BamA self-insertion drives inner to outer membrane remodelling in diderm Firmicutes
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ABSTRACT:
PROVIDER: PRJNA1314028 | ENA |
REPOSITORIES: ENA
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Project description:Endospore formation in Firmicutes is a complex morphological process triggered by environmental stress. At the end of this process, a mature spore containing the condensed genome is released through lysis of the mother cell. The mature spore is encased by several protective layers, including an inner and an outer spore membrane (IsM and OsM, respectively), both derived from the inner membrane (IM) of the mother cell. While this process is well understood in monoderm bacteria like Bacillus subtilis, less is known about diderm spore-formers such as Acetonema longum, which retain and remodel their OsM into a true outer membrane (OM) during germination. Our results show that outer membrane proteins (OMPs), including the essential β-barrel Assembly Machinery (BAM) component BamA, are present in vegetative and germinating cells but absent from mature spores. These observations highlight an unprecedented mechanism of inner to outer membrane remodelling in the absence of BamA. Expression profiles of cells at different growth stages and phylogenomic analyses identified two novel proteins, co-expressed and in an operon with BamA, that were highly conserved among diderm Firmicutes: a β-barrel OMP containing three polypeptide transport-associated (POTRA) domains (SonA) and a putative OM lipoprotein (SonB). In vitro experiments further demonstrated that both BamA and SonA can self-insert into liposomes under physiological conditions. Together, we propose a model in which BamA and its functional partners, SonA and SonB, drive membrane remodelling during germination. The process is initiated by self-insertion of BamA, which then assists the folding and insertion of other OMPs, including LptD, progressively shifting the protein and lipid composition of the OsM to produce a true OM. Collectively, our findings provide new insights into the functional role of BamA in membrane remodelling during OM biogenesis.
2025-09-06 | GSE306945 | GEO
Project description:The symbiotic relationship between the alpha-proteobacterium S. meliloti and the legume M. truncatula has been extensively studied. However, stress response mechanisms vital to bacterial survival and adaptation are as yet poorly understood. In this study, we sought to characterize the response of S. meliloti to depletion of essential outer membrane protein BamA, which is predicted to cause a loss of outer membrane integrity and an accumulation of unfolded proteins in the periplasm. A total of 132 differentially expressed genes were detected, with 74 genes up-regulated and 58 genes down-regulated. The transcriptomic response to BamA depletion shares similarities to those observed during activation of the RSI circuit, which plays a role in mitigating loss of viability in response to this stress. Induction of genes in the RSI regulon is likely due to a loss of negative regulator ExoR during BamA depletion. The two-component stress response system CpxRA and regulator CpxP also were found to affect the response to BamA depletion.
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Project description:In this paper we investigated the mechanism of action of Maganin-2 (Mag-2), a well-known antimicrobial peptide isolated from the African clawed frog Xenopus laevis, by functional proteomic approaches. Several proteins belonging to E. coli macromolecular membrane complexes were identified as Mag-2 putative interactors. Among these, we focused our attention on BamA a membrane protein belonging to the BAM complex responsible for the folding and insertion of nascent β‐barrel Outer Membrane Proteins (OMPs) in the outer membrane. In silico predictions by molecular modelling and in vitro fluorescence binding and Light Scattering experiments carried out using a recombinant form of BamA confirmed the formation of a stable Mag-2/BamA complex and indicated a high affinity of the peptide for BamA. The functional implications of these interactions were investigated by two alternative and complementary approaches. The number of outer membrane proteins OmpA and OmpF produced in E. coli following Mag-2 incubation was evaluated by both western blot analysis and quantitative tandem mass spectrometry in MRM scan mode. In both experiments, a gradual decrease in outer membrane protein production with time was observed because of Mag-2 treatment.
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Project description:Antimicrobial peptides (AMPs) are promising alternatives to classical antibiotics against multidrug resistant pathogens. TAT-RasGAP (317-326) is an AMP with broad range antibacterial activity, but its mechanism of action is unknown. Escherichia coli can become partially resistant to TAT-RasGAP (317-326) in vitro when passaged eight times with increasing concentrations of this peptide via mutations of the two-component system EnvZ/OmpR. To test whether additional mutations can cause further increase in resistance, we analysed a strain of E. coli showing high level of specific resistance to TAT-RasGAP (317-326) that we obtained after twenty passages of in vitro resistance selection. This strain bears, in addition to an envZ point mutation, a point mutation in bamA, an essential gene encoding an insertase involved in the insertion of outer membrane proteins. This mutation modifies the charge in a negatively charged loop (Q495-T505) at the surface of BamA. In silico docking simulations predict that binding affinity between TAT-RasGAP (317-326) and BamA varies depending on the charge of the Q495-T505 loop. We show here using CRISPR-Cas9-based targeted mutagenesis that mutations lowering the negative charge of the Q495-T505 loop decrease sensitivity of E. coli to TAT-RasGAP (317-326). Interestingly, BamA activity was not affected by TAT-RasGAP (317-326), indicating that BamA may function as a specific receptor for the AMP TAT-RasGAP (317-326). Our results indicate that binding and entry of TAT-RasGAP (317-326) may involve different mechanisms compared to other AMPs, which is in line with limited cross-resistance observed between different AMPs. This limited cross-resistance is important for the clinical application of AMPs towards multidrug resistant pathogens.
2024-12-02 | PXD051962 | Pride
Project description:The transcriptome is the complete set of all RNA transcripts produced by the genome in a cell and reflects the genes that are being actively expressed. Transcriptome analysis is essential for understanding the genetic mechanism controlling the phenotype of a cell. Using DNA microarray technique we examined transcriptomes of 2,000 individually collected inner (IHCs) and outer hair cells (OHCs), two types of auditory sensory cells critical for hearing. Among approximately 16,645 and 17,711 transcripts considered to be expressed, 1,296 and 256 genes showed significant differential expression in IHCs and OHCs, respectively. The top ten differentially expressed genes include Slc17a8, Dnajc5b, Slc1a3, Atp2a3, Osbpl6, Slc7a14, Bcl2, Bin1, Prkd1, Map4k4 in IHCs, and Slc26a5, C1ql1, Strc, Dnm3, Plbd1, Lbh, Olfm1, Plce1, Tectb, Ankrd22 in OHCs. Many unknown sequences and non-coding RNAs were also expressed in hair cells. The differentially expressed genes underlie the genetic mechanism for unique functions of IHCs and OHCs. The total RNA was extracted from the collected pools of single outer hair cell (OHC) and inner hair cell (IHC). Their whole-genome transcriptome expression was detected using GeneChip microarray analysis. The analysis and comparison between OHC and IHC allow us to determine what genes are expressed and what genes are uniquely or differential expressed in each population.
2014-08-12 | E-GEOD-56866 | biostudies-arrayexpress
Project description:To understand molecular pathways responding to loss of essential proteins in the inner- or outer-membrane, we knocked down essential genes in the inner- and outer membrane using a CRISPRi approach in Pseudomonas aeruginosa. CRISPRi is an inducible system with which even essential genes can be targeted by a specifig sg-RNA. In this study, we investigated the transcriptomic changes upon loss of BamA, LptD and FtsH. Loss of BamA and LptD, both outer membrane proteins, resulted in strong upregulation of genes involved in LipidA modification and upregulation of several two component systems. Depletion of FtsH resulted in strong upregulation in genes responsible for amino acid biosynthesis and regulatory genes. Surprisngly, knockdown of bamA and lptD resulted in (mild) upregulation of H1-T6SS genes.
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