Lipid modification of Escherichia coli penicillin-binding protein 3

Hayashi, Shigeru; Hara, Hiroshi; Suzuki, Hirotoshi; Hirota, Yukinori

doi:10.1128/jb.170.11.5392-5395.1988

Cited by 35 publications

(32 citation statements)

References 38 publications

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“…The construction of a water-soluble active PBP 2x derivative (PBP 2x*) proves once more that the only membraneanchoring region of high-Mr PBPs is the N-terminal hydrophobic peptide domain, and that this peptide is apparently not necessary for enzymatic activity with the possible exception of E. coli PBP 3 (Hayashi et al, 1988). That the water-soluble PBP 2x* appeared perfectly stable over several months is further support for the structural independence of the hydrophilic main part of the PBP.…”

Section: Discussionmentioning

confidence: 97%

Penicillin‐binding protein 2x of Streptococcus pneumoniae

Laible

Keck

Mottl

et al. 1992

European Journal of Biochemistry

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A 2.5-kb DNA fragment including the structural gene coding for the penicillin-binding protein 2x (PBP 2x) of Streptococcus pneumoniue has been cloned into the vector pJDC9 and expressed in Escherichia coli. Mapping of RNA polymerase binding sites by electron microscopy indicated that the pbpX promoter is well recognized by the E. coli enzyme. However, high-level expression occurred mainly under the control of the lac promoter upstream of the pJDC9 multiple cloning site. After induction with isopropyl P-d-thiogalactopyranoside, PBP 2x was expressed as one of the major cellular proteins. PBP 2x produced in E. coli corresponded to the pneumococcal PBP 2x in terms of electrophoretic mobility, fractionation with the cytoplasmic membrane, and penicillin-binding capacity. Deletion of 30 hydrophobic N-terminal amino acid residues at positions 19 -48 resulted in high-level expression of a cytoplasmic, soluble PBP 2x derivative (PBP 2x*) which still retained full p-lactam-binding activity. A two-step procedure involving dye affinity chromatography was established for obtaining large amounts of highly purified enzymatically active PBP 2x*.

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Section: Discussionmentioning

confidence: 97%

Penicillin‐binding protein 2x of Streptococcus pneumoniae

Laible

Keck

Mottl

et al. 1992

European Journal of Biochemistry

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“…On the other hand, if we assume multiple acylations of a fraction of spiralin, the other major fraction of spiralin molecules will stay integrated without the help of any acyl chain since, in S. melliferum, the whole spiralin fraction is bound to the cell membrane. Interestingly, in E. coli, less than 15% of the lipoprotein PBP3 (penicillin-binding protein 3) in the cytoplasmic membrane is modified with acyl chains (16). Whatever the actual situation, it is very probable that the covalent binding of hydrophobic chain(s) to spiralin strengthens the anchoring of the protein within the lipid bilayer.…”

Section: Resultsmentioning

confidence: 99%

“…This would explain the water-insoluble character of this part of the protein. The 21-kDa membrane-associated protein p21 of the ras oncogene family is water-soluble without its C-terminal ester-bound single 16:0 acyl chain but insoluble (or membrane attached) with the chain (13,26).…”

Section: Resultsmentioning

confidence: 99%

Topology and acylation of spiralin

et al. 1989

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Of the 51 polypeptides detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the plasma membrane of the helical mollicute Spiroplasma melliferum, 21 are acylated, predominantly with myristic (14:0) and palmitic (16:0) chains. This is notably the case for spiralin, the major membrane protein of this bacterium, which contains an average of 0.7 acyl chains per polypeptide, attached very probably by ester bonds to alcohol amino acids. The amphiphilicity of spiralin was demonstrated by the behavior of the protein in charge-shift electrophoresis, its incorporation into liposomes, and its ability to form in the absence of lipids and detergents, globular protein micelles (diameter, -15 nm). The presence of epitopes on the two faces of the cell membrane, as probed by antibody adsorption and crossed immunoelectrophoresis, and the strong interaction between spiralin and the intracytoplasmic fibrils show that spiralin is a transmembrane protein. The mean hydropathy of the amino acid composition of spiralin (-0.30) is on the hydrophilic side of the scale. Surprisingly, the water-insoluble core of spiralin micelles, which is the putative membrane anchor, has a still more hydrophilic amino acid composition (mean hydropathy, -0.70) and is enriched in glycine and serine residues. Taking into account all these properties, we propose a topological model for spiralin featuring a transbilayer localization with hydrophilic domains protruding on the two faces of the membrane and connected by a small domain embedded within the apolar region of the lipid bilayer. In this model, the membrane anchoring of the protein is strengthened by a covalently bound acyl chain.As the smallest and simplest cells known thus far (32, 33), mollicutes (trivial name, mycoplasmas) are of fundamental interest in cell biology. Indeed, the mycoplasma cell is bounded by only the plasma membrane, and its genome, which is the smallest cellular genome recorded to date, contains a low proportion of guanine and cytosine (33). Mollicutes are thus the bacteria which illustrate best the concept of the minimal cell (33). According to recent phylogenetic studies based on sequence comparisons of rRNAs and tRNAs, the mollicutes arose by degenerative evolution from the Clostridium spp. (46). Most, if not all, mollicutes are parasites, and a large number of species are pathogenic for plants, animals, or humans (32). Moreover, several species are also frequent contaminants of eucaryotic cell cultures (28).Spiroplasmas are a group of mollicutes characterized by a helical cell morphology and motility (7). Spiralin, the major membrane protein of the phytopathogenic species Spiroplasma citri, has been purified to homogeneity from three distinct strains (52,53). This amphiphilic protein cannot be solubilized without recourse to detergents (47) and forms homo-oligomers in the spiroplasmal membrane (48). A similar protein has also been purified from the membrane of the honeybee spiroplasma Spiroplasma melliferum (53). The four spiralins analyzed to date share very...

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“…The N-terminal region seemed to serve for insertion into the membrane: artificial removal of the N-terminal 40 residues resulted in accumulation of the protein in the cytoplasm (3). In the N-terminal region there is a pentapeptide with an amino acid sequence similar to the consensus for bacterial lipoproteins; this pentapeptide was shown, for a minor fraction of the molecules, to actually undergo the lipid modification and probably the processing (22). However, further investigations with gene manipulation to produce hybrid and truncated PBP 3 molecules (20) and with peptide mapping and amino acid sequence analysis of purified precursor and mature forms (42) revealed that cleavage of the C-terminal 11 residues, rather than that of the N-terminal signal peptide, is responsible for the maturation detected as a change in electrophoretic mobility.…”

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confidence: 99%

Cloning, mapping, and characterization of the Escherichia coli prc gene, which is involved in C-terminal processing of penicillin-binding protein 3

et al. 1991

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The prc gene, which is involved in cleavage of the C-terminal peptide from the precursor form of penicillin-binding protein 3 (PBP 3) of Escherichia coli, was cloned and mapped at 40.4 min on the chromosome. The gene product was identified as a protein of about 80 kDa in maxicell and in vitro systems. Fractionation of the maxicells producing the product suggested that the product was associated with the periplasmic side of the cytoplasmic membrane. This was consistent with the notion that the C-terminal processing of PBP 3 probably occurs outside the cytoplasmic membrane: the processing was found to be dependent on the secY and secA functions, indicating that the prc product or PBP 3 or both share the translocation machinery with other extracytoplasmic proteins. DNA sequencing analysis of the prc gene region identified an open reading frame, with two possible translational starts 6 bp apart from each other, that could code for a product with a calculated molecular weight of 76,667 or 76,432. The prc mutant was sensitive to thermal and osmotic stresses. Southern analysis of the chromosomal DNA of the mutant unexpectedly revealed that the mutation was a deletion of the entire prc gene and thus that the prc gene is conditionally dispensable. The mutation resulted in greatly reduced heat shock response at low osmolarity and in leakage of periplasmic proteins.

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Lipid modification of Escherichia coli penicillin-binding protein 3

Cited by 35 publications

References 38 publications

Penicillin‐binding protein 2x of Streptococcus pneumoniae

Penicillin‐binding protein 2x of Streptococcus pneumoniae

Topology and acylation of spiralin

Cloning, mapping, and characterization of the Escherichia coli prc gene, which is involved in C-terminal processing of penicillin-binding protein 3

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