Illumination of growth, division and secretion by metabolic labeling of the bacterial cell surface

Abstract

The cell surface is the essential interface between a bacterium and its surroundings. Composed primarily of molecules that are not directly genetically encoded, this highly dynamic structure accommodates the basic cellular processes of growth and division as well as the transport of molecules between the cytoplasm and the extracellular milieu. In this review, we describe aspects of bacterial growth, division and secretion that have recently been uncovered by metabolic labeling of the cell envelope. Metabolite derivatives can be used to label a variety of macromolecules, from proteins to non-genetically-encoded glycans and lipids. The embedded metabolite enables precise tracking in time and space, and the versatility of newer chemoselective detection methods offers the ability to execute multiple experiments concurrently. In addition to reviewing the discoveries enabled by metabolic labeling of the bacterial cell envelope, we also discuss the potential of these techniques for translational applications. Finally, we offer some guidelines for implementing this emerging technology.

DivIVA concentrates mycobacterial cell envelope assembly for initiation and stabilization of polar growth

“…In contrast to staining by antibiotic conjugates, labeling by tagged precursors of peptidoglycan or mycomembrane can reveal the presence of nascent envelope within minutes (Foley et al, 2016;Siegrist et al, 2015;Siegrist et al, 2013) and does not obviously interfere with bacterial growth (Siegrist et al, 2015). In contrast to staining by antibiotic conjugates, labeling by tagged precursors of peptidoglycan or mycomembrane can reveal the presence of nascent envelope within minutes (Foley et al, 2016;Siegrist et al, 2015;Siegrist et al, 2013) and does not obviously interfere with bacterial growth (Siegrist et al, 2015).…”

“…In contrast to staining by antibiotic conjugates, labeling by tagged precursors of peptidoglycan or mycomembrane can reveal the presence of nascent envelope within minutes (Foley et al, 2016;Siegrist et al, 2015;Siegrist et al, 2013) and does not obviously interfere with bacterial growth (Siegrist et al, 2015). Single residue D-amino acid probes have been hypothesized to incorporate into peptidoglycan in part or in whole via periplasmic transpeptidases (Siegrist et al, 2015). Derivatives of D-alanine and D-alanine-D-alanine have been used in many bacterial species, including mycobacteria, to detect cell wall metabolism (Botella et al, 2017;Fura et al, 2015;Hayashi et al, 2018;Kuru et al, 2012;Liechti et al, 2014;Lebar et al, 2014;Meniche et al, 2014;Siegrist et al, 2013;Siegrist et al, 2015).…”

“…We examined the effect of DivIVA depletion on peptidoglycan assembly by labeling cells with an alkyne-bearing dipeptide probe (alkDADA) (Liechti et al, 2014), which was detected by coppercatalyzed azide-alkyne cycloaddition (CuAAC) (Siegrist et al, 2015) to (Figure 3b,d). After 8 hours of depletion, the cells developed large bulges that were evenly labeled, and after 12 hours, they were primarily spherical, bounded by homogenous labeling (Figure 3b,d).…”

Visualization of mycobacterial membrane dynamics in live cells

“…This deficit likely reflects a lack of tools for probing cell envelope metabolites in live cells, and with subcellular resolution, a problem that chemists have tackled in recent years with the development of new imaging methods. 11 For example, early work using lipophilic fluorophore conjugates that nonspecifically intercalate into the mycomembrane suggested a heterogeneous cell surface landscape. 12,13 Progress toward defining the dynamic properties of Mtb ’s cell envelope, however, requires more refined methods for imaging its specific cell envelope components.…”

Imaging Bacterial Cell Wall Biosynthesis

“…For the purpose of this review, we first provide a general introduction to PG imaging techniques and then focus on PG labeling through the lens of fluorescent microscopy and FDAAs. For additional information regarding cell wall labeling, the reader is referred to the thorough review by Siegrist et al (7) as well as to the in-depth study of cell surface display and biotechnological applications by Gautam et al (8). Although a number of labeling methods are currently available, FDAAs have profoundly impacted studies on the bacterial cell wall (see Table 1).…”