LptM promotes oxidative maturation of the lipopolysaccharide translocon by substrate binding mimicry

Insertion of lipopolysaccharide (LPS) into the bacterial outer membrane (OM) is mediated by a druggable OM translocon consisting of a β-barrel membrane protein, LptD, and a lipoprotein, LptE. The β-barrel assembly machinery (BAM) assembles LptD together with LptE at the OM. In the enterobacterium Escherichia coli, formation of two native disulfide bonds in LptD controls translocon activation. Here we report the discovery of LptM (formerly YifL), a lipoprotein conserved in Enterobacteriaceae, that assembles together with LptD and LptE at the BAM complex. LptM stabilizes a conformation of LptD that can efficiently acquire native disulfide bonds, whereas its inactivation makes disulfide bond isomerization by DsbC become essential for viability. Our structural prediction and biochemical analyses indicate that LptM binds to sites in both LptD and LptE that are proposed to coordinate LPS insertion into the OM. These results suggest that, by mimicking LPS binding, LptM facilitates oxidative maturation of LptD, thereby activating the LPS translocon

Interaction of an autotransporter passenger domain with BamA during its translocation across the bacterial outer membrane

Autotransporters are a superfamily of virulence factors produced by Gram-negative bacteria consisting of a large N-terminal extracellular domain (“passenger domain”) and a C-terminal β barrel domain (“β domain”). The mechanism by which the passenger domain is translocated across the outer membrane (OM) is unknown. Here we show that the insertion of a small linker into the passenger domain of the Escherichia coli O157:H7 autotransporter EspP effectively creates a translocation intermediate by transiently stalling translocation near the site of the insertion. Using a site-specific photocrosslinking approach, we found that residues adjacent to the stall point interact with BamA, a component of a heterooligomeric complex (Bam complex) that catalyzes OM protein assembly, and that residues closer to the EspP N terminus interact with the periplasmic chaperones SurA and Skp. The EspP–BamA interaction was short-lived and could be detected only when passenger domain translocation was stalled. These results support a model in which molecular chaperones prevent misfolding of the passenger domain before its secretion and the Bam complex catalyzes both the integration of the β domain into the OM and the translocation of the passenger domain across the OM in a C- to N-terminal direction

Mitochondrial presequence import: Multiple regulatory knobs fine-tune mitochondrial biogenesis and homeostasis

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Bacterial machineries for the assembly of membrane-embedded β-barrel proteins

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The Iron-Responsive Regulator Fur Is Transcriptionally Autoregulated and Not Essential in Neisseria meningitidis

Fur is a well-known iron-responsive repressor of gene transcription, which is used by many bacteria to respond to the low-iron environment that pathogens encounter during infection. The fur gene in Neisseria meningitidis has been described as an essential gene that may regulate a broad array of genes. We succeeded in obtaining an N. meningitidis mutant with the fur gene knocked out and used it to undertake studies of fur-mediated iron regulation. We show that expression of both Fur and the transferrin binding protein Tbp2 is iron regulated and demonstrate that this regulation is Fur mediated for the Tbp2 protein. Footprinting analysis revealed that Fur binds to two distinct sites upstream of its coding region with different affinities and that these binding sites overlap two promoters that differentially control transcription of the fur gene in response to iron. The presence of two independently regulated fur promoters may allow meningococcus to fine-tune expression of this regulator controlling iron homeostasis, possibly during infection

Secretion of a bacterial virulence factor is driven by the folding of a C-terminal segment

Autotransporters are bacterial virulence factors consisting of an N-terminal “passenger domain” that is secreted in a C- to-N-terminal direction and a C-terminal “β domain” that resides in the outer membrane (OM). Although passenger domain secretion does not appear to use ATP, the energy source for this reaction is unknown. Here, we show that efficient secretion of the passenger domain of the Escherichia coli O157:H7 autotransporter EspP requires the stable folding of a C-terminal ≈17-kDa passenger domain segment. We found that mutations that perturb the folding of this segment do not affect its translocation across the OM but impair the secretion of the remainder of the passenger domain. Interestingly, an examination of kinetic folding mutants strongly suggested that the ≈17-kDa segment folds in the extracellular space. By mutagenizing the ≈17-kDa segment, we also fortuitously isolated a unique translocation intermediate. Analysis of this intermediate suggests that a heterooligomer that facilitates the membrane integration of OM proteins (the Bam complex) also promotes the surface exposure of the ≈17-kDa segment. Our results provide direct evidence that protein folding can drive translocation and help to clarify the mechanism of autotransporter secretion

In Vitro Analysis of Protein-Operator Interactions of the NikR and Fur Metal-Responsive Regulators of Coregulated Genes in Helicobacter pylori

Two important metal-responsive regulators, NikR and Fur, are involved in nickel and iron homeostasis and controlling gene expression in Helicobacter pylori. To date, they have been implicated in the regulation of sets of overlapping genes. We have attempted here dissection of the molecular mechanisms involved in transcriptional regulation of the NikR and Fur proteins, and we investigated protein-promoter interactions of the regulators with known target genes. We show that H. pylori NikR is a tetrameric protein and, through DNase I footprinting analysis, we have identified operators for NikR to which it binds with different affinities in a metal-responsive way. Mapping of the NikR binding site upstream of the urease promoter established a direct role for NikR as a positive regulator of transcription and, through scanning mutagenesis of this binding site, we have determined two subsites that are important for the binding of the protein to its target sequence. Furthermore, by alignment of the operators for NikR, we have shown that the H. pylori protein recognizes a sequence that is distinct from its well-studied orthologue in Escherichia coli. Moreover, we show that NikR and Fur can bind independently at distinct operators and also compete for overlapping operators in some coregulated gene promoters, adding another dimension to the previous suggested link between iron and nickel regulation. Finally, the importance of an interconnection between metal-responsive gene networks for homeostasis is discussed

CrgA Is an Inducible LysR-Type Regulator of Neisseria meningitidis, Acting both as a Repressor and as an Activator of Gene Transcription

The crgA gene of Neisseria meningitidis, which codes for a LysR-type regulator, is divergently oriented with respect to the mdaB gene, which codes for a hypothetical NADPH-quinone oxidoreductase. Transcriptional studies of the intergenic region between crgA and mdaB showed that two overlapping and divergent promoters, P(crgA) and P(mdaB), control transcription of these genes. Deletion of the crgA gene led to a strong increase in transcription from the P(crgA) promoter and a concomitant strong decrease in transcription from the P(mdaB) promoter, indicating that CrgA acts both as an autorepressor of transcription at its own promoter and as an activator of transcription at the mdaB promoter. Addition of α-methylene-γ-butyrolactone (MBL), an inducer of NADPH-quinone oxidoreductase, to wild-type N. meningitidis cells specifically resulted in further activation of transcription of the P(mdaB) promoter and more repression of transcription of the P(crgA) promoter. No such regulation was observed when MBL was added to crgA-deficient cells, indicating that the transcriptional response to MBL is CrgA mediated. Under the same experimental conditions, no regulation of transcription by either CrgA or MBL was detected at the pilus and capsule genes. The role of CrgA in the regulation of gene expression during the infectious cycle of N. meningitidis is discussed

The Mgr2 subunit of the TIM23 complex regulates membrane insertion of marginal stop‐transfer signals in the mitochondrial inner membrane

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

International audienceIn 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 (σ E ) transcriptional response. σ E upregulates OMP biogenesis factors, including the β-barrel assembly machinery (BAM) that catalyses OMP folding. Here we report that DolP (formerly YraP), a σ E -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