CLONING AND CHARACTERIZATION OF FULL LENGTH GCD AND INTERNAL TRUNCATIONS OF TGCD

Abstract

<p>The increase in antibiotic resistance has accelerated the search for novel antibacterial agents. As proteins with toxic properties appear to be less susceptible to common resistance mechanisms, they may serve as potential substitutes for current antibiotics. The present study focuses on a toxic peptide called tGcd derived from the <em>Escherichia coli</em> genome that can cause cell death when it is expressed. To better understand the mechanism of tGcd and to decipher the sequence determinants for its toxic phenotype, sequence analyses of this peptide were conducted. First, we conducted growth analysis for two full-length Gcd constructs to test whether the observed growth suppression was caused by the overexpression of tGcd in <em>E. coli</em> cells. We also generated a series of truncation mutants to determine the minimal sequence required for toxicity. Finally, to test whether tGcd’s toxicity was due to differences in expression levels, Western blot analysis was conducted. This study reveals that full length Gcd does not have an effect on bacterial growth when overexpressed. We also observed that some internal amino acids of tGcd can be truncated without the loss of the toxicity to the <em>E. coli</em> cells. Finally, Western blot analysis showed that the toxicity of this peptide is independent on its expression levels. These findings offer additional insights into properties common to membrane targeting antimicrobial peptides in Gram negative microbes, and contribute to the discovery of small bacterial peptides that may be useful for combating multidrug-resistant bacteria.</p>