Dataset Information

Understanding the interaction of SurA and BAM in the outer membrane of E. coli

ABSTRACT: The outer membrane (OM) is a formidable barrier that protects Gram-negative bacteria against environmental threats. The correct folding and insertion of outer membrane proteins (OMPs) into the OM requires the essential OM-embedded β-barrel assembly machinery (BAM), a heptameric protein complex in E. coli. OMPs are delivered to BAM by the periplasmic chaperone SurA. However, how the activity of SurA and BAM are coordinated to ensure successful OMP delivery to BAM for folding into the OM remained unclear. We have trapped SurA in the act of OMP delivery to BAM and, via cryoEM, solved the structures of this assembly. By mutating key interaction sites we validate this interaction and use proteomics to determine how this perturbs the proteome of E. coli.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Escherichia Coli

SUBMITTER: Antonio Calabrese

LAB HEAD: Antonio Calabrese

PROVIDER: PXD046606 | Pride | 2024-10-01

REPOSITORIES: pride

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Dataset's files

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			Action	DRS
	23107_Ecl01_Jim_DDA_IT50_TRAP_50cm_120min_AR_208_01_01.raw	Raw
	23107_Ecl01_Jim_DDA_IT50_TRAP_50cm_120min_AR_208_02_01.raw	Raw
	23107_Ecl01_Jim_DDA_IT50_TRAP_50cm_120min_AR_208_03_01.raw	Raw
	23107_Ecl01_Jim_DDA_IT50_TRAP_50cm_120min_JEH_108_01_01.raw	Raw
	23107_Ecl01_Jim_DDA_IT50_TRAP_50cm_120min_JEH_108_02_01.raw	Raw

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Publications

Outer membrane protein assembly mediated by BAM-SurA complexes.

Fenn Katherine L KL Horne Jim E JE Crossley Joel A JA Böhringer Nils N Horne Romany J RJ Schäberle Till F TF Calabrese Antonio N AN Radford Sheena E SE Ranson Neil A NA

Nature communications 20240901 1

The outer membrane is a formidable barrier that protects Gram-negative bacteria against environmental threats. Its integrity requires the correct folding and insertion of outer membrane proteins (OMPs) by the membrane-embedded β-barrel assembly machinery (BAM). Unfolded OMPs are delivered to BAM by the periplasmic chaperone SurA, but how SurA and BAM work together to ensure successful OMP delivery and folding remains unclear. Here, guided by AlphaFold2 models, we use disulphide bond engineering ...[more]

PMID: 39218969

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Project description:Clostridium acetobutylicum, the endospore-forming anaerobe best known for its ABE (acetone-butanol-ethanol) fermentation, has received renewed attention recently for the biological production of butanol, both for bulk chemical production and as a potential biofuel. With butanol production in mind, most of the recent research on C. acetobutylicum has focused on increasing butanol production, tolerance to butanol, and optimizing it for various substrates. However, an equally important trait, though less understood, is its sporulation program, which it coupled to solvent formation. The model organism for endospore formation is Bacillus subtilis, but significant physiological, metabolic, and genomic differences exist between the two organisms. Despite these differences, the major sporulation-related transcription/sigma factors are conserved between the two species. These transcription/sigma factors are activated in a cascade manner such that Spo0A becomes active first, followed by M-OM-^CF, then M-OM-^CE, then M-OM-^CG, and finally M-OM-^CK. The goal of this study is to determine the regulons of 4 of these transcription/sigma factors (Spo0A, M-OM-^CF, M-OM-^CE, and M-OM-^CG) and compare them to those in B. subtilis. To accomplish this goal, individual mutant strains were created for Spo0A, M-OM-^CF, M-OM-^CE, and M-OM-^CG, in which the transcription/sigma factor is silenced. These mutants were then compared transcriptionally using microarrays to determine the regulon of each transcription/sigma factor. To help avoid false positives, comparisons were made between strains in which the downstream transcription/sigma factor is silenced (e.g., for the Spo0A regulon, the Spo0A mutant was compared against the M-OM-^CF mutant and the M-OM-^CE mutant, since they are both upregulated by Spo0A) rather than just the WT. For each regulon, 4 timepoints were taken, since it is very difficult to synchronize sporulation in C. acetobutylicum cultures, and dye-swaps were prepared for each timepoint. Four transcription/sigma factor regulons were investigated: Spo0A, M-OM-^CF, M-OM-^CE, and M-OM-^CG. For Spo0A, two comparison were made: Spo0A mutant vs M-OM-^CF mutant and Spo0A mutant vs M-OM-^CE mutant. For M-OM-^CF, two comparison were also made: M-OM-^CF mutant vs M-OM-^CE mutant and M-OM-^CF mutant vs M-OM-^CG mutant. Finally for M-OM-^CE and M-OM-^CG, only one comparison for each was made: M-OM-^CE mutant vs M-OM-^CG mutant and M-OM-^CG mutant vs wild-type, respectively. For each comparison, 4 timepoints were analyzed, and dye-swaps were prepared for each timepoint comparison. Timepoints were chosen based on when each transcription/sigma factor was expected to be active, based on a previous study [Jones, SW, et al. Genome Biol. 2008;9(7):R114.].