Genetic interaction mapping highlights key roles of the <scp>Tol‐Pal</scp> complex

Tan, Wee Boon; Chng, Shu‐Sin

doi:10.1111/mmi.14882

Cited by 5 publications

(5 citation statements)

References 75 publications

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“…2C). These mutants perturb the cell envelope in different ways, with opgG and opgH encoding osmo-regulated periplasmic glucans [33], tolB encoding a key component of the Tol-Pal trans- envelope machinery that regulates OM constriction and homeostasis [34–36], and pgm encoding a phosphoglucomutase that has been implicated in cell division [37, 38]. Each of these mutants had a larger mean cell width than wild type (Fig.…”

Section: Resultsmentioning

confidence: 99%

Bacterial cell widening alters periplasmic size and activates envelope stress responses

Zietek

Miguel

Khusainov

et al. 2022

Preprint

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The Rcs signal transduction system is a phosphorelay responsible for sensing a wide variety of enterobacterial cell envelope stresses. In Escherichia coli, the Rcs system is required to survive A22 and mecillinam treatment, two drugs that perturb cell size. To test whether cell size changes might be correlated with envelope damage and thereby sensed by the Rcs system, we tuned E. coli cell size via drug inhibition with A22, point mutations to the cell-shape determinant MreB, and mechanically confined growth. In all conditions, cell width was strongly correlated with Rcs activation, with wider cells exhibiting more activation than wild-type. In all conditions, RcsF, the outer membrane-localized upstream component of the Rcs system, was essential for responding to cell width changes. Consistently, several envelope gene deletions known to induce the Rcs system via RcsF resulted in cells that were wider than wild-type. Cryo- electron microscopy revealed that the periplasm of a wide MreB mutant was on average ∼3 nm thinner than wild-type, thereby bringing RcsF closer to the downstream components of the signaling cascade in the inner membrane. Conversely, extending the flexible linker region of RcsF by ∼3 nm increased Rcs activity in wild-type cells. In summary, we propose that the Rcs system responds to changes in cell width because of altered periplasmic thickness.

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Section: Resultsmentioning

confidence: 99%

Bacterial cell widening alters periplasmic size and activates envelope stress responses

Zietek

Miguel

Khusainov

et al. 2022

Preprint

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“…Interestingly, the Tol-Pal complex may also have roles in regulating septal cell wall separation [90]. Recently, a transposon insertion screen revealed strong negative interactions between tolQ / A predominantly with cell wall synthesis/remodelling and OM biogenesis/homeostasis pathways [91], reinforcing the idea that the Tol-Pal complex plays key roles in these processes.…”

Section: The Tol-pal Complexmentioning

confidence: 99%

Of zones, bridges and chaperones – phospholipid transport in bacterial outer membrane assembly and homeostasis

Yeow

Chng

2022

Microbiology

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The outer membrane (OM) is a formidable permeability barrier that protects Gram-negative bacteria from detergents and antibiotics. It possesses exquisite lipid asymmetry, requiring the placement and retention of lipopolysaccharides (LPS) in the outer leaflet, and phospholipids (PLs) in the inner leaflet. To establish OM lipid asymmetry, LPS are transported from the inner membrane (IM) directly to the outer leaflet of the OM. In contrast, mechanisms for PL trafficking across the cell envelope are much less understood. In this review, we summarize and discuss recent advances in our understanding of PL transport, making parallel comparisons to well-established pathways for OM lipoprotein (Lol) and LPS (Lpt). Insights into putative PL transport systems highlight possible connections back to the ‘Bayer bridges’, adhesion zones between the IM and the OM that had been observed more than 50 years ago, and proposed as passages for export of OM components, including LPS and PLs.

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“…Pal is an OM lipoprotein that can bind the periplasmic protein TolB or peptidoglycan (but not simultaneously) (25, 26), with the latter interaction contributing to OM-cell wall tethering (26–28). Strains lacking any Tol-Pal component are known to have pleiotropic OM defects, including hypersensitivity to detergents and antibiotics, leakage of periplasmic contents, and OM vesiculation (19, 29–32). These phenotypes indicate substantial perturbations to envelope integrity, which is consistent with lipid dyshomeostasis observed in the OM (13, 31, 33).…”

Section: Introductionmentioning

confidence: 99%

“…It is also unclear if, and how, septal localized Tol-Pal contributes to OM lipid homeostasis. Yet, the involvement of Tol-Pal in both pathways is evident from synthetic genetic interactions between the tol-pal locus and genes involved in either cell wall remodeling or OM biogenesis (32). Interestingly, alleviating cell wall separation and division defects in tol-pal mutants does not appear to rescue OM instability (37).…”

Section: Introductionmentioning

confidence: 99%

“…OM lipid composition analysis. Experiment to measure PL:LPS ratio at the OM had been previously described (13,31,32). Briefly, 10 ml cultures were grown in the presence of [ 32 P]-phosphate (final concentration of 1 µCi ml -1 ; Perkin Elmer NEX060001MC) for until OD600 reached ~1.5-2, after which the cell pellets were harvested and washed twice with Tris-buffered saline (TBS).…”

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Primary role of the Tol-Pal complex in bacterial outer membrane lipid homeostasis

Tan,

Chng

2024

Preprint

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Gram-negative bacteria are defined by an outer membrane (OM) that contributes to cell envelope integrity, and functions as an effective permeability barrier. The OM is thus critical for fitness and antibiotic resistance. Building this bilayer require proper assembly of lipopolysaccharides, proteins, and phospholipids, yet how the intricate balance of these components is achieved to ensure a stable OM is not well understood. One system long known to be important for OM stability is the Tol-Pal complex, which has recently been implicated in the maintenance of OM lipid homeostasis, possibly via retrograde phospholipid transport. However, assignment of its primary function has been challenging, owing to septal localization of Tol-Pal and its associated role(s) during cell division. Here, we uncouple the function of the Tol-Pal complex in OM lipid homeostasis from its impact on cell division inEscherichia coli, by preventing recruitment to mid-cell. The Tol-Pal complex comprises the inner membrane TolQRA and OM TolB-Pal subcomplexes. We engineer a TolA variant that forms a chimeric complex with ExbBD, which are homologous to TolQR, resulting in loss of septal enrichment of Tol-Pal. We demonstrate that this peripherally-localized complex cannot rescue division defects in strains lacking TolQRA, but restores OM integrity and barrier. Importantly, this chimeric complex is fully capable of maintaining lipid balance in the OM, independent of the role of Tol-Pal in cell division. Our work establishes the primary function of the Tol-Pal complex in OM lipid homeostasis, and provides novel insights on how this conserved system also contributes to cell division.

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Genetic interaction mapping highlights key roles of the Tol‐Pal complex

Cited by 5 publications

References 75 publications

Bacterial cell widening alters periplasmic size and activates envelope stress responses

Bacterial cell widening alters periplasmic size and activates envelope stress responses

Of zones, bridges and chaperones – phospholipid transport in bacterial outer membrane assembly and homeostasis

Primary role of the Tol-Pal complex in bacterial outer membrane lipid homeostasis

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