Cloning, mapping, and characterization of the Escherichia coli prc gene, which is involved in C-terminal processing of penicillin-binding protein 3

Abstract

The prc gene, which is involved in cleavage of the C-terminal peptide from the precursor form of penicillin-binding protein 3 (PBP 3) of Escherichia coli, was cloned and mapped at 40.4 min on the chromosome. The gene product was identified as a protein of about 80 kDa in maxicell and in vitro systems. Fractionation of the maxicells producing the product suggested that the product was associated with the periplasmic side of the cytoplasmic membrane. This was consistent with the notion that the C-terminal processing of PBP 3 probably occurs outside the cytoplasmic membrane: the processing was found to be dependent on the secY and secA functions, indicating that the prc product or PBP 3 or both share the translocation machinery with other extracytoplasmic proteins. DNA sequencing analysis of the prc gene region identified an open reading frame, with two possible translational starts 6 bp apart from each other, that could code for a product with a calculated molecular weight of 76,667 or 76,432. The prc mutant was sensitive to thermal and osmotic stresses. Southern analysis of the chromosomal DNA of the mutant unexpectedly revealed that the mutation was a deletion of the entire prc gene and thus that the prc gene is conditionally dispensable. The mutation resulted in greatly reduced heat shock response at low osmolarity and in leakage of periplasmic proteins.

The lone S41 family C-terminal processing protease in Staphylococcus aureus is localized to the cell wall and contributes to virulence

“…If this hypothesis holds true for Gram-positive bacteria then it seems likely that the proteolytic targets of S. aureus CtpA-mediated hydrolysis are located in the bacterial cell wall. In Gram-negative bacteria, many of the phenotypes associated with ctpA 2 mutants, including altered cell morphology and increased sensitivity to heat and osmotic shock, are proposed to be a consequence of decreased cell-wall integrity (Hara et al, 1991;Kumru et al, 2011;Ostberg et al, 2004;Seoane et al, 1992). Studies in E. coli have demonstrated periplasmic protein leakage in a prc mutant, suggesting increased permeability of the outer membrane in this strain (Hara et al, 1991).…”

“…Results demonstrate that it is membrane anchored, with the majority of the protein, including the catalytic site, located in the bacterial cell wall. Known targets of Gram-negative CTPs, including PBP-3 and P13, are located either in the bacterial periplasm or in the outer membrane (Hara et al, 1991;Noppa et al, 2001). This suggests that in Gram-negative species CTPs colocalize with their target substrates (Hoge et al, 2011).…”

“…Although CTP mutants in Gram-negative bacteria display a variety of phenotypes, certain commonalities exist. Altered cell morphology, differences in osmotic/thermal stress resistance and altered susceptibility to antibiotics are common phenotypes of mutants from different species (and may result from alterations in the cell-wall integrity) (Hara et al, 1991;Kumru et al, 2011;Seoane et al, 1992). In addition, a number of Gram-negative bacteria, including E. coli, Brucella suis, Chlamydia trachomatis, Salmonella typhimurium, Pseudomonas aeruginosa and Burkholderia mallei, demonstrate reduced virulence upon inactivation of CTP (Bandara et al, 2005(Bandara et al, , 2008Bäumler et al, 1994;Lad et al, 2007;Seo & Darwin, 2013;Wang et al, 2012).…”

“…Typically, CTPs in Gram-negative bacteria are located in the periplasm (Hara et al, 1991;Hoge et al, 2011). The Escherichia coli CTP (called Prc or Tsp) was originally identified as a periplasmic protease responsible for C-terminal processing of penicillin-binding protein 3 (PBP-3) (Hara et al, 1991).…”

Transcriptional Analysis of Butanol Stress and Tolerance in Clostridium acetobutylicum

“…A truncated rlpA gene in Escherichia coli (4) was able to rescue a conditionally lethal mutation in the prc gene (involved in Cterminal processing of penicillin-binding protein-3). prc mutants are sensitive to heat and osmotic stress (19). A conserved membrane protein (CAC2582) is in this same cluster of oppositely regulated genes (Fig.…”