After cultivation under inducing conditions (i e , addition of 30

After cultivation under inducing conditions (i.e., addition of 30 μM CuSO4), the strain was mixed with 100 ng of pVI1056 and plated on selective medium. Experiments were performed under various conditions: i) glucose concentration at 1% or 0.1%, ii) growth in microaerobiosis or aeration, and induction at early, middle or late exponential phase iii) addition of MgCl2 (80 mM) during contact between cells and DNA, after middle phase induction in microaerobiosis or aeration; in addition, chromosomal L. sakei DNA (1 μg) was also used as exogenous DNA. None of the tested conditions resulted in DNA transformation. Development of natural transformation see more may be strain-dependent [30, 38, 39]. We therefore used a second strategy (independent

of sigH overexpression) to test different L. sakei isolates for competence, using a protocol where DNA and strains are deposited on solid medium. In addition to 23 K, four strains (64 K, 332 F, 160 K and LTH675) were chosen based on their different genotypes and genome sizes, and known capacities to be transformed by electroporation [20, 58]; Chaillou and Anba, personal communication]. Two replicative plasmids and chromosomal L. sakei DNA were used. In spite of varying media (MRS or MCD) and incubation temperatures (4°C, 30°C or 37°C), no colonies CP690550 were recovered on

selective medium. Among the Lactobacillales, natural genetic transformation has been reported for many species of the genus Streptococcus [40] and has been suspected for one Methane monooxygenase Lactobacillus [41]. In recent years, natural transformation has been demonstrated in several Gram-positive or Gram-negative species, previously unsuspected

to develop genetic competence [42, 43]. Overproduction of the activator protein has proven to be an efficient way to trigger genetic transformation in various bacteria, e.g., TfoX in Vibrio cholerae [42] or ComK in Bacillus species [14, 44]. However, artificially raising transcription of the ComX master regulator gene initially failed to induce efficient genetic transformation for S. thermophilus strain LMD-9 [30], which was very recently shown to be efficiently naturally transformable [37]. In the present and previous studies, a failure to achieve a competent state in bacteria (either spontaneously or triggered by artificial overexpression of a master activator) may be due to the use of inappropriate growth conditions, which might not allow the detection by the cells of a needed specific triggering factor [38, 42] or the full activation of multiple converging regulatory pathways [30]. As such, in the case of L. lactis [21], S. pyogenes [45], S. aureus [12], or L. sakei (this paper), only the activation of several competence genes, but not genetic transformation, could be obtained after ectopic expression of the activating sigma factor. Our results suggest that some of the genes induced in other naturally competent Firmicutes are not activated by the sole sigH Lsa overexpression in L. sakei.

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