Revision as of 23:11, 22 October 2020

Outer membrane protein A, aa 46-66

Sequence and Features

This part codes for amino acids #46-66 of outer membrane protein A (OmpA). It corresponds to a single transmembrane domain of OmpA, crossing the outer membrane of E. coli.

This was fused with the lipoprotein signal peptide upstream and streptavidin downstream in the project to express streptavidin on the outer surface of E. coli.

Contribution

• Group: Nanjing-China
• Author: Peiqing Sun, Kuisong Song, Wenyin Su
• Summary: We cloned and characterized OmpA and fused it with a heavy metal binding protein called PbrR. We successfully extended the application of OmpA into the cell surface display system of E.coli.

• Group: William_and_Mary
• Author: Bradley, Avery
• Summary: We performed a literature review of OmpA.

Usage and Biology

As we all know, improving the function or characterization of previously existing parts in the part registry of iGEM is not only important for maintenance of the part registry, but essential for other teams to utilize the part properly as well. Part BBa_J36836 encodes outer membrane protein (OmpA) of E. coli, which corresponds to a single transmembrane domain of OmpA. As far as we are concerned, this protein can be applied to the cell surface display system, which is to fix other proteins onto the surface of E.coli. This year we extended the application of OmpA in the construction of our artificial PS system. In our project, a metal binding protein PbrR is fused with OmpA so as to induce precipitation of CdS nanoparticles on the surface of E.coli cells. Protein PbrR is a special metal protein found in Cupriavidus metallidurans that specifically binds to Pb2+ ions. In realistic research we further examined that this protein also bears a high affinity to Cd2+ ions. We demonstrated in our project that when we fused PbrR with OmpA, the binding of PbrR with Cd2+ is greatly enhanced. (Figure 1).

Figure 1. OmpA-PbrR fusion protein. (Left) Structure of OmpA-PbrR fusion protein; (right) Metal-binding specificity of PbrR

Characterisation of OmpA

Protein PbrR is a special metal protein found in Cupriavidus metallidurans that specifically binds to Pb2+ ions. In realistic research we further examined that this protein also bears a high affinity to Cd2+ ions(Figure 1). We demonstrated in our project that when we fused PbrR with OmpA, the binding of PbrR with Cd2+ is greatly enhanced.

Figure 2. Photocatalytic reduction of MV by TiO2 and induced CdS nanoparticles

For organism M.thermoacetica, this kind of bacteria can produce S2- ions from cysteine and forms a higher sulfur concentration around the cell which then induces the precipitation of CdS nanoparticles when Cd2+ ions are added into the media. We assume that if we form a same local high concentration of Cd2+ with fused protein OmpA-PbrR on the outer cell membrane, we can also achieve a similar precipitation of CdS nanoparticles on to the walls of E.coli, the well model bacteria. To confirm the capability of our CdS system based on OmpA-PbrR, we conducted the same photo-catalytic assay. Bacteria were divided into three groups. Bacteria were induced to express OmpA-PbrR protein and cultured with both Cd2+ and S2- in the experiment group. Groups that either lacked induced expression or necessary ions to build semiconductors were negative controls. We found that illumination resulted in a same increasing trend in experiment group (Figure 2). This confirmed the photo-catalytic capability of our PbrR-based precipitation of semiconductors.

Figure 3. TEM image of native E.coli cells (right) and cells with in situ formed CdS nanoparticles (left)

We also did a TEM imaging of the CdS particles formed on bacteria surface and demonstrated that the CdS particles are nanoparticles. It indicated that OmpA works efficiently on the cell surface display system of E.coli. To conclude, we utilized the part BBa_J36836 and extended its application to the cell surface display system in E.coli. We successfully displayed a kind of metal binding protein PbrR to the surface of E.coli and identified enhanced function of PbrR after fusion with OmpA. To learn more details about our project design, please see the following project overview part.

OmpA Information Contribution by team William_and_Mary 2020

Usage and Biology

OmpA, an outer membrane protein, consists of 325 residues (1). This protein consists of a N-terminal region with an eight stranded beta-barrel that anchors four loops which extend into the extracellular area (2). A fifteen-nucleotide long region of this protein connects the N-terminal region to the C-terminal region, which is located in the periplasm (3). OmpA has been shown to form dimers and it is hypothesized to exist in a hybrid state between its monomer and dimer forms (3). While there is conclusive evidence of the existence of the OmpA dimer, the function of this dimer is unclear (3, 4). OmpA has been shown to be heat-modifiable and its molecular mass ranges from 28 kDa to 36 kDa based on the temperature of the protein before the weight has been calculated. There are about 100,000 copies of OmpA per cell (2,5). OmpA is most commonly located in gram-negative bacteria and is best characterized in E. coli (5). However, OmpA has homologs in many different species such as P. aeruginosa and C. trachomatis (5).
OmpA transcription has been shown to be dependent on environmental conditions (2). Factors such as nitrogen availability, adhesion to surfaces, and growth rate all impact the transcription of OmpA (6,7,8). For example, when the cell is growing at a higher rate, it maintains a mRNA half-life of about 15 minutes; however, with a slower growth rate, the mRNA half-life decreases to about 4 minutes (9). OmpA has many functions within E. coli. OmpA assists the cell in holding together the outer membrane and the peptidoglycan layer (1). While there has been documented nucleotide variability within the loop portion of the protein, the beta-barrel region remains consistent across many different E. coli species, hinting at this region's role in structural support (1). Further, these beta-barrels also play an important role in regulating environmental stress. When mutant bacterial strains without OmpA were formed, they showed greater responses to environmental stress and a greater death rate when exposed to acidic environments or osmotic shock compared to bacterial strains containing OmpA (1). In addition to providing E. coli with structural support, OmpA acts as a non-specific porin with a pore size of roughly 1 nm (10).
While OmpA has many functions in E. coli, it plays a large role in the pathogenesis of E. coli. OmpA is simultaneously a way by which pathogenic bacteria can avoid host immune system defenses and become a target of the immune system (2,5). OmpA provides bacterial cells with methods to avoid the immune system. By providing a binding site for protein C4, OmpA is thought to avoid antibody detection (5). Further, OmpA enables E. coli cells to enter macrophages, replicate, and eventually lyse the macrophages (11). While OmpA helps the cell to avoid immune system responses, it is also an identifier of infection and triggers an immune response. For example, the immune system uses an enzyme called neutrophil elastase to destroy pathogenic bacteria (2,5). Normally, neutrophil elastase is able to effectively kill E. coli. However, in mutant bacterial strains without OmpA, neutrophil elastase is no longer effective, suggesting that OmpA acts as a target of the immune system (12). Furthermore, OmpA is thought to bind to a receptor on macrophages, alerting the infected cell to a potential bacterial invasion (13).