Abstract
A strategy was developed to mutate and genetically identify exported proteins in Streptococcus pneumoniae. Vectors were created and used to screen pneumococcal DNA in Escherichia coli and S. pneumoniae for translational gene fusions to alkaline phosphatase (PhoA). Twenty five PhoA+ pneumococcal mutants were isolated and the loci from eight of these mutants showed similarity to known exported or membrane-associated proteins. Homologues were found to: (i) protein-dependent peptide permeases, (ii) penicillin-binding proteins, (iii) Clp proteases, (iv) two-component sensor regulators, (v) the phosphoenolpyruvate: carbohydrate phosphotransferases permeases, (vi) membrane-associated dehydrogenases, (vii) P-type (E1E2-type) cation transport ATPases, (viii) ABC transporters responsible for the translocation of the RTX class of bacterial toxins. Unexpectedly one PhoA+ mutant contained a fusion to a member of the DEAD protein family of ATP-dependent RNA helicases suggesting export of these proteins.
Publication types
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Comparative Study
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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ATP-Dependent Proteases
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Alkaline Phosphatase / biosynthesis*
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Alkaline Phosphatase / genetics
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Amino Acid Sequence
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Bacterial Proteins / biosynthesis*
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Bacterial Proteins / genetics
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Base Sequence
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Carrier Proteins / biosynthesis
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Carrier Proteins / genetics
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Cloning, Molecular
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Enzymes / biosynthesis*
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Enzymes / genetics
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Escherichia coli / metabolism
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Genes, Bacterial
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Genetic Vectors
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Heat-Shock Proteins / biosynthesis
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Heat-Shock Proteins / genetics
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Hexosyltransferases*
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Membrane Transport Proteins / biosynthesis
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Membrane Transport Proteins / genetics
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Molecular Sequence Data
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Muramoylpentapeptide Carboxypeptidase / biosynthesis
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Muramoylpentapeptide Carboxypeptidase / genetics
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Mutagenesis, Insertional
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Oligodeoxyribonucleotides
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Oxidoreductases / biosynthesis
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Oxidoreductases / genetics
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Penicillin-Binding Proteins
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Peptidyl Transferases*
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Protein Biosynthesis
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Recombinant Fusion Proteins / biosynthesis
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Sequence Homology, Amino Acid
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Serine Endopeptidases / biosynthesis
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Serine Endopeptidases / genetics
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Streptococcus pneumoniae / genetics
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Streptococcus pneumoniae / metabolism*
Substances
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Bacterial Proteins
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Carrier Proteins
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Enzymes
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Heat-Shock Proteins
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Membrane Transport Proteins
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Oligodeoxyribonucleotides
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Penicillin-Binding Proteins
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Recombinant Fusion Proteins
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Oxidoreductases
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Peptidyl Transferases
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Hexosyltransferases
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Alkaline Phosphatase
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Muramoylpentapeptide Carboxypeptidase
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ATP-Dependent Proteases
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Serine Endopeptidases
Associated data
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GENBANK/L20556
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GENBANK/L20557
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GENBANK/L20558
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GENBANK/L20559
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GENBANK/L20560
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GENBANK/L20561
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GENBANK/L20562
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GENBANK/L20563
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GENBANK/L20564