Biomedical subjects

Kevin D Young

Publications and source records attributed to Kevin D Young.

5 recordsLinked to original sources

PubMed2003

Bacterial shape.

In free-living eubacteria an external shell of peptidoglycan opposes internal hydrostatic pressure and prevents membrane rupture and death. At the same time, this wall imposes on each cell a shape. Because shape is both stable and heritable, as is the ability of many organisms to execute defined morphological transformations, cells must actively choose from among a large repertoire of available shapes. How they do so has been debated for decades, but recently experiment has begun to catch up with theory. Two discoveries are particularly informative. First, specific protein assemblies, nucleated by FtsZ, MreB or Mbl, appear to act as internal scaffolds that influence cell shape, perhaps by correctly localizing synthetic enzymes. Second, defects in cell shape are correlated with the presence of inappropriately placed, metabolically inert patches of peptidoglycan. When combined with what we know about mutants affecting cellular morphology, these observations suggest that bacteria may fabricate specific shapes by directing the synthesis of two kinds of cell wall: a long-lived, rigid framework that defines overall topology, and a metabolically plastic peptidoglycan whose shape is directed by internal scaffolds.

Adenosine Triphosphatases↗

PubMed2003

Branching of Escherichia coli cells arises from multiple sites of inert peptidoglycan.

Some strains of Escherichia coli defective for dacA, the gene coding for penicillin-binding protein 5, exhibit a strong branching phenotype when cell division is blocked. Since such branch formation implies a differentiation of polar caps at ectopic locations in the cell envelope, we analyzed murein segregation and observed a strong correlation between areas of inert murein and these morphological anomalies. In particular, the tips of branches exhibited the same properties as those described for polar caps of wild-type cells, i.e., the synthesis and turnover of murein were inhibited. Also, the mobility of cell envelope proteins was apparently constrained in areas with morphological defects. Polar regions of branching cells and sacculi had aberrant morphologies with a very high frequency. Of special interest was that areas of inert murein at polar caps were often split by areas of active synthesis, a situation unlike that observed in wild-type cells. These observations suggest that in dacA mutants, branches and other morphological anomalies may arise from split polar caps or by de novo generation of new poles built around inert peptidoglycan patches in the side walls of the cell.

Bacterial Proteins↗

PubMed2003

Sequences near the active site in chimeric penicillin binding proteins 5 and 6 affect uniform morphology of Escherichia coli.

Penicillin binding protein (PBP) 5, a DD-carboxypeptidase that removes the terminal D-alanine from peptide side chains of peptidoglycan, plays an important role in creating and maintaining the uniform cell shape of Escherichia coli. PBP 6, a highly similar homologue, cannot substitute for PBP 5 in this respect. Previously, we localized the shape-maintaining characteristics of PBP 5 to the globular domain that contains the active site (domain I), where PBPs 5 and 6 share substantial identity. To identify the specific segment of domain I responsible for shape control, we created a set of hybrids and determined which ones complemented the aberrant morphology of a misshapen PBP mutant, E. coli CS703-1. Fusion proteins were constructed in which 47, 199 and 228 amino-terminal amino acids of one PBP were fused to the corresponding carboxy-terminal amino acids of the other. The morphological phenotype was reversed only by hybrid proteins containing PBP 5 residues 200 to 228, which are located next to the KTG motif of the active site. Because residues 220 to 228 were identical in these proteins, the morphological effect was determined by alterations in amino acids 200 to 219. To confirm the importance of this segment, we constructed mosaic proteins in which these 20 amino acids were grafted from PBP 5 into PBP 6 and vice versa. The PBP 6/5/6 mosaic complemented the aberrant morphology of CS703-1, whereas PBP 5/6/5 did not. Site-directed mutagenesis demonstrated that the Asp(218) and Lys(219) residues were important for shape maintenance by these mosaic PBPs, but the same mutations in wild-type PBP 5 did not eliminate its shape-promoting activity. Homologous enzymes from five other bacteria also complemented the phenotype of CS703-1. The overall conclusion is that creation of a bacterial cell of regular diameter and uniform contour apparently depends primarily on a slight alteration of the enzymatic activity or substrate accessibility at the active site of E. coli PBP 5.

Amino Acid Sequence↗

PubMed2002

Contribution of membrane-binding and enzymatic domains of penicillin binding protein 5 to maintenance of uniform cellular morphology of Escherichia coli.

Four low-molecular-weight penicillin binding proteins (LMW PBPs) of Escherichia coli are closely related and have similar DD-carboxypeptidase activities (PBPs 4, 5, and 6 and DacD). However, only one, PBP 5, has a demonstrated physiological function. In its absence, certain mutants of E. coli have altered diameters and lose their uniform outer contour, resulting in morphologically aberrant cells. To determine what differentiates the activities of these LMW PBPs, we constructed fusion proteins combining portions of PBP 5 with fragments of other DD-carboxypeptidases to see which hybrids restored normal morphology to a strain lacking PBP 5. Functional complementation occurred when truncated PBP 5 was combined with the terminal membrane anchor sequences of PBP 6 or DacD. However, complementation was not restored by the putative carboxy-terminal anchor of PBP 4 or by a transmembrane region of the osmosensor protein ProW, even though these hybrids were membrane bound. Site-directed mutagenesis of the carboxy terminus of PBP 5 indicated that complementation required a generalized amphipathic membrane anchor but that no specific residues in this region seemed to be required. A functional fusion protein was produced by combining the N-terminal enzymatic domain of PBP 5 with the C-terminal beta-sheet domain of PBP 6. In contrast, the opposite hybrid of PBP 6 to PBP 5 was not functional. The results suggest that the mode of PBP 5 membrane anchoring is important, that the mechanism entails more than a simple mechanical tethering of the enzyme to the outer face of the inner membrane, and that the physiological differences among the LMW PBPs arise from structural differences in the DD-carboxypeptidase enzymatic core.

Amino Acid Sequence↗

PubMed1998

Comparative analysis of Pseudomonas aeruginosa penicillin-binding protein 7 in the context of its membership in the family of low-molecular-mass PBPs.

The Pseudomonas aeruginosa pbpG gene encoding penicillin-binding protein 7, a homologue of the Escherichia coli gene encoding a DD-endopeptidase, was cloned and sequenced, pbpG was located immediately downstream of the phenylalanine hydroxylase (phh) operon. DNA sequencing revealed an open reading frame of 936 bp (starting with a GTG codon) which encodes a protein of 34,115 Da. N-terminal amino acid sequencing confirmed the presence of a cleavable N-terminal signal peptide of 23 amino acids. Verification that the protein is a penicillin-binding protein was directly demonstrated by labelling with 125I-labelled penicillin X. Inactivation of P. aeruginosa pbpG by interposon mutagenesis resulted in no obvious phenotypic changes, but when P. aeruginosa PbpG was overexpressed in E. coli using a T7 expression system, cell lysis resulted. P. aeruginosa PbpG resembled E. coli PbpG in being associated with the membrane fraction. Two additional members of the PbpG subfamily were identified in the database. P. aeruginosa PbpG shows 63% identity with E. coli penicillin-binding protein 7 (PbpG) and 60% identity with Vibrio cholerae PbpG, but only 23% identity with Haemophilus influenzae PbpG. The PbpG subfamily and three other subfamilies constituting the low-molecular-mass PBP protein family were analysed by multiple alignment of 26 sequences. PbpG exhibited the consensus motifs of other penicillin-binding proteins. Ten anchor residues were identified that are conserved at the family level within the superfamily of serine-active-site penicillin-interacting proteins.

Amino Acid Sequence↗