Intracellular expression of Peptide fusions for demonstration of protein essentiality in bacteria

Abstract

We describe a "protein knockout" technique that can be used to identify essential proteins in bacteria. This technique uses phage display to select peptides that bind specifically to purified target proteins. The peptides are expressed intracellularly and cause inhibition of growth when the protein is essential. In this study, peptides that each specifically bind to one of seven essential proteins were identified by phage display and then expressed as fusions to glutathione S-transferase in Escherichia coli. Expression of peptide fusions directed against E. coli DnaN, LpxA, RpoD, ProRS, SecA, GyrA, and Era each dramatically inhibited cell growth. Under the same conditions, a fusion with a randomized peptide sequence did not inhibit cell growth. In growth-inhibited cells, inhibition could be relieved by concurrent overexpression of the relevant target protein but not by coexpression of an irrelevant protein, indicating that growth inhibition was due to a specific interaction of the expressed peptide with its target. The protein knockout technique can be used to assess the essentiality of genes of unknown function emerging from the sequencing of microbial genomes. This technique can also be used to validate proteins as drug targets, and their corresponding peptides as screening tools, for discovery of new antimicrobial agents.

FIG. 1.

Expression vectors. (A) Peptide intracellular expression vector pKBU-Tet/GST is based on the Clontech pPROTet.E332-lacZ vector. SalI and NheI sites were introduced on a cassette, and then the gene for GST and a 5′ hinge region were inserted into the HindIII and PstI sites. Subsequently, peptides were cloned into the SalI and NheI sites. (B) Protein intracellular expression vector pKBU-Pro-Lar is based on the Clontech pPROLar.A222 vector. NdeI, BspHI, and SmaI sites were introduced on a cassette cloned into the KpnI and PacI sites. Subsequently, target genes of interest were cloned into the NdeI or BspHI and SmaI sites. RBS, ribosome binding site.

FIG. 2.

Intracellular peptide expression. In the presence of the inducer anhydrotetracycline, peptide specific for an essential cellular protein is produced inside the cell as an N-terminal fusion to GST. Specific peptide binding to the target protein inhibits the essential function and causes growth inhibition.

FIG. 3.

Intracellular peptide expression inhibits cell growth. Cells with the plasmid carrying an inducible target-specific peptide fused to GST were grown in the absence (striped bars) or presence (solid bars) of the inducer; the absorbance at 600 nm was read 44 to 48 h after induction. Data are expressed relative to the uninduced control for a given peptide, with the absorbance of the uninduced control at 600 nm representing 100%. Peptide identification numbers are given above each data set; the peptide target is given below each data set. The peptide RND is a noninhibitory peptide, using amino acids generally like that of inhibitory peptides but with a randomized primary sequence. Data shown are representative of several such experiments.

FIG. 4.

Specific rescue of peptide-dependent growth inhibition. Dotted bars, protein (LpxA or DnaN) induction; striped bars, peptide (920 or 1337) induction; cross-hatched bars: coinduction. Peptide 920 was originally selected in vitro on LpxA; peptide 1337 was selected on DnaN. The absorbance of cultures at 600 nm was read 48 h after induction of protein and/or peptide in liquid medium. Data are expressed relative to those obtained with uninduced control cultures grown in parallel; error bars show standard deviations.

FIG. 5.

Specific rescue of peptide-dependent growth inhibition. Cultures were plated on selective medium containing appropriate inducers 3 h after induction of protein and/or peptide in liquid medium and then grown for 16 h at 37°C. (A) No induction; (B) peptide induction alone; (C) protein induction alone; (D) peptide and protein induction. Each quadrant is labeled with the protein and peptide, respectively, whose expression is induced. Peptide 920 was selected on LpxA; peptide 1337 was selected on DnaN.