Title

ClpXP Modulates Cell Growth and Morphology in Cell Shape Mutants of E.coli

Author(s)

Ryann MurphyFollow

Major

Microbiology

Advisor

Camberg, Jodi, L.

Advisor Department

Cell and Molecular Biology

Advisor

Camberg, Jodi, L.

Advisor Department

Cell and Molecular Biology

Date

5-2014

Keywords

Penicillin binding proteins; cell morphology; cell division

Abstract

ClpXP modulates cell growth and morphology in cell shape mutants of E. coli

Ryann Murphy¹ and Jodi L. Camberg¹

¹University of Rhode Island, Department of Cell and Molecular Biology, Kingston, RI, 02881

Penicillin Binding Proteins (PBPs) are a family of prokaryotic membrane proteins named for their propensity to bind the antibiotic penicillin and are involved in remodeling and deposition of peptidoglycan. In wild type Escherichia coli cells, the uniform rod shape is conserved across generations. E.coli cells containing multiple deletions of Low Molecular Weight (LMW) PBPs exhibit irregular shapes. LMW PBP5 (dacA) is a potential substrate for degradation by ClpXP, an ATP-dependent protease that recognizes proteins with specific recognition sequences, unfolds and degrades them. To determine if changes in cellular ClpXP levels perturb cell shape through the PBP pathway, we investigated cell growth and morphology in PBP deletion strains (PBPD4,5,7 and PBPD4,7) overexpressing ClpXP from a plasmid. Results showed that PBPD4,7 deletion cells containing the ClpXP expression plasmid were significantly impaired for cell growth compared to PBPD4,7 cells containing the control vector. However, wild type cells and PBPD4,5,7 were modestly impaired for growth when ClpXP was expressed from a plasmid. Cellular morphology of mutant strains were observed using differential interference contrast microscopy. Both PBP double and triple mutants exhibited irregular morphology in comparison to the parental strain. Branching was associated with PBPD4,5,7. Median cell length of wild type cells containing the ClpXP expression plasmid is 75% longer than cells containing the control vector; however, the median cell length of PBPD4,7 deletions cells containing the ClpXP expression plasmid is 45% longer than cells containing the control vector. PBPD4,5,7 cells were highly irregular in shape in the presence and absence of ClpXP expression plasmid, therefore lengths could not be determined. Our results indicate that overexpression of ClpXP causes reduced cell growth in all strains, with PBPD4,7 cells being most severely affected. PBPD4,7 cells are more dependent on PBP5 for normal cell morphology since PBP4 and PBP7 are absent. Differences in cell growth and morphology when ClpXP is overexpressed may suggest that ClpXP is involved in regulating cell shape determinants or that deletion of PBPs makes cells less able to cope with reduced FtsZ levels, since ClpXP is also known to degrade FtsZ, an essential cell division protein.