Lipoprotein DolP supports proper folding of BamA in the bacterial outer membrane promoting fitness upon envelope stress

In Proteobacteria, integral outer membrane proteins (OMPs) are crucial for the maintenance of the envelope permeability barrier to some antibiotics and detergents. In Enterobacteria, envelope stress caused by unfolded OMPs activates the sigmaE (σE) transcriptional response. σE upregulates OMP biogenesis factors, including the β-barrel assembly machinery (BAM) that catalyses OMP folding. Here we report that DolP (formerly YraP), a σE-upregulated and poorly understood outer membrane lipoprotein, is crucial for fitness in cells that undergo envelope stress. We demonstrate that DolP interacts with the BAM complex by associating with outer membrane-assembled BamA. We provide evidence that DolP is important for proper folding of BamA that overaccumulates in the outer membrane, thus supporting OMP biogenesis and envelope integrity. Notably, mid-cell recruitment of DolP had been linked to regulation of septal peptidoglycan remodelling by an unknown mechanism. We now reveal that, during envelope stress, DolP loses its association with the mid-cell, thereby suggesting a mechanistic link between envelope stress caused by impaired OMP biogenesis and the regulation of a late step of cell division.

Download Full-text

Outer membrane lipoprotein DolP interacts with the BAM complex and promotes fitness during envelope stress response

10.1101/2020.09.23.308296 ◽

2020 ◽

Author(s):

David Ranava ◽

Yiying Yang ◽

Luis Orenday-Tapia ◽

François Rousset ◽

Catherine Turlan ◽

...

Keyword(s):

Cell Division ◽

Outer Membrane ◽

Detrimental Effect ◽

Critical Determinant ◽

Cell Recruitment ◽

Bam Complex ◽

Outer Membrane Lipoprotein ◽

Envelope Stress ◽

Late Step ◽

Membrane Lipoprotein

AbstractIn Gram-negative bacteria, coordinated remodelling of the outer membrane (OM) and the peptidoglycan is crucial for envelope integrity. Envelope stress caused by unfolded OM proteins (OMPs) activates sigmaE (σE) in Enterobacteria. σE upregulates OMP biogenesis factors, including the β-barrel assembly machinery (BAM) that catalyzes OMP-folding. Elevated σE activity, however, can be detrimental for OM integrity. Here we report that DolP (YraP), a σE-upregulated OM lipoprotein important for envelope integrity, is a novel interactor of BAM and we demonstrate that OM-assembled BamA is a critical determinant of the BAM-DolP complex. Mid-cell recruitment of DolP had been previously associated to activation of septal peptidoglycan remodelling during cell division, but its role during envelope stress was unknown. We now show that DolP promotes cell fitness upon stress-induced activation of σE and opposes a detrimental effect caused by the overaccumulation of BAM in the OM. During envelope stress, DolP loses its association with the mid-cell, thus suggesting a possible link between envelope stress caused by impaired OMP biogenesis and the regulation of a late step of cell division.

Download Full-text

The CpxQ sRNA Negatively Regulates Skp To Prevent Mistargeting of β-Barrel Outer Membrane Proteins into the Cytoplasmic Membrane

mBio ◽

10.1128/mbio.00312-16 ◽

2016 ◽

Vol 7 (2) ◽

Cited By ~ 27

Author(s):

Marcin Grabowicz ◽

Daria Koren ◽

Thomas J. Silhavy

Keyword(s):

Stress Response ◽

Membrane Proteins ◽

Outer Membrane ◽

Protein Misfolding ◽

Outer Membrane Proteins ◽

Inner Membrane ◽

Bam Complex ◽

Envelope Stress ◽

Periplasmic Chaperone

ABSTRACT The promoter most strongly induced upon activation of the Cpx two-component envelope stress response is the cpxP promoter. The 3′ untranscribed region (UTR) of the cpxP transcript is shown to produce a small RNA (sRNA), CpxQ. We investigated the role of CpxQ in combating envelope stress. Remarkably, the two effectors specified by the transcript are deployed to combat distinct stresses in different cellular compartments. CpxP acts in both a regulatory negative-feedback loop and as an effector that combats periplasmic protein misfolding. We find that CpxQ combats toxicity at the inner membrane (IM) by downregulating the synthesis of the periplasmic chaperone Skp. Our data indicate that this regulation prevents Skp from inserting β-barrel outer membrane proteins (OMPs) into the IM, a lethal event that likely collapses the proton motive force. Our findings suggest that Skp can fold and directly insert OMPs into a lipid bilayer in vivo without the aid of the Bam complex. IMPORTANCE Skp is a well-characterized periplasmic chaperone that binds unfolded OMPs. Surprisingly, we find that Skp can catalyze the folding and mistargeting of OMPs into the inner membrane without the aid of the other cellular proteins that normally assemble OMPs. Several OMPs function as diffusion pores. Accordingly, their mistargeting is lethal because it depolarizes the inner membrane. We show that the most highly expressed transcript of the Cpx stress response produces an sRNA from the 3′ UTR, CpxQ, which combats this potential toxicity by downregulating Skp production. Defects in OMP assembly trigger the σ E response to upregulate factors, including Skp, that promote OMP folding. The Cpx response downregulates σ E . Our findings reveal that this heretofore puzzling hierarchy exists to protect the inner membrane.

Download Full-text

Classifying β-Barrel Assembly Substrates by Manipulating Essential Bam Complex Members

Journal of Bacteriology ◽

10.1128/jb.00263-16 ◽

2016 ◽

Vol 198 (14) ◽

pp. 1984-1992 ◽

Cited By ~ 30

Author(s):

Tara F. Mahoney ◽

Dante P. Ricci ◽

Thomas J. Silhavy

Keyword(s):

Escherichia Coli ◽

Outer Membrane ◽

Mechanistic Model ◽

Outer Membrane Proteins ◽

Permeability Barrier ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Content Type ◽

Bam Complex ◽

Periplasmic Chaperones

ABSTRACTThe biogenesis of the outer membrane (OM) ofEscherichia coliis a conserved and vital process. The assembly of integral β-barrel outer membrane proteins (OMPs), which represent a major component of the OM, depends on periplasmic chaperones and the heteropentameric β-barrel assembly machine (Bam complex) in the OM. However, not all OMPs are affected by null mutations in the same chaperones or nonessential Bam complex members, suggesting there are categories of substrates with divergent requirements for efficient assembly. We have previously demonstrated two classes of substrates, one comprising large, low-abundance, and difficult-to-assemble substrates that are heavily dependent on SurA and also Skp and FkpA, and the other comprising relatively simple and abundant substrates that are not as dependent on SurA but are strongly dependent on BamB for assembly. Here, we describe novel mutations inbamDthat lower levels of BamD 10-fold and >25-fold without altering the sequence of the mature protein. We utilized these mutations, as well as a previously characterized mutation that lowers wild-type BamA levels, to reveal a third class of substrates. These mutations preferentially cause a marked decrease in the levels of multimeric proteins. This susceptibility of multimers to lowered quantities of Bam machines in the cell may indicate that multiple Bam complexes are needed to efficiently assemble multimeric proteins into the OM.IMPORTANCEThe outer membrane (OM) of Gram-negative bacteria, such asEscherichia coli, serves as a selective permeability barrier that prevents the uptake of toxic molecules and antibiotics. Integral β-barrel proteins (OMPs) are assembled by the β-barrel assembly machine (Bam), components of which are conserved in mitochondria, chloroplasts, and all Gram-negative bacteria, including many clinically relevant pathogenic species. Bam is essential for OM biogenesis and accommodates a diverse array of client proteins; however, a mechanistic model that accounts for the selectivity and broad substrate range of Bam is lacking. Here, we show that the assembly of multimeric OMPs is more strongly affected than that of monomeric OMPs when essential Bam complex components are limiting, suggesting that multiple Bam complexes are needed to assemble multimeric proteins.

Download Full-text

Fine-Tuning of σEActivation Suppresses Multiple Assembly-Defective Mutations inEscherichia coli

Journal of Bacteriology ◽

10.1128/jb.00745-18 ◽

2019 ◽

Vol 201 (11) ◽

Cited By ~ 4

Author(s):

Elizabeth M. Hart ◽

Aileen O’Connell ◽

Kimberly Tang ◽

Joseph S. Wzorek ◽

Marcin Grabowicz ◽

...

Keyword(s):

Stress Response ◽

Cell Viability ◽

Outer Membrane ◽

Stress Responses ◽

Expression Profiles ◽

Outer Membrane Proteins ◽

Fine Tuning ◽

Permeability Barrier ◽

Gram Negative ◽

Envelope Stress

ABSTRACTThe Gram-negative outer membrane (OM) is a selectively permeable asymmetric bilayer that allows vital nutrients to diffuse into the cell but prevents toxins and hydrophobic molecules from entering. Functionally and structurally diverse β-barrel outer membrane proteins (OMPs) build and maintain the permeability barrier, making the assembly of OMPs crucial for cell viability. In this work, we characterize an assembly-defective mutant of the maltoporin LamB, LamBG439D. We show that the folding defect of LamBG439Dresults in an accumulation of unfolded substrate that is toxic to the cell when the periplasmic protease DegP is removed. Selection for suppressors of this toxicity identified the novel mutantdegSA323Eallele. The mutant DegSA323Eprotein contains an amino acid substitution at the PDZ/protease domain interface that results in a partially activated conformation of this protein. This activation increases basal levels of downstream σEstress response signaling. Furthermore, the enhanced σEactivity of DegSA323Esuppresses a number of other assembly-defective conditions without exhibiting the toxicity associated with high levels of σEactivity. We propose that the increased basal levels of σEsignaling primes the cell to respond to envelope stress before OMP assembly defects threaten cell viability. This finding addresses the importance of envelope stress responses in monitoring the OMP assembly process and underpins the critical balance between envelope defects and stress response activation.IMPORTANCEGram-negative bacteria, such asEscherichia coli, inhabit a natural environment that is prone to flux. In order to cope with shifting growth conditions and the changing availability of nutrients, cells must be capable of quickly responding to stress. Stress response pathways allow cells to rapidly shift gene expression profiles to ensure survival in this unpredictable environment. Here we describe a mutant that partially activates the σEstress response pathway. The elevated basal level of this stress response allows the cell to quickly respond to overwhelming stress to ensure cell survival.

Download Full-text

Characterization of Immunodominant and Potentially Protective Epitopes of Mannheimia haemolytica Serotype 1 Outer Membrane Lipoprotein PlpE

Infection and Immunity ◽

10.1128/iai.72.12.7265-7274.2004 ◽

2004 ◽

Vol 72 (12) ◽

pp. 7265-7274 ◽

Cited By ~ 20

Author(s):

Sahlu Ayalew ◽

Anthony W. Confer ◽

Emily R. Blackwood

Keyword(s):

Outer Membrane ◽

Outer Membrane Proteins ◽

Mannheimia Haemolytica ◽

Cellulose Membrane ◽

Outer Membrane Lipoprotein ◽

Serotype 1 ◽

Shipping Fever ◽

Membrane Lipoprotein ◽

Nested Deletions

ABSTRACT Mannheimia haemolytica serotype 1 (S1) is the most common bacterial isolate found in shipping fever pneumonia in beef cattle. Currently used vaccines against M. haemolytica do not provide complete protection against the disease. Research with M. haemolytica outer membrane proteins (OMPs) has shown that antibodies to one particular OMP from S1, PlpE, may be important in immunity. In a recently published work, members of our laboratory showed that recombinant PlpE (rPlpE) is highly immunogenic when injected subcutaneously into cattle and that the acquired immunity markedly enhanced resistance to experimental challenge (A. W. Confer, S. Ayalew, R. J. Panciera, M. Montelongo, L. C. Whitworth, and J. D. Hammer, Vaccine 21:2821-2829, 2003). The objective of this work was to identify epitopes of PlpE that are responsible for inducing the immune response. Western blot analysis of a series of rPlpE with nested deletions on both termini with bovine anti-PlpE hyperimmune sera showed that the immunodominant region is located close to the N terminus of PlpE. Fine epitope mapping, in which an array of overlapping 13-mer synthetic peptides attached to a derivatized cellulose membrane was probed with various affinity-purified anti-PlpE antibodies, identified eight highly reactive regions, of which region 2 (R2) was identified as the specific epitope. The R2 region is comprised of eight imperfect repeats of a hexapeptide (QAQNAP) and is located between residues 26 and 76. Complement-mediated bactericidal activity of affinity-purified anti-PlpE bovine antibodies confirmed that antibodies directed against the R2 region are effective in killing M. haemolytica.

Download Full-text

Outer membrane lipoprotein biogenesis: Lol is not the end

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2015.0030 ◽

2015 ◽

Vol 370 (1679) ◽

pp. 20150030 ◽

Cited By ~ 67

Author(s):

Anna Konovalova ◽

Thomas J. Silhavy

Keyword(s):

Outer Membrane ◽

Signal Sequence ◽

Cysteine Residue ◽

Lipid Modification ◽

Mature Protein ◽

Bam Complex ◽

Beta Barrel ◽

Outer Membrane Lipoprotein ◽

Lipid Moiety ◽

Membrane Lipoprotein

Bacterial lipoproteins are lipid-anchored proteins that contain acyl groups covalently attached to the N-terminal cysteine residue of the mature protein. Lipoproteins are synthesized in precursor form with an N-terminal signal sequence (SS) that targets translocation across the cytoplasmic or inner membrane (IM). Lipid modification and SS processing take place at the periplasmic face of the IM. Outer membrane (OM) lipoproteins take the localization of lipoproteins (Lol) export pathway, which ends with the insertion of the N-terminal lipid moiety into the inner leaflet of the OM. For many lipoproteins, the biogenesis pathway ends here. We provide examples of lipoproteins that adopt complex topologies in the OM that include transmembrane and surface-exposed domains. Biogenesis of such lipoproteins requires additional steps beyond the Lol pathway. In at least one case, lipoprotein sequences reach the cell surface by being threaded through the lumen of a beta-barrel protein in an assembly reaction that requires the heteropentomeric Bam complex. The inability to predict surface exposure reinforces the importance of experimental verification of lipoprotein topology and we will discuss some of the methods used to study OM protein topology.

Download Full-text

The YfgL Lipoprotein Is Essential for Type III Secretion System Expression and Virulence of Salmonella enterica Serovar Enteritidis

Infection and Immunity ◽

10.1128/iai.00716-06 ◽

2006 ◽

Vol 75 (1) ◽

pp. 358-370 ◽

Cited By ~ 32

Author(s):

Yann Fardini ◽

Kamel Chettab ◽

Olivier Grépinet ◽

Sandrine Rochereau ◽

Jérôme Trotereau ◽

...

Keyword(s):

Outer Membrane ◽

Salmonella Enterica ◽

Outer Membrane Proteins ◽

Intestinal Barrier ◽

Effector Proteins ◽

Open Reading Frames ◽

Secretion Systems ◽

Reverse Transcription Pcr ◽

Outer Membrane Lipoprotein ◽

Membrane Lipoprotein

ABSTRACT Salmonella enterica, like many gram-negative pathogens, uses type three secretion systems (TTSS) to infect its hosts. The three TTSS of Salmonella, namely, TTSS-1, TTSS-2, and flagella, play a major role in the virulence of this bacterium, allowing it to cross the intestinal barrier and to disseminate systemically. Previous data from our laboratory have demonstrated the involvement of the chromosomal region harboring the yfgL, engA, and yfgJ open reading frames in S. enterica serovar Enteritidis virulence. Using microarray analysis and real-time reverse transcription-PCR after growth of bacterial cultures favorable for either TTSS-1 or TTSS-2 expression, we show in this study that the deletion in S. enterica serovar Enteritidis of yfgL, encoding an outer membrane lipoprotein, led to the transcriptional down-regulation of most Salmonella pathogenicity island 1 (SPI-1), SPI-2, and flagellar genes encoding the TTSS structural proteins and effector proteins secreted by these TTSS. In line with these results, the virulence of the ΔyfgL mutant was greatly attenuated in mice. Moreover, even if YfgL is involved in the assembly of outer membrane proteins, the regulation of TTSS expression observed was not due to an inability of the ΔyfgL mutant to assemble TTSS in its membrane. Indeed, when we forced the transcription of SPI-1 genes by constitutively expressing HilA, the secretion of the TTSS-1 effector protein SipA was restored in the culture supernatant of the mutant. These results highlight the crucial role of the outer membrane lipoprotein YfgL in the expression of all Salmonella TTSS and, thus, in the virulence of Salmonella. Therefore, this outer membrane protein seems to be a privileged target for fighting Salmonella.

Download Full-text