The complete genome sequence of Lactobacillus bulgaricus reveals extensive and ongoing reductive evolution.

Lactobacillus delbrueckii ssp. bulgaricus (L. bulgaricus) is a representative of the group of lactic acid-producing bacteria, mainly known for its worldwide application in yogurt production. The genome sequence of this bacterium has been determined and shows the signs of ongoing specialization, with a substantial number of pseudogenes and incomplete metabolic pathways and relatively few regulatory functions. Several unique features of the L. bulgaricus genome support the hypothesis that the genome is in a phase of rapid evolution. (i) Exceptionally high numbers of rRNA and tRNA genes with regard to genome size may indicate that the L. bulgaricus genome has known a recent phase of important size reduction, in agreement with the observed high frequency of gene inactivation and elimination; (ii) a much higher GC content at codon position 3 than expected on the basis of the overall GC content suggests that the composition of the genome is evolving toward a higher GC content; and (iii) the presence of a 47.5-kbp inverted repeat in the replication termination region, an extremely rare feature in bacterial genomes, may be interpreted as a transient stage in genome evolution. The results indicate the adaptation of L. bulgaricus from a plant-associated habitat to the stable protein and lactose-rich milk environment through the loss of superfluous functions and protocooperation with Streptococcus thermophilus.

Apparent and real recombination frequencies in multicopy plasmids: the need for a novel approach in frequency determination.

Recombination studies of bacteria are often carried out with multicopy plasmids, and recombination frequencies are often deduced from the proportion of cells in the population that express a recombinant phenotype. These frequencies should however be called apparent frequencies, since detection of the recombinant cells requires not only the formation of a rearranged plasmid but also its establishment in the cell. The establishment of the recombinant plasmid can possibly be affected by its interaction with the parental plasmids. To test this hypothesis, we have used a plasmid system enabling the study of deletion formation between short direct repeats (18 bp) in Bacillus subtilis and developed a method by which deletion frequencies are measured under conditions under which interaction is abolished. Real deletion frequencies were thus determined and compared with apparent deletion frequencies. Real frequencies were underestimated by a factor ranging from 4- to 500-fold, depending upon the plasmid under study. This implies that a large majority of the recombinant molecules that are formed are generally not detected. We show that apparent deletion frequencies strongly depend upon (i) the parental plasmid copy number, (ii) the ability of the recombinant molecules to form heterodimeric plasmids, and (iii) the fitness of the recombinant molecules relative to that of parental molecules. Finally, we show that under conditions under which all recombinant molecules are scored, transcription can inhibit the deletion process 10-fold.

Frequency of deletion formation decreases exponentially with distance between short direct repeats.

The effect of distance between 18 bp direct repeats on deletion formation has been examined in Bacillus subtilis. The deletion frequency decreased exponentially by more than 1000-fold as the distance increased from 33 to 2313 bp. This decrease occurred in two distinct phases, which may be determined by DNA-duplex flexibility. A similar relationship between deletion formation and distance was observed in a theta-replicating plasmid and in the chromosome, indicating that this relationship might have a general validity.