Nuclear protein localization.
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Amino Acid Sequence↗
Biomedical subjects
J Garcia-Bustos
Publications and source records attributed to J Garcia-Bustos.
A biological price of antibiotic resistance: major changes in the peptidoglycan structure of penicillin-resistant pneumococci.
Pneumococcal strains with greatly elevated levels of resistance to penicillin have by now been described with increasing frequency worldwide. The mechanism of antibiotic resistance in these strains involves the molecular remodeling of cell wall synthetic enzymes (penicillin binding proteins). We have now analyzed the peptidoglycan structures of 10 penicillin-susceptible and 10 penicillin-resistant clinical isolates (4 of intermediate and 6 of high level resistance) with a high-resolution HPLC technique. Cell wall peptidoglycan of the susceptible strains contained monomeric and oligomeric forms of primarily (70% or more) linear stem peptides with the sequence of L-Ala-D-iGln-L-Lys-D-Ala (where iGln is isoglutamine). In contrast, the major peptide species (70% or more) of resistant cell walls were abnormal branched-stem peptides carrying Ala-Ser or Ala-Ala dipeptides on the epsilon-amino groups of the stem peptide lysine residues. The structural alteration in the peptidoglycan was not related to serotype, date, or site of isolation but showed strong correlation with penicillin resistance and was cotransformed with high-level penicillin resistance during genetic transformation. We suggest that the remodeling of the active site of penicillin binding proteins in the resistant bacteria, which results in the reduced affinity for penicillin, also changes the substrate preference of these enzymes for the more hydrophobic branched peptides (instead of linear peptides) for cell wall synthesis.
Amino Acid Sequence↗
Mechanism of pneumococcal cell wall degradation in vitro and in vivo.
We compared the products of autolytic amidase-catalyzed wall degradation in vivo (in penicillin-induced lysis) and in vitro. Pneumococci labeled in their cell wall stem peptides by radioactive lysine were treated with penicillin, and the nature of wall degradation products released to the medium during lysis of the bacteria was determined. At early times of lysis (20% loss of wall label), virtually all the radioactive peptides released (greater than 94%) were of high molecular size and were still attached to glycan and teichoic acid. At times of more extensive bacterial lysis (56%), progressively larger and larger fractions of the released peptides became free, i.e., detached from glycan and teichoic acid. Analysis of the nondegraded residual wall material by high-resolution high-pressure liquid chromatography revealed that this in vivo-triggered autolysis did not involve selective hydrolysis of some of the chemically distinct stem peptides. Parallel in vitro experiments yielded completely different results. Purified pneumococcal cell walls labeled with radioactive lysine were treated in vitro with low concentrations of pure amidase, and the nature of wall degradation products released during limited hydrolysis and after more extensive degradation was determined. In sharp contrast to the in vivo experiments, the main products of in vitro hydrolysis were free peptides. After a short treatment with amidase (resulting in a 20% loss of label), the material released was enriched for the monomeric stem peptides. At all times of hydrolysis (including the time of extensive degradation), only a relatively small fraction of the released wall peptides was covalently attached to glycan and teichoic acid components (17% as compared with 40% in the intact cell wall). We propose that the in vivo-triggered amidase activity first attacks the amide bonds in some strategically located (or unprotected) stem peptides that hold large segments of cell wall material together. The observations indicate that the in vivo activity of the pneumococcal autolysin is under topographic constraints.
Amidohydrolases↗