LytB, a novel pneumococcal murein hydrolase essential for cell separation.

Streptococcus pneumoniae is an important human pathogen that has an absolute nutritional requirement for choline. Replacement of this amino alcohol in a synthetic medium by structural analogues, such as ethanolamine (EA cells), leads to alterations in several physiological properties including cell separation (Tomasz, 1968, Proc Natl Acad Sci USA 59: 86–93). Identical changes including chain formation and loss of autolytic properties can also be induced by adding up to 2% choline chloride to the growth medium (Briese and Hakenbeck, 1985, Eur J Biochem 146: 417– 427). Choline has been shown to inhibit the LytA pneumo-coccal autolysin (an N-acetylmuramoyl-L-alanine amidase) by preventing its attachment to wall teichoic acids (Giudi-celli and Tomasz, 1984, J Bacteriol 158: 1188–1190). In addition, it has been shown that the pneumococcal surface protein A (PspA) is anchored to the choline residues of the membrane-associated lipoteichoic acids (Yother and White, 1994, J Bacteriol 176: 2976–2985). Pneumococcus synthesizes several proteins that recognize and bind to the choline residues of the teichoic and lipoteichoic acids through a specialized domain, the cho-line-binding domain (ChBD) (Sánchez-Puelles et al., 1990, Gene 89: 69–75). ChBD is built up of six or more well-conserved motifs, each about 20-amino-acid residues long Currently, there is an increasing interest in the study of pneumococcal genes coding for proteins that possess ChBDs. These proteins have been demonstrated to participate in a series of important biological functions such as cell adhesion and division. For many years, we have studied the molecular structure and biological role of the lytic enzymes of pneumococcus and its bacteriophages (Ló pez et al., 1997, Microb Drug Resist 3: 199–211). These enzymes, which exhibit different chemical substrate specificities (i.e. lysozymes, amidases and glucosamini-dases), display a modular organization in which the catalytic domain and the ChBD are located at the N-and C-terminal moieties of the protein respectively. It is likely that a defective autolytic system might explain the physiological alterations leading to chain formation in S. pneumoniae. Recently, two independent experimental approaches have generated pneumococcal mutants that do not require choline or analogues for growth (Severin These mutants form long chains when growing under choline-free conditions , and it has been claimed that the lack of an active LytA amidase, the main pneumococcal lytic enzyme, could be responsible for impaired cell separation at the end of cell division. Nevertheless, previous studies have demonstrated that the primary biological consequences of the lytA gene deletion were the formation of …