Partial characterization of a bacteriocin produced by Lactobacillus delbrueckii subsp. lactis UO004, an intestinal isolate with probiotic potential.

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 purified 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. CONCLUSION: 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. SIGNIFICANCE 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.

Autogenous transcriptional activation of a thiostrepton-induced gene in Streptomyces lividans.

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 purified 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.

Structure of the DNA of five bacteriophages infecting Micromonospora.

The physical maps of the DNA of five bacteriophages (Mm1, OM2, OM3, Mm4 and Mm5) which infect Micromonospora are presented. The restriction analyses showed that all of them had linear, double-stranded DNA, but only four (Mm1, OM2, Mm4 and Mm5) presented cohesive ends. The phages showed no relationship in terms of their restriction maps or of DNA-DNA hybridization, with the exception of Mm4 and Mm5, which resulted to be very similar. Phage Mm5 presented a high level of resistance to chelating agents, although deletion mutants, all of them showing a single detection of 1.4 kb, were obtained by using extremely selective conditions.

Transfection in Micromonospora spp.

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.

Characteristics of the developmental cycle of actinophage phi C31.

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.