Nucleotide sequences of the pbpX genes encoding the penicillin-binding proteins 2x from Streptococcus pneumoniae R6 and a cefotaxime-resistant mutant, C506
Laible, G.; Hakenbeck, R.; Sicard, M. A.et al.
1989 • In Molecular Microbiology, 3 (10), p. 1337-1348
[en] Development of penicillin resistance in Streptococcus pneumoniae is due to successive mutations in penicillin-binding proteins (PBPs) which reduce their affinity for beta-lactam antibiotics. PBP2x is one of the high-Mr PBPs which appears to be altered both in resistant clinical isolates, and in cefotaxime-resistant laboratory mutants. In this study, we have sequenced a 2564 base-pair chromosomal fragment from the penicillin-sensitive S. pneumoniae strain R6, which contains the PBP2x gene. Within this fragment, a 2250 base-pair open reading frame was found which coded for a protein having an Mr of 82.35kD, a value which is in good agreement with the Mr of 80-85 kD measured by SDS-gel electrophoresis of the PBP2x protein itself. The N-terminal region resembled an unprocessed signal peptide and was followed by a hydrophobic sequence that may be responsible for membrane attachment of PBP2x. The corresponding nucleotide sequence of the PBP2x gene from C504, a cefotaxime-resistant laboratory mutant obtained after five selection steps, contained three nucleotide substitutions, causing three amino acid alterations within the beta-lactam binding domain of the PBP2x protein. Alterations affecting similar regions of Escherichia coli PBP3 and Neisseria gonorrhoeae PBP2 from beta-lactam-resistant strains are known. The penicillin-binding domain of PBP2x shows highest homology with these two PBPs and S. pneumoniae PBP2b. In contrast, the N-terminal extension of PBP2x has the highest homology with E. coli PBP2 and methicillin-resistant Staphylococcus aureus PBP2'. No significant homology was detected with PBP1a or PBP1b of Escherichia coli, or with the low-Mr PBPs.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Laible, G.; Max-Planck Institut fur Molekulare Genetik
Hakenbeck, R.; Max-Planck Institut fur Molekulare Genetik
Sicard, M. A.; Université Paul Sabatier - Toulouse 3 - UPS
Joris, Bernard ; Université de Liège - ULiège > Departement de Microbiologie
Ghuysen, Jean-Marie ; Université de Liège - ULiège > Departement de Microbiologie
Language :
English
Title :
Nucleotide sequences of the pbpX genes encoding the penicillin-binding proteins 2x from Streptococcus pneumoniae R6 and a cefotaxime-resistant mutant, C506
Publication date :
01 October 1989
Journal title :
Molecular Microbiology
ISSN :
0950-382X
eISSN :
1365-2958
Publisher :
Blackwell Publishing, Oxford, United Kingdom
Volume :
3
Issue :
10
Pages :
1337-1348
Peer reviewed :
Peer Reviewed verified by ORBi
Funders :
FRSM - Fonds de la Recherche Scientifique Médicale
Asoh S., Matsuzawa H., Ishino F., Strominger J.L. (1986) Nucleotide sequence of the pbpA gene and characteristics of the deduced amino acid sequence of penicillin‐binding protein 2 of Escherichia coli K12. Eur J Biochem 160:231-238.
Bimboim H.C., Doly J. (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucl Acids Res 7:1513-1523.
Briese T., Ellerbrok H., Schier H.‐M., Hakenbeck R. (1988) Reactivity of anti‐β‐lactam antibodies with β‐lactam‐penicillin‐binding protein complexes. Antibiotic Inhibition of Bacterial Cell Surface Assembly and Function , Actor, P., Daneo‐Moore, L., Higgins, M.L., Salton, M.R.J., Shockman, G.D., (eds). Washington D.C.:, American Society for Microbiology; 404-409.
Broome‐Smith J.K., Edelman A., Spratt B.G. (1983) Sequence of penicillin‐binding protein 5 of Escherichia coli. The Target of Penicillin , Hakenbeck, R., Labischinski, H., Höltje, J.‐V., (eds). Berlin:, Walter de Gruyter; 403-408.
Broome‐Smith J.K., Edelman A., Yousif S., Spratt B.G. (1985) The nucleotide sequences of the ponA and ponB genes encoding penicillin‐binding proteins 1A and 1B of Escherichia coli K12. Eur J Biochem 147:147-437.
Brown M.G.M., Weston A., Saunders J.R., Humphreys G.O. (1979) Transformation of E. coli C600 by plasmid DNA at different phases of growth. FEMS Microbiol Lett 5:5-219.
Chen E.J., Seeburg P.H. (1985) Supercoil sequencing: a fast simple method for sequencing plasmid DNA. DNA 4:4-165.
Chen J.‐D., Morrison D.A. (1987) Cloning of Streptococcus pneumoniae DNA fragments in Escherichia coli requires vectors protected by strong transcriptional terminators. Gene 55:55-179.
Dowson C.G., Hutchison A., Spratt B.G. (1989) Extensive re‐modelling of the transpeptidase domain of penicillin‐binding protein 2B of a penicillin‐resistant South African isolate of Streptococcus pneumoniae. Mol Microbiol 3:3-95.
Duez C., Piron‐Fraipont C., Joris B., Dusart J., Urdea M.S., Martial J.A., Frere J.‐M., Ghuysen J.‐M. (1987) Primary structure of the Streptomyces R61 extracellular DD‐peptidase. 1. Cloning into Streptomyces lividans and nucleotide sequence of the gene. Eur J Biochem 162:162-509.
Edelman A., Bowler L.B., Broome‐Smith J.K., Spratt B.G. (1987) Use of a β‐lactamase fusion vector to investigate the organization of penicillin‐binding protein 1B in the cytoplasmic membrane of Escherichia coli. Mol Microbiol 1:1-101.
Engelman D.M., Steitz T.A., Goldman A. (1986) Identifying nonpolar transbilayer helices in amino acid sequences of membrane proteins. Annu Rev Biochem Biophys Chem 15:15-321.
Garcia P., Garcia J.L., Garcia E., López R. (1986) Nucleotide sequence and expression of the pneumococcal autolysin gene from its own promoter in Escherichia coli. Gene 43:43-265.
Garnier J., Osguthorpe D.J., Robson B. (1978) Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. J Mol Biol 120:120-197.
Goad W.B., Kanehisa M.I. (1982) Pattern recognition in nucleic acid sequences. I. A general method for finding local homologies and symmetries. Nucl Acids Res 10:10-247.
Guo L.H., Yang R.C.A., Wu R. (1983) An improved strategy for rapid direct sequencing of both strands of long DNA molecules cloned in a plasmid. Nucl Acids Res 11:11-5521.
Hakenbeck R., Kohiyama M. (1982) Purification of penicillin‐binding protein 3 from Streptococcus pneumoniae. Eur J Biochem 127:127-231.
Hakenbeck R., Tarpay M., Tomasz A. (1980) Multiple changes of penicillin‐binding proteins in penicillin‐resistant clinical isolates of Streptococcus pneumoniae. Antimicrob Agents Chemother 17:17-364.
Hakenbeck R., Ellerbrok H., Briese T. (1986) Antibodies against the benzylpenicillin moiety as a probe for penicillin‐binding proteins. Eur J Biochem 157:101-106.
Hakenbeck R., Tornette S., Adkinson N.F. (1987) Interaction of non‐lytic β‐lactams with penicillin‐binding proteins in Streptococcus pneumoniae. J Gen Microbiol 30:30-755.
Hakenbeck R., Briese T., Ellerbrok H., Laible G., Martin C., Metelmann C., Schier H.‐M., Tornette S. (1988) Targets of β‐lactams in Streptococcus pneumoniae. Antibiotic Inhibition of Bacterial Cell Surface Assembly and Function , Actor, P., Daneo‐Moore, L., Higgins, M.L., Salton, M.R.J., Shockman, G.D., (eds) Washington, D.C.:, American Society for Microbiology; 390-399.
Hayashi S., Hara H., Suzuki H., Hirota Y. (1988) Lipid modification of Escherichia coli penicillin‐binding protein 3. J Bacteriol 170:170-5392.
Hedge P.J., Spratt B.G. (1985) Amino acid sustitutions that reduce the affinity of penicillin‐binding protein 3 of Escherichia coli for cephalexin. Eur J Biochem 151:111-121.
Hedge P.J., Spratt B.G. (1985) Resistance to β‐lactam antibiotics by re‐modelling the active site of an E. coli penicillin‐binding protein. Nature 318:318-478.
Joris B., Ghuysen J.‐M., Dive G., Renard A., Dideberg O., Charlier P., Frère J.‐M., Kelly J.A., Boyington J.C., Moews P.C., Knox J.R. (1988) The active‐site‐serine penicillin‐recognizing enzymes as members of the Streptomyces R61 DD‐peptidase family. Biochem J 250:250-313.
Kelly J.A., Knox J.R., Moews P.C., Moring J., Zhao H.C. (1988) Molecular graphics: studying β‐lactam inhibition in three dimensions. Antibiotic inhibition of Bacterial Cell Surface Assembly and Function , Actor, P., Daneo‐Moore, L., Higgins, M.L., Salton, M.R.J., Shockman, G.D., (eds). Washington, D.C.:, American Society for Microbiology; 261-267.
Kyte J., Doolittle R.F. (1982) A simple method for displaying the hydropathic character of a protein. J Mol Biol 157:105-132.
Lacks S., Hotchkiss R.D. (1960) A study of the genetic material determining an enzyme activity in pneumococcus. Biochim Biophys Acta 39:39-508.
Laible G., Hakenbeck R. (1987) Penicillin‐binding proteins in β‐lactam‐resistant laboratory mutants of Streptococcus pneumoniae. Mol Microbioli 1:1-355.
Levèfre J.C., Claverys J.P., Sicard A.M. (1979) Donor deoxyribonucleic acid length and marker effect in pneumococcal transformation. J Bacteriol 138:138-180.
Maniatis T., Fritsch F.E., Sambrook F. Molecular Cloning. A Laboratory Manual, Cold Spring Harbor, New York:, Cold Spring Harbor Laboratory Press; 1982.
Méjean V., Claverys J.‐P., Vasseghi H., Sicard A.M. (1981) Rapid cloning of specific DNA fragments of Streptococcus pneumoniae by vector integration into the chromosome followed by endonucleolytic excision. Gene 15:15-289.
Messing I., Vieira J. (1982) A new pair of M13 vectors for selecting either strand of a double‐digest restriction fragment. Gene 19:19-269.
Messing I., Crea R., Seeburg P.H. (1981) A system for shotgun DNA sequencing. Nucl Acids Res 9:9-309.
Nakagawa J.I., Tamaki S., Tomioka S., Matsuhashi M. (1984) Functional biosynthesis of cell wall peptidoglycan by polymorphic bifunctional polypeptides: penicillin‐binding protein 1 Bs of Escherichia coli with activities of transglycosylase and transpeptidase. J Biol Chem 259:259-13937.
Nakamura M., Maruyama I.N., Soma M., Kato J.‐I., Suzuki H., Hirota Y. (1983) On the process of cellular division in Escherichia coli: nucleotide sequence of the gene for penicillin‐binding protein 3. MGG Molecular & General Genetics , 191–191; 191.
Ottolenghi E., Hotchkiss R.D. (1962) Release of genetic transforming agent from pneumococcal cultures during growth and disintegration. J Exp Med 116:116-491.
Prats H., Martin B., Pognonec P., Burger A.‐C., Claverys J.‐P. (1985) A plasmid vector allowing positive selection of recombinant plasmids in Streptococcus pneumoniae. Gene 39:39-41.
Sanger F., Nicklen S., Coulson A.R. (1977) DNA sequencing with chain‐terminating inhibitors. Proc Natl Acad Sci USA 74:74-5463.
Shockley T.E., Hotchkiss R.D. (1970) Stepwise introduction of transformable penicillin resistance in pneumococcus. Genetics 64:64-397.
Spratt B.G. (1988) Hybrid penicillin‐binding proteins in penicillin‐resistant strains of Neisseria gonorrhoeae. Nature 332:173-176.
Song M.D., Wachi M., Doi M., Ishino F., Matsuhashi M. (1987) Evolution of an inducible penicillin‐target protein in methicillin‐resistant Staphylococcus aureus by gene fusion. FEBS Lett 221:167-171.
Stassi D.L., Lacks S.A. (1982) Effect of strong promoters on the cloning in Escherichia coli of DNA fragments from Streptococcus pneumoniae. Gene 18:18-319.
Tabor S., Richardson C.C. (1987) DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc Natl Acad Sci USA 84:84-4767.
Tiraby J.‐G., Fox M.S. (1974) Marker discrimination and mutagen‐induced alterations in pneumococcal transformation. Genetics 77:77-449.
Todd J.A., Roberts A.N., Johnstone K., Piggot P.J., Winter G., Ellar D. (1986) Reduced heat resistance of mutant spores after cloning and mutagenesis of the Bacilius subtilis gene encoding penicillin‐binding protein 5. J Bacteriol 167:167-257.
Tomioka S., Ishino F., Tamaki S., Matsuhashi M. (1982) Formation of hypercrosslinked peptidoglycan with multiple crosslinkages by a penicillin‐binding protein, 1A, of Eschenchia coli. Biochem Biophys Res Commun 106:106-1175.
Vogelstein B., Gillespie D. (1979) Preparative and analytical purification of DNA from agarose. Proc Natl Acad Sci USA 76:76-615.
von Heijne G. (1985) Signal sequences: the limits of variation. J Mol Biol 184:184-199.
Zighelboim S., Tomasz A. (1980) Penicillin‐binding proteins of multiple resistant South African strains of Streptococcus pneumoniae. Antimicrob Agents Chemother 17:434-442.
