lact. delbrueckii is regarded as a suitable starter for the production of fermented milks.

No abstract

activities have been detected in culture supernatant fluids of several Lactobacillus plantarum strains. In the case of Lact. plantarum HER 1325 at least, the extracellular activity is accompanied by a cytoplasmic restriction endonuclease. Among the secreted nucleases, the greatest activity was from Lact. plantarum ATCC 10241. This strain secretes an enzyme, with a molecular mass in the range 10-40 kDa, that cuts double-stranded DNA in a sequenceindependent way by initially introducing single-strand nicks in supercoiled molecules, followed by linearization and complete degradation of the substrate.

Some characteristics of the lytic development of the temperate phage phi C31 in Streptomyces coelicolor A3(2) were studied using a thermoinducible lysogen. The physiological state of the host and the culture medium influenced the production of progeny virus after induction. The latent period lasted 45 min and the rise period 20-30 min. RNA synthesis in induced cultures was reduced with respect to controls. This reduction was restricted to cellular transcription as evidenced by: no stable RNA being synthesized in induced cultures, and the proportion of phage specific RNA increasing from 0.5% before induction to more than 30% in induced cultures. Host RNA synthesis proceeded throughout the lytic cycle. Protein synthesis was also reduced in induced cultures, although to a lesser extent than RNA synthesis. Phage DNA synthesis started at around 10 min postinduction, marking the division between the early and late periods of phage development. Host DNA synthesis occurred during the first 20 min after induction, and gradually decreased later.

Some general characteristics of five phages (Mrnl, 4M2, $M3, Mm4 and Mm5) infecting Micrornonospora are presented. All were naked, showing an icosahedral head and long noncontractile tail; they differed in their size and the presence of specific structures at the end of the tail. The phages were temperate, and four immunity groups were delimited (Mm4 and Mm5 were in the same group). Mm4 and Mm5 produced plaques on 13 of 20 strains of Micromonospora tested, whereas the remaining three phages could infect only Micromonospora sp. IMET 8002. No phage was able to infect any of four Streptomyces strains tested. Phages Mm4 and Mm5 both exhibited a buoyant density of 1.513 f 0.002 g ml-l in CsCl density gradients, and showed a similar pattern of structural polypeptides. All five phages had a single molecule of double-stranded DNA; their sizes (kb) were 50.6 (Mml), 18.9 (4M2), 65.5 ($M3), 44.0 (Mm4) and 44.4 (Mm5), as determined after digestion with 13 restriction enzymes. The restriction patterns of Mm4 and Mm5 showed the presence of common size fragments.

The introduction of bacteriophage DNA into Micromonospora protoplasts, resulting in the production of infective viral progeny, is reported. Transfection was affected by several factors. We observed that it reached a maximum when protoplasts from young mycelium (15 h old) were used. Maximum transfection took place when polyethylene glycol (PEG) was added to the mixtures at a final concentration of 20% (vol/vol) and did not occur at PEG concentrations under 10% or over 35%. The addition of positively charged liposomes to the mixtures was essential, since no transfectants were detected in the absence of liposomes at any PEG concentration. When DNA was present in nonlimiting amounts, a maximum efficiency of around 10(-3) to 10(-4) PFU per protoplast was obtained. The efficiency per DNA molecule showed a constant value of around 10(-4) to 10(-5) PFU, but the data suggest that transfection could be achieved by a single DNA molecule. The method proved to be equally efficient for the DNAs of at least five Micromonospora bacteriophages. On the contrary, we failed to transfect five of seven Micromonospora strains. These data suggest that only a minor subpopulation of protoplasts is competent and that the main factors influencing the transfection of Micromonospora protoplasts are neither the characteristics nor the origin of the DNA but the properties and status of the protoplasts.