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 pneumococcal autolysin (an N-acetylmuramoyl-L-alanine amidase) by preventing its attachment to wall teichoic acids (Giudicelli 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 choline-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 (Garcı́a et al., 1988, Proc Natl Acad Sci USA 85: 914– 918; Garcı́a et al., 1998, Microb Drug Resist 4: 25–36). 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 (López et al., 1997, Microb Drug Resist 3: 199–211). These enzymes, which exhibit different chemical substrate specificities (i.e. lysozymes, amidases and glucosaminidases), display a modular organization in which the catalytic domain and the ChBD are located at the Nand 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 et al., 1997, Microb Drug Resist 3: 391–400; Yother et al., 1998, J Bacteriol 180: 2093–2101). 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 small chains (six to ten cells) and the absence of lysis in the stationary phase of growth (Sánchez-Puelles et al., 1986, Eur J Biochem 158: 289–293; Ronda et al., 1987, Eur J Biochem 164: 621–624) (Fig. 2A). A recent study of ABC-type Mn permease-defective mutants of pneumococcus also reports chain formation in the stationary phase of growth (psaC and psaD mutants) or the appearance of small conglomerates or simply aberrant morphology (psaA and psaB mutants) (Novak et al., 1998, Mol Microbiol 29: 1285–1296). Nevertheless, a gene involved in the formation of long chains is not yet known in pneumococcus. The fact that most bacteria possess several lytic enzymes makes it difficult to determine the precise physiological role of these enzymes. Overcoming this limitation requires experiments with well-defined, single, or perhaps multiple, mutants with altered peptidoglycan hydrolase. Using a previously published procedure to characterize the pneumococcal glucosaminidase (Garcı́a et al., 1989, Biochem Biophys Res Commun 158: 251–256), we identified, by SDS–PAGE, four protein bands with apparently strong choline binding affinity. One of these bands was excised from the acrylamide gel, and the N-terminal amino acid sequence was found to be Ser-Asp-Gly-ThrTrp-Gln-Gly. This sequence was compared with the translated version of the partial nucleotide sequence of the S. pneumoniae genome (ftp://ftp.tigr.org/pub/data/s_pneumoniae), and a perfect match was found with an internal peptide of a gene product. This gene (hereafter designated lytB), located in the 10 373 bp contig no. 4117, was polymerase chain reaction (PCR) amplified, sequenced and preliminarily characterized (accession no. AJ010312). The putative 76.4 kDa LytB protein (658 amino acid residues) displays a modular organization different from all the ChBD proteins described previously in the pneumococcal system (Fig. 1A). Furthermore, this enzyme contains a 23-aminoacid-long, cleavable signal peptide (predicted Mr of the processed protein 73 800), as in the case of the S. pneumoniae LytC lysozyme, a new murein hydrolase recently Molecular Microbiology (1999) 31(4), 1275–1281