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β-barrel membrane proteins in Gram-negative bacteria, mitochondria, and chloroplasts are assembled by highly conserved multi-protein complexes. The mechanism by which these molecular machines fold and insert their substrates is poorly understood. It has not been possible to dissect the folding and insertion pathway because the process has not been reproduced in a biochemical system. We purified the components that fold and insert Escherichia coli outer membrane proteins and reconstituted β-barrel protein assembly in proteoliposomes using the enzymatic activity of a protein substrate to report on its folding state. The assembly of this protein occurred without an energy source but required a soluble chaperone in addition to the multi-protein assembly complex.

The protein complex that assembles integral membrane β-barrel proteins in the outer membranes of Gram-negative bacteria is an attractive target in the development of new antibiotics. This complex, the β-barrel assembly machine (Bam), contains two essential proteins, BamA and BamD. We have identified a peptide that inhibits the assembly of β-barrel proteins in vitro by characterizing the interaction of BamD with an unfolded substrate protein. This peptide is a fragment of the substrate protein and contains a conserved amino acid sequence. We have demonstrated that mutations of this sequence in the full-length substrate protein impair the protein’s assembly, implying that BamD’s interaction with this sequence is an important part of the assembly mechanism. Finally, we have found that in vivo expression of a peptide containing this sequence causes growth defects and sensitizes Escherichia coli to antibiotics to which they are normally resistant. Therefore, inhibiting the binding of substrates to BamD is a viable strategy for developing new antibiotics directed against Gram-negative bacteria. Significance New antibiotics are urgently needed to treat infections caused by Gram-negative bacteria. These bacteria possess an outer membrane, which is impermeable to most currently available antibiotics. We have studied how a protein that is essential for maintaining the integrity of the outer membrane of Escherichia coli interacts with its substrates. In so doing, we have identified a peptide that inhibits this protein’s function in vitro and accordingly has deleterious effects on growth and membrane permeability in vivo. This study thus provides a starting point for the development of new antibiotics that directly target outer membrane biogenesis.

The β-barrel assembly machine (Bam) complex folds and inserts integral membrane proteins into the outer membrane of Gram-negative bacteria. The two essential components of the complex, BamA and BamD, both interact with substrates, but how the two coordinate with each other during assembly is not clear. To elucidate aspects of this process we slowed the assembly of an essential β-barrel substrate of the Bam complex, LptD, by changing a conserved residue near the C terminus. This defective substrate is recruited to the Bam complex via BamD but is unable to integrate into the membrane efficiently. Changes in the extracellular loops of BamA partially restore assembly kinetics, implying that BamA fails to engage this defective substrate. We conclude that substrate binding to BamD activates BamA by regulating extracellular loop interactions for folding and membrane integration.

The outer membrane (OM) of Gram-negative bacteria such as Escherichia coli contains lipoproteins and integral β-barrel proteins (outer-membrane proteins, OMPs) assembled into an asymmetrical lipid bilayer. Insertion of β-barrel proteins into the OM is mediated by a protein complex that contains the OMP BamA and four associated lipoproteins (BamBCDE). The mechanism by which the Bam complex catalyzes the assembly of OMPs is not known. We report here the isolation and characterization of a temperature-sensitive lethal mutation, bamAE373K, which alters the fifth polypeptide transport-associated domain and disrupts the interaction between the BamAB and BamCDE subcomplexes. Suppressor mutations that map to codon 197 in bamD restore Bam complex function to wild-type levels. However, these suppressors do not restore the interaction between BamA and BamD; rather, they bypass the requirement for stable holocomplex formation by activating BamD. These results imply that BamA and BamD interact directly with OMP substrates.

The Bam complex assembles β-barrel proteins into the outer membrane (OM) of Gram-negative bacteria. These proteins comprise cylindrical β-sheets with long extracellular loops and create pores to allow passage of nutrients and waste products across the membrane. Despite their functional importance, several questions remain about how these proteins are assembled into the OM after their synthesis in the cytoplasm and secretion across the inner membrane. To understand this process better, we studied the assembly of an essential β-barrel substrate for the Bam complex, BamA. By mutating conserved residues in the β-barrel domain of this protein, we generated three assembly-defective BamA substrates that stall early in the folding process in the periplasm. Two of the three defective substrates, which harbor mutations within β-strands, fail to associate productively with the Bam complex. The third substrate, which harbors mutations in a conserved extracellular loop, accumulates on BamD during assembly, but does not integrate efficiently into the membrane. The assembly of all three substrates can be restored by artificially tethering a region of the substrate, which ultimately becomes an extracellular loop, to the lumen of the forming β-barrel. These results imply that a critical step in the folding process involves the interaction of residues on the interior of the nascent β-barrel wall with residues in one of the extracellular loops. We conclude that a prerequisite for membrane integration of β-barrel proteins is burial of the extracellular loops within the forming β-barrel.

The Bam complex assembles β-barrel proteins into the outer membrane (OM) of Gram-negative bacteria. These proteins comprise cylindrical β-sheets with long extracellular loops and create pores to allow passage of nutrients and waste products across the membrane. Despite their functional importance, several questions remain about how these proteins are assembled into the OM after their synthesis in the cytoplasm and secretion across the inner membrane. To understand this process better, we studied the assembly of an essential β-barrel substrate for the Bam complex, BamA. By mutating conserved residues in the β-barrel domain of this protein, we generated three assembly-defective BamA substrates that stall early in the folding process in the periplasm. Two of the three defective substrates, which harbor mutations within β-strands, fail to associate productively with the Bam complex. The third substrate, which harbors mutations in a conserved extracellular loop, accumulates on BamD during assembly, but does not integrate efficiently into the membrane. The assembly of all three substrates can be restored by artificially tethering a region of the substrate, which ultimately becomes an extracellular loop, to the lumen of the forming β-barrel. These results imply that a critical step in the folding process involves the interaction of residues on the interior of the nascent β-barrel wall with residues in one of the extracellular loops. We conclude that a prerequisite for membrane integration of β-barrel proteins is burial of the extracellular loops within the forming β-barrel.

The protein complex that assembles integral membrane β-barrel proteins in the outer membranes of Gram-negative bacteria is an attractive target in the development of new antibiotics. This complex, the β-barrel assembly machine (Bam), contains two essential proteins, BamA and BamD. We have identified a peptide that inhibits the assembly of β-barrel proteins in vitro by characterizing the interaction of BamD with an unfolded substrate protein. This peptide is a fragment of the substrate protein and contains a conserved amino acid sequence. We have demonstrated that mutations of this sequence in the full-length substrate protein impair the protein's assembly, implying that BamD's interaction with this sequence is an important part of the assembly mechanism. Finally, we have found that in vivo expression of a peptide containing this sequence causes growth defects and sensitizes Escherichia coli to antibiotics to which they are normally resistant. Therefore, inhibiting the binding of substrates to BamD is a viable strategy for developing new antibiotics directed against Gram-negative bacteria.

The outer membrane (OM) of Gram-negative bacteria such as Escherichia coli contains lipoproteins and integral β-barrel proteins (outer-membrane proteins, OMPs) assembled into an asymmetrical lipid bilayer. Insertion of β-barrel proteins into the OM is mediated by a protein complex that contains the OMP BamA and four associated lipoproteins (BamBCDE). The mechanism by which the Bam complex catalyzes the assembly of OMPs is not known. We report here the isolation and characterization of a temperature-sensitive lethal mutation, bamAE373K, which alters the fifth polypeptide transport-associated domain and disrupts the interaction between the BamAB and BamCDE subcomplexes. Suppressor mutations that map to codon 197 in bamD restore Bam complex function to wild-type levels. However, these suppressors do not restore the interaction between BamA and BamD; rather, they bypass the requirement for stable holocomplex formation by activating BamD. These results imply that BamA and BamD interact directly with OMP substrates. [PUBLICATION ABSTRACT]

The extent to which vaccinated persons who become infected with SARS-CoV-2 contribute to transmission is unclear. During a SARS-CoV-2 Delta variant outbreak among incarcerated persons with high vaccination rates in a federal prison, we assessed markers of viral shedding in vaccinated and unvaccinated persons. Consenting incarcerated persons with confirmed SARS-CoV-2 infection provided mid-turbinate nasal specimens daily for 10 consecutive days and reported symptom data via questionnaire. Real-time reverse transcription-polymerase chain reaction (RT-PCR), viral whole genome sequencing, and viral culture was performed on these nasal specimens. Duration of RT-PCR positivity and viral culture positivity was assessed using survival analysis. A total of 957 specimens were provided by 93 participants, of whom 78 (84 %) were vaccinated and 17 (16 %) were unvaccinated. No significant differences were detected in duration of RT-PCR positivity among vaccinated participants (median: 13 days) versus those unvaccinated (median: 13 days; p = 0.50), or in duration of culture positivity (medians: 5 days and 5 days; p = 0.29). Among vaccinated participants, overall duration of culture positivity was shorter among Moderna vaccine recipients versus Pfizer (p = 0.048) or Janssen (p = 0.003) vaccine recipients. In post-hoc analyses, Moderna vaccine recipients demonstrated significantly shorter duration of culture positivity compared to unvaccinated participants (p = 0.02). When restricted to participants without reported prior infection, the difference between Moderna vaccine recipients and unvaccinated participants was more pronounced (medians: 3 days and 6 days, p = 0.002). Infectious periods for vaccinated and unvaccinated persons who become infected with SARS-CoV-2 are similar and can be highly variable, though some vaccinated persons are likely infectious for shorter durations. These findings are critically important, especially in congregate settings where viral transmission can lead to large outbreaks. In such settings, clinicians and public health practitioners should consider vaccinated, infected persons to be no less infectious than unvaccinated, infected persons.

Zika virus (ZIKV) is an emerging pathogen causally associated with serious sequelae in fetuses, inducing fetal microcephaly and other neurodevelopment defects. ZIKV is primarily transmitted by mosquitoes, but can persist in human semen and sperm, and sexual transmission has been documented. Moreover, exposure of type-I interferon knockout mice to ZIKV results in severe damage to the testes, epididymis and sperm. Candidate ZIKV vaccines have shown protective efficacy in preclinical studies carried out in animal models, and several vaccines have entered clinical trials. Here, we report that administration of a synthetic DNA vaccine encoding ZIKV pre-membrane and envelope (prME) completely protects mice against ZIKV-associated damage to the testes and sperm and prevents viral persistence in the testes following challenge with a contemporary strain of ZIKV. These data suggest that DNA vaccination merits further investigation as a potential means to reduce ZIKV persistence in the male reproductive tract. Zika virus (ZIKV) can persist in human semen and sperm, which can result in sexual transmission. Here, Griffin et al . show that a DNA vaccine, expressing ZIKV pre-membrane and envelope proteins, protects mice from infection-associated damage to testes and sperm, and prevents viral persistence in testes.