Here we describe five tandemly arranged and converging ORFs in Acinetobacter sp. BD413, namely lytB, orfY, orfX, comB, and orfZ, located upstream of the previously identified competence gene comC. The N-termini of the deduced proteins OrfY and ComB exhibit the conserved endopeptidase cleavage motifs of prepilin proteins; the deduced protein ComB is similar to type IV pilins. LytB is similar to the Escherichia coli LytB, which has been implicated in the stringent response. No homologues of OrfX, OrfY and OrfZ could be identified. A mutation in orfY or orfZ led to 100-fold reduced transformation frequencies and a mutation in comB resulted in a non-competent phenotype. Disruption of lytB did not affect the natural transformation phenotype. Complementation studies clearly demonstrated that comB is involved in natural transformation, whereas the transformation-deficient phenotypes of orfY and orfZ mutants were due to polar effects on comB and comC, respectively. Analyses of the twitching motility phenotype and of the ultrastructure of the noncompetent comB mutant suggested that the competence gene comB is not essential for the biogenesis of type IV pili and expression of the type IV pili-associated property of twitching motility. Transcriptional fusions between comB and a promoter-free lacZ gene were constructed, and analysis of growth-phase-dependent transcription revealed increased expression of comB during prolonged exponential and stationary phases.