Imaging Bacterial Cell Wall Biosynthesis

Abstract

Peptidoglycan is an essential component of the cell wall that protects bacteria from environmental stress. A carefully coordinated biosynthesis of peptidoglycan during cell elongation and division is required for cell viability. This biosynthesis involves sophisticated enzyme machineries that dynamically synthesize, remodel, and degrade peptidoglycan. However, when and where bacteria build peptidoglycan, and how this is coordinated with cell growth, have been long-standing questions in the field. The improvement of microscopy techniques has provided powerful approaches to study peptidoglycan biosynthesis with high spatiotemporal resolution. Recent development of molecular probes further accelerated the growth of the field, which has advanced our knowledge of peptidoglycan biosynthesis dynamics and mechanisms. Here, we review the technologies for imaging the bacterial cell wall and its biosynthesis activity. We focus on the applications of fluorescent d-amino acids, a newly developed type of probe, to visualize and study peptidoglycan synthesis and dynamics, and we provide direction for prospective research.

Keywords: bacterial cell wall; bacterial morphogenesis; d-amino acids; fluorescent probes; microscopy; peptidoglycan.

Figure 1

Structure of the bacterial cell wall in different types of bacteria. Left: Gram-negative bacteria cell wall contains a thinner peptidoglycan (PG) layer (~5 nm) sandwiched between the cytoplasmic membrane and the outer membrane. Middle: Gram-positive bacteria cell wall contains a thicker PG layer (20–50 nm) and the cytoplasmic membrane. Right: Mycobacteria cell wall contains a PG layer, the cytoplasmic membrane, and a waxy surface layer made of lipids, mycolic acids, and arabinogalactan.

Figure 2

Biosynthesi pathway of PG in Escherichia coli. Some cell wall components are not shown in the figure (e.g., the outer membrane) for simplification. The biosynthesis pathway starts with the formation of Park’s nucleotide in cytoplasm, followed by binding to a lipid component to produce lipid II. Finally, lipid II is translocated across the cytoplasmic membrane and then inserted into the existing PG through transglycosylation and transpeptidation reactions. Abbreviations: PBPs, penicillin-binding proteins; PG, peptidoglycan; SEDS, shape, elongation, division, and sporulation enzyme family.

Figure 3

Applications of fluorescent d-amino acid (FDAA) labeling. (a) Comparison between short-and long-pulse labeling in Streptomyces venezuelae. (b) Pulse-and-chase labeling; scheme of labeling in S. venezuelae. (c) Virtual time-lapse labeling; scheme of labeling in S. venezuelae. (d) Peptidoglycan growth dynamics in Hyphomonas neptunium; example of FDAA short-pulse labeling. (e) Growth pattern at septal peptidoglycan; example of virtual time-lapse labeling in Bacillus subtilis division septum. (f) Long-pulse-chase-new-labeling; example using two FDAAs in Streptococcus pneumoniae for peptidoglycan turnover rate measurement.

Figure 4

Proposed incorporation pathway of FDAA (top) and EDA-DA (bottom). FDAA incorporation occurs in periplasm through transpeptidases activity (e.g., PBP activity). EDA-DA incorporation occurs in cytoplasm through MurF activity. Abbreviations: EDA-DA, ethynyl-d-alanyl-d-alanine; FDAA, fluorescent d-amino acid; HADA, hydroxycoumarin 3-amino-d-alanine; PBP, penicillin-binding protein; PG, peptidoglycan.