Dynamics of Braun Lipoprotein Tethering and Release in the Escherichia coli Cell Wall

Core Concepts

Newly synthesized Braun lipoprotein and peptidoglycan subunits are independently incorporated into the expanding cell wall, and the tethering of Braun lipoprotein is dynamically regulated by the hydrolytic activity of the YafK enzyme.

Abstract

The study investigates the mode of tethering of the Braun lipoprotein (Lpp) to the peptidoglycan (PG) cell wall in Escherichia coli. Using a heavy isotope labeling approach coupled with mass spectrometry, the authors tracked the kinetics of incorporation of newly synthesized Lpp and PG subunits into the expanding cell wall.

The key findings are:

1. Lpp and PG subunits are independently incorporated into the lateral cell walls, suggesting that Lpp tethering is not coupled to PG polymerization.
2. Newly synthesized Lpp appears to be incorporated in limited amounts into the septal PG, which is predominantly composed of newly synthesized subunits.
3. The hydrolytic enzyme YafK actively cleaves the covalent bond between Lpp and the tripeptide stem of PG, contributing to the dynamic equilibrium between the PG-tethered and free forms of Lpp.
4. Deletion of YafK slows down the shuffling of Lpp between old and new PG stems, indicating that YafK-mediated hydrolysis is important for this process.
5. Estimates suggest that at least 50% of the Lpp-PG bonds are hydrolyzed in one generation, highlighting the dynamic nature of Lpp tethering.

The authors conclude that the dynamic equilibrium between free and PG-tethered Lpp may be physiologically relevant for spatially controlling the formation of outer membrane vesicles.

Stats

The relative abundance of the Tri→KR muropeptide isotopologues containing new (light) and old (heavy) Lpp and PG moieties decreased by approximately 50% in one generation in the wild-type strain, but not in the ΔyafK mutant.
The relative abundance of old Lpp moieties decreased by approximately 50% in one generation in both the wild-type and ΔyafK strains.
The relative abundance of old tripeptide moieties decreased by approximately 50% more in the wild-type strain compared to the ΔyafK mutant in one generation.

Quotes

"Newly synthesized Lpp and stem peptides are independently incorporated into PG"
"Hydrolysis of tripeptide→Lpp bonds by YafK contributes to the shuffling of Lpp between old and new peptidoglycan stems"
"At least 50% of the tripeptide→Lpp bonds are hydrolyzed in one generation"

Key Insights Distilled From

Peptidoglycan-tethered and free forms of the Braun lipoprotein are in dynamic equilibrium in Escherichia coli

by LIANG,Y., HU... at www.biorxiv.org 09-26-2023

https://www.biorxiv.org/content/10.1101/2023.09.26.559517v3

Deeper Inquiries

How might the dynamic equilibrium between free and PG-tethered Lpp be regulated in response to different environmental or physiological conditions?

The dynamic equilibrium between free and PG-tethered Lpp could be regulated in response to different environmental or physiological conditions through various mechanisms. One way is through the regulation of LDT enzymes that catalyze the tethering and hydrolysis of Lpp to PG. Environmental cues or stressors could modulate the expression or activity of these enzymes, thereby influencing the balance between free and PG-tethered Lpp. For example, changes in osmotic pressure, nutrient availability, or exposure to antimicrobial agents could impact the activity of LDTs, leading to alterations in the distribution of Lpp between the outer membrane and the peptidoglycan layer.
Additionally, the turnover rate of Lpp molecules could be regulated in response to specific conditions. For instance, under conditions of membrane stress or damage, there may be an increased turnover of Lpp molecules, leading to a higher proportion of free Lpp. On the other hand, during periods of cell growth or envelope stability, the turnover rate of Lpp tethering to PG may be reduced, favoring the PG-tethered form.
Furthermore, signaling pathways or regulatory networks within the cell could sense changes in the environment and modulate the expression or localization of Lpp-binding proteins. These proteins could act as chaperones or regulators of Lpp tethering, influencing the equilibrium between free and PG-tethered forms. Overall, the dynamic equilibrium between free and PG-tethered Lpp is likely tightly regulated to adapt to varying environmental conditions and maintain the integrity of the bacterial cell envelope.

What other cellular processes or structures could be affected by the disruption of Lpp tethering and shuffling, beyond the formation of outer membrane vesicles?

Disruption of Lpp tethering and shuffling could have broader implications beyond the formation of outer membrane vesicles. One significant impact could be on the structural integrity and stability of the bacterial cell envelope. Lpp plays a crucial role in bridging the outer membrane to the peptidoglycan layer, contributing to the overall stability of the cell envelope. Disruption of Lpp tethering could compromise the structural integrity of the cell envelope, making the cell more susceptible to environmental stresses or mechanical forces.
Moreover, alterations in Lpp tethering and shuffling could affect the localization and function of other envelope proteins. Lpp is involved in maintaining the distance between the inner and outer membranes, which is essential for proper functioning of membrane proteins involved in transport, signaling, or cell division. Changes in Lpp distribution could impact the localization and activity of these membrane proteins, leading to disruptions in essential cellular processes.
Additionally, the dynamic equilibrium between free and PG-tethered Lpp could influence the immune response of the host during bacterial infections. Lpp is a known immunostimulatory molecule, and changes in its distribution on the bacterial surface could affect the recognition and response of the host immune system. Disruption of Lpp tethering and shuffling could alter the host-pathogen interaction dynamics, influencing the outcome of infection.

Could the insights from this study on peptidoglycan remodeling and lipoprotein dynamics be leveraged to develop novel antimicrobial strategies targeting the bacterial cell envelope?

The insights gained from this study on peptidoglycan remodeling and lipoprotein dynamics offer valuable information that could be harnessed to develop novel antimicrobial strategies targeting the bacterial cell envelope. One potential strategy could involve targeting the enzymes involved in Lpp tethering and shuffling, such as LDTs and YafK. By designing inhibitors that specifically disrupt the interactions between Lpp and PG or inhibit the hydrolysis of these bonds, it may be possible to destabilize the bacterial cell envelope and enhance the susceptibility of bacteria to existing antibiotics.
Furthermore, understanding the spatial and temporal dynamics of peptidoglycan synthesis and Lpp incorporation could inform the development of combination therapies that target multiple steps in cell envelope biogenesis. By exploiting the vulnerabilities in the process of Lpp tethering and shuffling, synergistic antimicrobial approaches could be designed to disrupt bacterial envelope integrity and enhance the efficacy of existing antibiotics.
Moreover, the findings on the regulation of Lpp distribution in response to environmental cues could inspire the development of antimicrobial agents that exploit specific stress conditions to selectively target bacterial cells. By modulating the equilibrium between free and PG-tethered Lpp under certain environmental conditions, novel antimicrobial strategies could be designed to disrupt bacterial envelope integrity and combat antibiotic resistance mechanisms.