Abstract

The bacterial cell wall, which is comprised of a mesh of polysaccharide strands crosslinked via peptide bridges (peptidoglycan, PG), is critical for maintenance of cell shape and survival. PG assembly is mediated by a variety of Penicillin Binding Proteins (PBP) whose fundamental activities have been characterized in great detail; however, there is limited knowledge of the factors that modulate their activities in different environments or growth phases. In Vibrio cholerae, the cause of cholera, PG synthesis during the transition into stationary phase is primarily mediated by the bifunctional enzyme PBP1A. Here, we screened an ordered V. cholerae transposon library for mutants that are sensitive to growth inhibition by non-canonical D-amino acids (DAA), which prevent growth and maintenance of cell shape in PBP1A-deficient V. cholerae. In addition to PBP1A and its lipoprotein activator LpoA, we found that CsiV, a small periplasmic protein with no previously described function, is essential for growth in the presence of DAA. Deletion of csiV, like deletion of lpoA or the PBP1A–encoding gene mrcA, causes cells to lose their rod shape in the presence of DAA or the beta-lactam antibiotic cefsulodin, and all three mutations are synthetically lethal with deletion of mrcB, which encodes PBP1B, V. cholerae's second key bifunctional PBP. CsiV interacts with LpoA and PG but apparently not with PBP1A, supporting the hypothesis that CsiV promotes LpoA's role as an activator of PBP1A, and thereby modulates V. cholerae PG biogenesis. Finally, the requirement for CsiV in PBP1A-mediated growth of V. cholerae can be overcome either by augmenting PG synthesis or by reducing PG degradation, thereby highlighting the importance of balancing these two processes for bacterial survival.

Highlights

  • The bacterial cell wall is a remarkably sturdy, web-like structure composed mainly of peptidoglycan (PG), a polysaccharide mesh whose approximately parallel strands are crosslinked via peptide sidechains [1,2,3]

  • We show that CsiV interacts with LpoA, a lipoprotein activator of PBP1A, as well as with PG

  • CsiV, LpoA, or PBP1A are all required for survival of V. cholerae lacking PBP1B, and mutants lacking any of these factors show marked changes in PG content in stationary phase

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Summary

Introduction

The bacterial cell wall is a remarkably sturdy, web-like structure composed mainly of peptidoglycan (PG), a polysaccharide mesh whose approximately parallel strands are crosslinked via peptide sidechains [1,2,3]. It forms a relatively thin layer between the inner and outer membranes of gram-negative bacteria, and a thicker layer in gram-positive bacteria, for which it is often the outermost bacterial structure. PBP1A and PBP1B are largely functionally redundant and conditionally essential, i.e., in the absence of one, the other becomes strictly required for growth [10,11]

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