The aggregation-promoting factor in Lactobacillus delbrueckii ssp. bulgaricus: confirmation of the presence and expression of the apf gene and in silico analysis of the corresponding protein.

In lactobacilli the aggregation phenotype is linked to their ability to colonize the intestinal and urogenital tracts and to counteract pathogenic bacteria. In all available complete genome sequences of Lactobacillus delbrueckii ssp. bulgaricus there are at least two genes putatively related to aggregation, one of which is annotated as aggregation-promoting factor (apf). Here we report the results from the in silico analysis of this gene and its product. The apf gene was present in the genome of all 70 tested L. delbr. ssp. bulgaricus strains. Its expression was confirmed for a selection of five strains with aggregation phenotype and two aggregation-negative strains. The mature Apf protein had a length of 257-284 amino acids with predicted molecular weight in the range of 28.64-30.36 kDa and isoelectric point of 10.6 ± 0.1, showing some similarity to Apf1 and Apf2 from L. johnsonii NCC533 and Apf1 and Apf2 from L. gasseri which are similar in size (28-35 kDa) and share a similar high isoelectric point (pI > 9). Predictive analyzes have indicated that Apf is a secretory protein. The 30 amino acid signal peptide and the predicted cleavage site in the pre-protein suggested that it was processed by Type I Signal protease. In the mature Apf protein a glutamine-rich N-terminal region was followed by an unusual lysine/alanine-rich region with variable length, supposed to be positively charged under physiological conditions, interacting with bacterial teichoic acids. The alignment of the C-termini of the Apf proteins showed similarity to conserved C-terminal domains in aggregation-related proteins in other lactobacilli such as Apf1 of Lactobacillus johnsonii ATCC 11506 and the secretory protein Sep of L. fermentum BR11, that may be involved in non-covalent binding to carbohydrates. The C-terminal anchor and the cationic domain in Apf may serve as mediators of physical cell-to-cell interaction in L. delbr. ssp. bulgaricus.