scholarly journals Pal stabilises the bacterial outer membrane during constriction by a mobilisation-and-capture mechanism

2019 ◽  
Author(s):  
Joanna Szczepaniak ◽  
Peter Holmes ◽  
Karthik Rajasekar ◽  
Renata Kaminska ◽  
Firdaus Samsudin ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Outer Membrane Lipoprotein ◽  
Capture Mechanism ◽  
Dividing Cells ◽  
Bacterial Outer Membrane ◽  
Membrane Lipoprotein ◽  
Inner Membrane Protein

SummaryCoordination of outer membrane constriction with septation is critical to faithful division in Gram-negative bacteria and vital to the barrier function of the membrane. Recent studies suggest this coordination is through the active accumulation of the peptidoglycan-binding outer membrane lipoprotein Pal at division sites by the Tol system, but the mechanism is unknown. Here, we show that Pal accumulation at Escherichia coli division sites is a consequence of three key functions of the Tol system. First, Tol mobilises Pal molecules in dividing cells, which otherwise diffuse very slowly due to their binding of the cell wall. Second, Tol actively captures mobilised Pal molecules and deposits them at the division septum. Third, the active capture mechanism is analogous to that used by the inner membrane protein TonB to dislodge the plug domains of outer membrane TonB-dependent nutrient transporters. We conclude that outer membrane constriction is coordinated with cell division by active mobilisation-and-capture of Pal at division septa by the Tol system.

scholarly journals The inner membrane protein YhdP modulates the rate of anterograde phospholipid flow in Escherichia coli

2020 ◽  
Author(s):  
Jacqueline Grimm ◽  
Handuo Shi ◽  
Wei Wang ◽  
Angela M. Mitchell ◽  
Ned S. Wingreen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Permeability Barrier ◽  
Gram Negative Bacteria ◽  
High Flux ◽  
Inner Membrane ◽  
Gram Negative ◽  
Selective Permeability ◽  
Delayed Cell Death ◽  
Inner Membrane Protein

AbstractThe outer membrane (OM) of Gram-negative bacteria is a selective permeability barrier that allows uptake of nutrients while simultaneously protecting the cell from harmful compounds. The basic pathways and molecular machinery responsible for transporting lipopolysaccharides (LPS), lipoproteins, and β-barrel proteins to the OM have been identified, but very little is known about phospholipid (PL) transport. To identify genes capable of affecting PL transport, we screened for genetic interactions with mlaA*, a mutant in which anterograde PL transport causes the inner membrane (IM) to shrink and eventually rupture; characterization of mlaA*-mediated lysis suggested that PL transport can occur via a high-flux, diffusive flow mechanism. We found that YhdP, an IM protein involved in maintaining the OM permeability barrier, modulates the rate of PL transport during mlaA*-mediated lysis. Deletion of yhdP from mlaA* reduced the rate of IM transport to the OM by 50%, slowing shrinkage of the IM and delaying lysis. As a result, the weakened OM of ΔydhP cells was further compromised and ruptured before the IM during mlaA*-mediated death. These findings demonstrate the existence of a high-flux, diffusive pathway for PL flow in Escherichia coli that is modulated by YhdP.Significance StatementThe outer membrane (OM) of Gram-negative bacteria serves as a barrier that protects cells from harmful chemical compounds, including many antibiotics. Understanding how bacteria build this barrier is an important step in engineering strategies to circumvent it. A long-standing mystery in the field is how phospholipids (PLs) are transported from the inner membrane (IM) to the OM. We previously discovered that a mutation in the gene mlaA causes rapid flow of PLs to the OM, eventually resulting in IM rupture. Here, we found that deletion of the gene yhdP delayed cell death in the mlaA mutant by slowing flow of PLs to the OM. These findings reveal a high-flux, diffusive pathway for PL transport in Gram-negative bacteria modulated by YhdP.

scholarly journals Pyocin S5 Import into Pseudomonas aeruginosa Reveals a Generic Mode of Bacteriocin Transport

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
Author(s):  
Hannah M. Behrens ◽  
Edward D. Lowe ◽  
Joseph Gault ◽  
Nicholas G. Housden ◽  
Renata Kaminska ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Outer Membrane ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Content Type ◽  
Animal Infection ◽  
Inner Membrane Protein ◽  
Biochemical Analyses ◽  
Functional Analyses

ABSTRACT Pyocin S5 (PyoS5) is a potent protein bacteriocin that eradicates the human pathogen Pseudomonas aeruginosa in animal infection models, but its import mechanism is poorly understood. Here, using crystallography, biophysical and biochemical analyses, and live-cell imaging, we define the entry process of PyoS5 and reveal links to the transport mechanisms of other bacteriocins. In addition to its C-terminal pore-forming domain, elongated PyoS5 comprises two novel tandemly repeated kinked 3-helix bundle domains that structure-based alignments identify as key import domains in other pyocins. The central domain binds the lipid-bound common polysaccharide antigen, allowing the pyocin to accumulate on the cell surface. The N-terminal domain binds the ferric pyochelin transporter FptA while its associated disordered region binds the inner membrane protein TonB1, which together drive import of the bacteriocin across the outer membrane. Finally, we identify the minimal requirements for sensitizing Escherichia coli toward PyoS5, as well as other pyocins, and suggest that a generic pathway likely underpins the import of all TonB-dependent bacteriocins across the outer membrane of Gram-negative bacteria. IMPORTANCE Bacteriocins are toxic polypeptides made by bacteria to kill their competitors, making them interesting as potential antibiotics. Here, we reveal unsuspected commonalities in bacteriocin uptake pathways, through molecular and cellular dissection of the import pathway for the pore-forming bacteriocin pyocin S5 (PyoS5), which targets Pseudomonas aeruginosa. In addition to its C-terminal pore-forming domain, PyoS5 is composed of two tandemly repeated helical domains that we also identify in other pyocins. Functional analyses demonstrate that they have distinct roles in the import process. One recognizes conserved sugars projected from the surface, while the other recognizes a specific outer membrane siderophore transporter, FptA, in the case of PyoS5. Through engineering of Escherichia coli cells, we show that pyocins can be readily repurposed to kill other species. This suggests basic ground rules for the outer membrane translocation step that likely apply to many bacteriocins targeting Gram-negative bacteria.

scholarly journals Inhibition of Escherichia coli lipoprotein diacylglyceryl transferase is insensitive to resistance caused by deletion of Braun’s lipoprotein

2021 ◽  
Author(s):  
Jingyu Diao ◽  
Rie Komura ◽  
Tatsuya Sano ◽  
Homer Pantua ◽  
Kelly M. Storek ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Bacterial Growth ◽  
Signal Peptidase ◽  
Common Mechanism ◽  
Unexpected Finding ◽  
Gram Negative ◽  
Outer Membrane Lipoprotein ◽  
Membrane Lipoprotein ◽  
Bacterial Lipoprotein

Lipoprotein diacylglyceryl transferase (Lgt) catalyzes the first step in the biogenesis of Gram-negative bacterial lipoproteins which play crucial roles in bacterial growth and pathogenesis. We demonstrate that Lgt depletion in a clinical uropathogenic Escherichia coli strain leads to permeabilization of the outer membrane and increased sensitivity to serum killing and antibiotics. Importantly, we identify G2824 as the first described Lgt inhibitor that potently inhibits Lgt biochemical activity in vitro and is bactericidal against wild-type Acinetobacter baumannii and E. coli strains. While deletion of the major outer membrane lipoprotein, lpp, leads to rescue of bacterial growth after genetic depletion or pharmacologic inhibition of the downstream type II signal peptidase, LspA, no such rescue of growth is detected after Lgt depletion or treatment with G2824. Inhibition of Lgt does not lead to significant accumulation of peptidoglycan-linked Lpp in the inner membrane. Our data validate Lgt as a novel antibacterial target and suggest that, unlike downstream steps in lipoprotein biosynthesis and transport, inhibition of Lgt may not be sensitive to one of the most common resistance mechanisms that invalidate inhibitors of bacterial lipoprotein biosynthesis and transport. Importance As the emerging threat of multidrug-resistant (MDR) bacteria continues to increase, no new classes of antibiotics have been discovered in the last fifty years. While previous attempts to inhibit the lipoprotein biosynthetic (LspA) or transport (LolCDE) pathways have been made, most efforts have been hindered by the emergence of a common mechanism leading to resistance; namely, the deletion of the major Gram-negative outer membrane lipoprotein, lpp. Our unexpected finding that inhibition of Lgt is not susceptible to lpp deletion-mediated resistance uncovers the complexity of bacterial lipoprotein biogenesis and the corresponding enzymes involved in this essential outer membrane biogenesis pathway, and potentially points to new antibacterial targets in this pathway.

scholarly journals MexAB-OprM Efflux Pump Interaction with the Peptidoglycan of Escherichia coli and Pseudomonas aeruginosa

2021 ◽  
Vol 22 (10) ◽  
pp. 5328
Author(s):  
Miao Ma ◽  
Margaux Lustig ◽  
Michèle Salem ◽  
Dominique Mengin-Lecreulx ◽  
Gilles Phan ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Cell Division ◽  
Membrane Proteins ◽  
Outer Membrane ◽  
Efflux Pump ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Active Efflux

One of the major families of membrane proteins found in prokaryote genome corresponds to the transporters. Among them, the resistance-nodulation-cell division (RND) transporters are highly studied, as being responsible for one of the most problematic mechanisms used by bacteria to resist to antibiotics, i.e., the active efflux of drugs. In Gram-negative bacteria, these proteins are inserted in the inner membrane and form a tripartite assembly with an outer membrane factor and a periplasmic linker in order to cross the two membranes to expulse molecules outside of the cell. A lot of information has been collected to understand the functional mechanism of these pumps, especially with AcrAB-TolC from Escherichia coli, but one missing piece from all the suggested models is the role of peptidoglycan in the assembly. Here, by pull-down experiments with purified peptidoglycans, we precise the MexAB-OprM interaction with the peptidoglycan from Escherichia coli and Pseudomonas aeruginosa, highlighting a role of the peptidoglycan in stabilizing the MexA-OprM complex and also differences between the two Gram-negative bacteria peptidoglycans.

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