3 HIGHLY HOMOLOGOUS MEMBRANE-BOUND LIPOPROTEINS PARTICIPATE IN OLIGOPEPTIDE TRANSPORT BY THE AMI SYSTEM OF THE GRAM-POSITIVE STREPTOCOCCUS-PNEUMONIAE

Oligopeptides are an important source of nutrients, but can serve also as signals for intercellular communication. Oligopeptide-binding prot eins seem likely to play a role both in oligopeptide transport and in communication processes. One such protein, AmiA, has been identified i n Streptococcus pneumoniae. amiA is the first gene of an operon, ami, which encodes a multicomponent oligopeptide transporter belonging to t he family of ABC transporters (or traffic ATPases). This transporter w as the first system of this type described in Gram-positive bacteria. To investigate the role and the subcellular location of the putative o ligopeptide-binding protein in a bacterium devoid of periplasm, AmiA n ull mutants were first constructed. None was affected for oligopeptide uptake by the Ami system. Since this apparent dispensability of AmiA could result from a functional redundancy, we looked for chromosomal g enes encoding homologues of AmiA. Two homologous genes were identified by DNA-DNA hybridization at low stringency with an amiA probe. Both g enes (aliA and aliB) were cloned and shown to encode putative lipoprot eins highly homologous to AmiA (close to 60% amino acid identity). Exa mination of all combinations of amiA, aliA and aliB mutations indicate d that these proteins have overlapping specificities toward oligopepti des. The triple mutant is as deficient for oligopeptide transport as m utants in the amiCBE or F genes, which demonstrates that an oligopepti de-binding component is absolutely required for transport by the Ami s ystem. Metabolic labelling with [H-3]palmitic acid and cell fractionat ion were used to demonstrate that the three proteins are indeed membra ne-bound lipoproteins in S. pneumoniae. This supports our previous hyp othesis that substrate-binding lipoproteins are functionally equivalen t to the periplasmic substrate-binding component of ABC transporters o f Gram-negative bacteria. Finally, the observation that competence for genetic transformation was drastically reduced in a particular AliB m utant suggests that oligopeptide sensing is important for triggering c ompetence.