~Acinetobacter calcoaceticus BD413 develops competence for natural transformation immediately after the start of the exponential growth-phase and remains competent up to a few hours into the stationary phase, after which competence gradually declines. The transformation frequencies obtained strongly depend on the kind of transforming DNA and the incubation time with DNA. Up to 25% of the cells in a culture can be transformed. DNA uptake in Acinetobacter does not display sequence specificity, is Mg2+-, Mn2+-or Ca2+dependent and is uncoupler sensitive. The transforming DNA enters the cells in single-stranded form. These properties constitute a unique combination, not previously observed in other bacteria, and make A. calcoaceticus ideally suited for detailed studies of the bioenergetics of DNA translocation.

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We develop two novel transition path sampling (TPS) algorithms for harvesting ensembles of rare event trajectories using non-equilibrium dynamics. These methods have the advantage that no predefined reaction coordinate is needed. Instead, an instantaneous reaction coordinate is based on the current path. Constituting a Monte Carlo random walk in trajectory space, the algorithms can be viewed as bridging between the original TPS methodology and the Rosenbluth based forward flux sampling methodology. We illustrate the new methods on toy models undergoing equilibrium and non-equilibrium dynamics, including an active Brownian particle system. For the latter, we find that transitions between steady states occur via states that are locally ordered but globally disordered.

Acinetobacter calcoaceticus induced competence for natural transformation maximally after dilution of a stationary culture into fresh medium. Competence was gradually lost during prolonged exponential growth and after entrance into the stationary state. Growth cessation and nutrient upshift were involved in the induction of competence. The level of competence of a chemostat culture of A. calcoaceticus was dependent on the nature of the growth limitation. Under potassium limitation a transformation frequency of + 1 x 10 .4 was obtained. This frequency was independent of the specific growth rate. In phosphate-, nitrogen-, and carbon-limited chemostat cultures, in contrast, the transformation frequency depended on the specific growth rate; the transformation frequency equalled _+ 10.4 at dilution rates close to g,~a• of 0.6h -1 and decreased to _+ 10 -7 at a dilution rate of 0.1 h-L We conclude that (1) DNA uptake for natural transformation in A.calcoaceticus does not serve a nutrient function and (2) competence induction is regulated via a promoter(s) that resembles the fis promoter from Escherichia coli.

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