Abstract
Oligonucleotide-directed mutagenesis of a plasmid containing the lytA gene coding for the pneumococcal amidase has allowed the separation of the coding sequence of the gene. This sequence has been placed in plasmid pIN-III(lppP-5)-A3 downstream from both a modified lipoprotein promoter and the lactose promoter to construct the recombinant plasmid pGL100. When Escherichia coli RB 791 (pGL100) was grown in the presence of lactose, the pneumococcal amidase accounted for 7% of the total protein present in this strain after 18 h incubation at 37°C. The overproduced amidase was purified in a single-step procedure using a choline-Sepharose 6B column taking advantage of the fact that this enzyme was the unique protein with affinity for choline present in extracts obtained from E. coli RB791 (pGL100). The development of the above design opens up the possibility of studying the mechanism that regulates the activity of this important autolysin by using physicochemical techniques that require the availability of high amounts of purified amidase.
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References
Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucl Acids Res 7:1513–1523
Boyer HW, Rouland-Dussoix D (1969) A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol 41:459–474
Brent R, Ptashne M (1981) Mechanism of action of the lexA gene product. Proc Natl Acad Sci USA 78:4204–4208
Briese T, Hakenbeck R (1985) Interaction of the pneumococcal amidase with lipoteichoic acid and choline. Eur J Biochem 146:417–427
García P, Méndez E, García E, Ronda C, López R (1984) Biochemical characterization of a murein hydrolase induced by bacteriophage Dp-1 in Streptococcus pneumoniae: comparative study between bacteriophage-associated lysin and the host amidase. J Bacteriol 159:793–796
García E, Ronda C, García JL, López R (1985a) A rapid procedure to detect the autolysin phenotype in Streptococcus pneumoniae. FEMS Microbiol Lett 29:77–81
García E, García JL, Ronda C, García P, López R (1985b) Cloning and expression of the pneumococcal autolysin gene in Escherichia coli. Mol Gen Genet 201:225–230
García P, García JL, García E, López R (1986a) Nucleotide sequence and expression of the pneumococcal autolysin gene from its own promoter in Escherichia coli. Gene 43:265–272
García JL, Sánchez-Puelles JM, García P, López R, Ronda C, García E (1986b) Molecular characterization of an autolysin-defective mutant of Streptococcus pneumoniae. Biochem Biolphys Res Commun 137:614–619
Höltje JV, Tomasz A (1976) Purification of the pneumococcal N-acetylmuramyl-l-alanine amidase to biochemical homogeneity. J Biol Chem 251:4199–4207
Inouye S, Inouye M (1985) Up-promoter mutations in the lpp gene of Escherichia coli. Nucl Acids Res 13:3101–3110
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680–685
López R, Sánchez-Puelles JM, García E, García JL, Ronda C, García P (1986) Isolation, characterization and physiological properties of an autolytic-deficient mutant of Streptococcus pneumoniae. Mol Gen Genet 204:237–242
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory Press, New York
Messing J (1983) New M13 vectors for cloning. Methods Enzymol 101:20–78
Morinaga Y, Franceschini T, Inouye S, Inouye M (1984) Improvement of oligonucleotide-directed site-specific mutagenesis using double stranded plasmid DNA. Bio/Technology 2:636–639
Mosser JL, Tomasz A (1970) Choline-containing teichoic acid as a structural component of pneumococcal cell wall and its role in sensitivity of lysis by an autolytic enzyme. J Biol Chem 245:287–298
Ronda C, García JL, García E, Sánchez-Puelles JM, López R (1987) Biological role of the pneumococcal amidase. Cloning of the lytA gene in Streptococcus pneumoniae. Eur J Biochem. 164: 621–624
Sánchez-Puelles JM, Ronda C, García JL, García P, López R, García E (1986) Searching for autolysin functions. Characterization of a pneumococcal mutant deleted in the lytA gene. Eur J Biochem 158:289–293
Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467
Shine J, Dalgarno L (1974) The 3′-terminal sequence of E. coli 16S rRNA: complementarity to nonsense triplets and ribosome binding sites. Proc Natl Acad Sci USA 71:1342–1346
Stassi DL, Lacks S (1982) Effect of strong promoters on the cloning in Escherichia coli of DNA fragments from Streptococcus pneumoniae Gene 18:319–328
Tomasz A, Westphal M (1971) Abnormal autolytic enzyme in a pneumococcus with altered teichoic acid composition. Proc Natl Acad Sci USA 68:2627–2630
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García, J.L., García, E. & López, R. Overproduction and rapid purification of the amidase of Streptococcus pneumoniae . Arch. Microbiol. 149, 52–56 (1987). https://doi.org/10.1007/BF00423136
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DOI: https://doi.org/10.1007/BF00423136