Dataset Information

Cross-species chimeras reveal BamA POTRA and ?-barrel domains must be fine-tuned for efficient OMP insertion.

ABSTRACT: BAM is a conserved molecular machine, the central component of which is BamA. Orthologues of BamA are found in all Gram-negative bacteria, chloroplasts and mitochondria where it is required for the folding and insertion of ?-barrel containing integral outer membrane proteins (OMPs) into the outer membrane. BamA binds unfolded ?-barrel precursors via the five polypeptide transport-associated (POTRA) domains at its N-terminus. The C-terminus of BamA folds into a ?-barrel domain, which tethers BamA to the outer membrane and is involved in OMP insertion. BamA orthologues are found in all Gram-negative bacteria and appear to function in a species-specific manner. Here we investigate the nature of this species-specificity by examining whether chimeric Escherichia coli?BamA fusion proteins, carrying either the ?-barrel or POTRA domains from various BamA orthologues, can functionally replace E.?coli?BamA. We demonstrate that the ?-barrel domains of many BamA orthologues are functionally interchangeable. We show that defects in the orthologous POTRA domains can be rescued by compensatory mutations within the ?-barrel. These data reveal that the POTRA and barrel domains must be precisely aligned to ensure efficient OMP insertion.

SUBMITTER: Browning DF

PROVIDER: S-EPMC4950039 | biostudies-literature | 2015 Aug

REPOSITORIES: biostudies-literature

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Publications

Cross-species chimeras reveal BamA POTRA and β-barrel domains must be fine-tuned for efficient OMP insertion.

Browning Douglas F DF Bavro Vassiliy N VN Mason Jessica L JL Sevastsyanovich Yanina R YR Rossiter Amanda E AE Jeeves Mark M Wells Timothy J TJ Knowles Timothy J TJ Cunningham Adam F AF Donald James W JW Palmer Tracy T Overduin Michael M Henderson Ian R IR

Molecular microbiology 20150606 4

BAM is a conserved molecular machine, the central component of which is BamA. Orthologues of BamA are found in all Gram-negative bacteria, chloroplasts and mitochondria where it is required for the folding and insertion of β-barrel containing integral outer membrane proteins (OMPs) into the outer membrane. BamA binds unfolded β-barrel precursors via the five polypeptide transport-associated (POTRA) domains at its N-terminus. The C-terminus of BamA folds into a β-barrel domain, which tethers BamA ...[more]

PMID: 25943387

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Project description:Most integral outer membrane proteins (OMPs) of Gram-negative bacteria, such as Escherichia coli, assume a ?-barrel structure. The ?-barrel assembly machine (Bam), a five-member complex composed of ?-barrel OMP BamA and four associated lipoproteins, BamB, BamC, BamD, and BamE, folds and inserts OMPs into the outer membrane. The two essential proteins BamA and BamD interact to stabilize two subcomplexes, BamAB and BamCDE, and genetic and structural evidence suggests that interactions between BamA and BamD occur via an electrostatic interaction between a conserved aspartate residue in a periplasmic domain of BamA and a conserved arginine in BamD. In this work, we characterize charge-change mutations at these key BamA and BamD residues and nearby charged residues in BamA with respect to OMP assembly and Bam complex stability. We show that Bam complex stability does not correlate with function, that BamA and BamD must adopt at least two active conformational states during OMP assembly, and that these charged residues are not required for function. Rather, these charged residues are important for coordinating the activities of BamA and BamD to allow efficient OMP assembly. We present a model of OMP assembly wherein recognition and binding of unfolded OMP substrate by BamA and BamD induce a signaling interaction between the two proteins, causing conformational changes necessary for the assembly reaction to proceed. By analogy to signal sequence recognition by SecYEG, we believe these BamA-BamD interactions ensure that both substrate and complex are competent for OMP assembly before the assembly reaction commences.IMPORTANCE Conformational changes in the proteins of the ?-barrel assembly machine (Bam complex) are associated with the folding and assembly of outer membrane proteins (OMPs) in Gram-negative bacteria. We show that electrostatic interactions between the two essential proteins BamA and BamD coordinate conformational changes upon binding of unfolded substrate that allow the assembly reaction to proceed. Mutations affecting this interaction are lethal not because they destabilize the Bam complex but rather because they disrupt this coordination. Our model of BamA-BamD interactions regulating conformation in response to proper substrate interaction is reminiscent of conformational changes the secretory (Sec) machinery undergoes after signal sequence recognition that ensure protein quality control.

Project description:A diversity of arthropods (myrmecophiles) thrives within ant nests, many of them unmolested though some, such as the specialized Eucharitidae parasitoids, may cause direct damage to their hosts. Ants are known to discriminate between nestmates and non-nestmates, but whether they recognize the strength of a threat and their capacity to adjust their behavior accordingly have not been fully explored. We aimed to determine whether Ectatomma tuberculatum ants exhibited specific behavioral responses to potential or actual intruders posing different threats to the host colony and to contribute to an understanding of complex ant-eucharitid interactions. Behavioral responses differed significantly according to intruder type. Ants evicted intruders that represented a threat to the colony's health (dead ants) or were not suitable as prey items (filter paper, eucharitid parasitoid wasps, non myrmecophilous adult weevils), but killed potential prey (weevil larvae, termites). The timing of detection was in accordance with the nature and size of the intruder: corpses (a potential source of contamination) were detected faster than any other intruder and transported to the refuse piles within 15 min. The structure and complexity of behavioral sequences differed among those intruders that were discarded. Workers not only recognized and discriminated between several distinct intruders but also adjusted their behavior to the type of intruder encountered. Our results confirm the previously documented recognition capabilities of E. tuberculatum workers and reveal a very fine-tuned intruder discrimination response. Colony-level prophylactic and hygienic behavioral responses through effective removal of inedible intruders appears to be the most general and flexible form of defense in ants against a diverse array of intruders. However, this generalized response to both potentially lethal and harmless intruders might have driven the evolution of ant-eucharitid interactions, opening a window for parasitoid attack and allowing adult parasitoid wasps to quickly leave the natal nest unharmed.

Dataset Information

Cross-species chimeras reveal BamA POTRA and ?-barrel domains must be fine-tuned for efficient OMP insertion.

Publications

Cross-species chimeras reveal BamA POTRA and β-barrel domains must be fine-tuned for efficient OMP insertion.

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