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Journal of Bacteriology, April 2003, p. 2178-2186, Vol. 185, No. 7
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.7.2178-2186.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Sequences near the Active Site in Chimeric Penicillin Binding Proteins 5 and 6 Affect Uniform Morphology of Escherichia coli

Anindya S. Ghosh and Kevin D. Young*

Department of Microbiology and Immunology, University of North Dakota School of Medicine, Grand Forks, North Dakota 58202-9037

Received 4 September 2002/ Accepted 6 January 2003

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 Asp218 and Lys219 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.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, University of North Dakota School of Medicine, Grand Forks, ND 58202-9037. Phone: (701) 777-2624. Fax: (701) 777-2054. E-mail: kyoung{at}medicine.nodak.edu.

Journal of Bacteriology, April 2003, p. 2178-2186, Vol. 185, No. 7
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.7.2178-2186.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Meberg, B. M., Paulson, A. L., Priyadarshini, R., Young, K. D. (2004). Endopeptidase Penicillin-Binding Proteins 4 and 7 Play Auxiliary Roles in Determining Uniform Morphology of Escherichia coli. J. Bacteriol. 186: 8326-8336 [Abstract] [Full Text]  
  • Antignac, A., Boneca, I. G., Rousselle, J.-C., Namane, A., Carlier, J.-P., Vazquez, J. A., Fox, A., Alonso, J.-M., Taha, M.-K. (2003). Correlation between Alterations of the Penicillin-binding Protein 2 and Modifications of the Peptidoglycan Structure in Neisseria meningitidis with Reduced Susceptibility to Penicillin G. J. Biol. Chem. 278: 31529-31535 [Abstract] [Full Text]  


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