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When Lactobacillus planfarum ATCC 8014 was maintained in LCM broth (which consists of buffered tryptone and is sufficient to support the growth of some species of Lactobacillus) for long periods (1120 d), viable bacteria persisted. Rifampicin-, streptomycin-and sodium-fusidate-resistant mutants were recovered from parallel LCM broth cultures following a stochastic pattern. Individual cultures appeared to yield mutants intermittently. One culture in particular yielded rifampicin-resistant colonies at a frequency of 1 in I00 viable bacteria after 20 d incubation and these persisted until the experiment was terminated a t 115 d. In a separate experiment two parallel cultures yielded mutants resistant to low concentrations of streptomycin a t a similar frequency. Using a chemostat it was shown that in continuous culture in LCM at slow growth rates the highest frequency of recovery of antibiotic-resistant mutants was achieved when the bacteria exhibited doubling times of 90 h or greater. The frequency of recovery of mutants was as high as 1 in 1000 viable bacteria. Thus, mutations to antibiotic resistance in L. plantarum ATCC 8014 can take place in the absence of measurable cell division. The data are consistent with the notion that populations of starved bacteria in stationary phase can be genetically dynamic.

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Cointegrates generated between a plasmid pIP501 deletion derivative (pVA797) and nonconjugative shuttle vector pSA3 were confirmed as capable of exconjugation from lactococci into a range of strains ofLactobacillus helveticus with the concomitant expression of a recombinant gene. The plasmid cointegrate that was formed appeared to be segregationally stable at 37°C in some host strains. In all strains, however, the plasmid became increasingly unstable as the incubation temperature was raised. The technique offers not only a generalized method for the introduction of novel genetic material into this important industrial microbe but also the possibility of exploiting the thermal sensitivity of the plasmid to enable it to act as a delivery system for the integration of cloned genes into the bacterial chromosome, at restrictive temperatures, by recombination at regions of homology.