RESEARCH PROJECTS OF THE DEPARTMENT OF MOLECULAR MICROBIOLOGY


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Research program

In our department we investigate protein secretion pathways in bacteria, mainly Gram-negative bacteria like E. coli. In these bacteria some proteins have to be incorporated into the innner membrane, whereas other proteins pass the inner membrane and end up in the periplasm or in the outer membrane. Furthermore, bacteria have evolved several mechanism by which proteins (enzymes, virulence factors) are secreted into the extracelluar environment. Knowledge about these protein translocation processes is important in the understanding of the complex structure and functioning of a living bacterial cell, but also for the biotechnological applications in industrial as well as in medical processes.

There are three different lines of research in the department:



Project I The biogenesis of innner membrane proteins

Project II Protein secretion routes and bacterial infections in human (peritonitis, tuberculosis)

Project III Bacterial cell division


Project I. Biogenesis of inner membrane proteins in E. coli (Dr. Luirink).

Aim of this project is to study the biogenesis of inner membrane proteins of bacteria, in particular the mechanisms by which these so-called IMPs are targeted and inserted into the inner membrane of E. coli , and how they fold into their native structure and form complexes. Most inner membrane proteins are targeted to the membrane via an interaction with the so-called signal recognition particle (SRP) and its receptor FtsY. The SRP binds to a hydrophobic signal anchors or transmembrane segment (TM's) of a nascent inner membrane protein as it emerges from the ribosome. The interaction of various types of signal anchor sequences or TM's of truncated nascent chains of different length with the SRP is studied, mostly by protein cross-linking techniques. The binding of SRP and trigger factor to ribosomal protein (L23, for instance) is also studied in this context. Furthermore, the role of the SRP receptor FtsY is investigated as well as the possible role of cytoplasmic folding factors like SecB, DnaK, etc.
Upon release of the SRP from the nascent chains by FtsY at the E. coli inner membrane, the nascent inner membrane proteins are transferred to a common translocation site containing at least SecY, SecE and SecG, and most of the times also SecA. Periplasmic and outer membrane proteins also use SecYEG and SecA as well as other Sec translocase components for their translocation across the inner membrane. These latter proteins depend on cytoplasmic SecB for their (post-translational) targeting to the translocation site. Thus, both proteins that depend on SecB for their routing to the inner membrane as well as SRP-dependent proteins are initially targeted to the core SecYEG/A-translocase.

Little is known about the consecutive interactions of nascent inner membrane chains during their membrane integration. Using cross-linking approaches we have demonstrated that hydrophilic portions of co-translationally targeted inner membrane proteins (like FtsQ and Lep) are close to SecA and SecY, while the transmembrane domain is close to lipids and to a newly characterized protein, YidC. YidC is homologous to the yeast mitochondrial Oxa1p, and appears to be specificaly involved in inner membrane protein assembly. Much of our efforts are now aimed at understanding the structure and function of YidC. In the biogenesis of some, Sec independent, inner membrane proteins YidC plays a critical and essential role; there is probably a direct interaction of nascent chains and YidC.

To study the membrane insertion process of inner membrane proteins is we use several approaches. In one af these approaches, insertion intermediates are generated by in vitro translation of truncated mRNA in the presence of inverted inner membrane vesicles (IMVs). Since the truncated mRNAs lack a termination codon, the targeted nascent chains remain attached to the ribosome as peptidyl-tRNA. To explore the molecular environment of the nascent chain, stop codons (TAG) are introduced at specific sites in the nascent chain coding region and these stop codons are then suppressed during synthesis by addition of 4-(-3-trifluoromethyl)phenylalanyl-tRNAsup, which is charged with a modified phenylalanine that carries a highly reactive photocross-linking agent. Following cross-linking, cross-linking adducts are identified by immunoprecipitation, fosforimaging, etc. This type of approach has given us detailed information about the direct molecular environment of newly synthesized short amino acid chains in the ribosomal tunnel as well as with chaperones, folding and translocation factors just outside the ribosomal tunnel and in the cytosol of E. coli and in the inner membrane.


Recent projects for students, PhD students and postdocs focus on the structure-function relationship of YidC, the specificity of TMs that bind YidC and the formation of large protein complexes in the innner membrane.



Project II. Protein secretion routes and bacterial infections in human (peritonitis, tuberculosis), especially the secretion of a heme-binding autotransporter protein in E. coli and its role in heme uptake, pathogenicity, intra-abdominal infections and abscess formation (Dr. Otto, dr. Luirink, dr. Oudega).

In the past we have focussed on the secretion routes of bacteriocins and adhesins like pili and fimbriae. In this project we now study strains isolated from intra-abdominal infections containing two different bacterial strains (mixed infection, Escherichia coli/Bacteroides fragilis). This type of infection often leads to abscess formation which can be lethal. The molecular mechanisms that are important for these mixed infections are not clear. The E. coli strain we study produces a relatively large extracellular protein that plays an important role in iron/heme acquisition during the infection process. The molecular structure of this protein (designated Hbp) is studied, as well as its mechanism of excretion, it is a so-called autotransporter. This protein possesses hemoglobinase activity; this activity may lead to the release of heme groups. These liberated heme groups may then bind to the HBP, which might then shuttle the heme into the E. coli and B. fragilis cells. The final goal of these studies will be a complete understanding of this heme acquisition mechanism and the development of chemicals/pharmaceuticals that interfere with this heme uptake system, and therefore block the pathogenicity of these E. coli strains and prevent intra-abdominal infections and abscess formation.

We have recently published the structure of the hemoglobin/heme binding protein. The structure is very unusual in that it contains a long helical stack of beta strands. We can now use the detailed structural information to study the secretion and release processes.

Research projects for (Msc and PhD students) deal with the role of the unusually long signal sequence in the targeting to the inner membrane and the insertion and transport through the Sec translocon, the folding or unfolding processes of the processed protein in the periplasm, the formation of the outer membrane translocation pore (beta barrel), and the various interactions with other cell factors.


Below is one of the putative models for the release of autotransporter proteins by E. coli cells.







Project III. Bacterial cell division (Dr. Scheffers).