PhD projects


Structural Biology of Membrane Proteins

PhD project:

Structure - function of sugar transporters in Streptococcus and Lactococcus

Principal Investigator:

Margarida Archer, (Membrane Protein Crystallography Group), ITQB, Av. República, EAN, Oeiras Portugal; archer|

In collaboration with:

Ana Rute Neves, (Lactic Acid Bacteria & in vivo NMR Group, ITQB).

Helena Santos, (Cell Physiology and NMR Group, ITQB).

Frank Bernhard, Institute of Biophysical Chemistry, Centre of Biomolecular Magnetic Ressonance, J.W. Goethe University of Frankfurt, Max-von-Laue-Str. 9, D-60438 Frankfurt am Main, Germany; fbern|

A. Structural characterization of sugar transporters in the bacterial family Streptococcaceae

Bacilli form a class of rod-shaped or spherical low-GC Gram-positive Eubacteria that share numerous genetic, morphological, metabolic and physiological characteristics, yet are found in diverse habitats ranging from soil to milk, from the surfaces of plants to compartments within insects and mammals. Bacilli in the family Streptococcaceae are, for very different reasons, of enormous importance to humans, both in health and industry. The Streptococcaceae are strictly fermentative, and thereby rely on glycolytic metabolism of anaerobic sugars for energy production. Therefore, the mechanisms governing sugar uptake and degradation are central to their physiology. The chief goal of this work is to gain insight into these mechanisms. For that purpose we use as model organisms, Streptococcus pneumoniae, a major human pathogen, and Lactococcus lactis a closely related organism (the non-pathogen with the most similar set of proteins), which is a crucial player in the dairy industry.

1. Structural characterization of the mannose-PTS in Streptococcus pneumoniae

Streptococcus pneumoniae is an important human pathogen that causes a range of infectious diseases including the life-threatening pneumonia, meningitis and septicaemia, as well as other less severe but highly prevalent diseases, such as otitis media and sinusitis. This bacterium is also a commensal organism found in the nasopharynx, which serves as the reservoir for streptococci to cause infections in children, the elderly and immuno-compromised individuals.

The PTS consists of two general proteins, enzyme I (ptsI) and the heat-stable phosphocarrier protein HPr (ptsH), and sugar-specific permeases, which are known as enzyme II complexes. Sugar-specific permeases catalyze the translocation and concomitant phosphorylation of several different sugars. The mannose-PTS, a sugar-sepcific permease in the PTS Mannose-Fructose-Sorbose (Man) family, is composed of four domains, IIA, IIB, IIC and IID. IIA and IIB domains are soluble and constitute a single polypeptide, whereas IIC, the permease porter, and IID are membrane spanning domains. The function of IID, a domain only present in Man family, remains elusive. Phosphoryl relay proceeds sequentially from PEP to EI, HPr, IIA, IIB, and finally to the incoming sugar, which is transported across the membrane via the integral membrane IIC porter. Beyond the primary function of sugar transport, the PTS plays roles in various processes central to the physiology of the cell, including a wide number of mechanisms for metabolic and transcriptional regulation(1). In streptococci, the PTS was proposed to regulate the expression of known virulence genes(2, 3). A recent study revealed that the lactococci mannose-PTS is the main target/receptor of class II bacteriocins(4). The PTS proteins are unique to bacteria and thus good targets for drug design. The demonstration that the PTS indeed serves as a target for a number of membrane-permeabilizing peptides supports that claim. Therefore, it is timely to obtain the three-dimensional structures of these proteins.

2. Structural characterization of glucose transporters in Lactococcus lactis

Lactococcus lactis is widely used in industrial food fermentation contributing to the flavour, texture, and preservation of fermented products. The economical importance of this microorganism has prompted an extensive number of studies on its physiology and genetics. As a result, a large array of efficient tools for the genetic manipulation has been developed, making tasks like gene deletion or overexpression generally straightforward. The availability of technology for genetic engineering of L. lactis combined with a long history of safe usage opened a range of new opportunities for applications even beyond the food industry. However, its utilization as a cell factory demands thorough characterization of metabolic and regulatory networks. Despite the wealth of information on sugar fermentation, the first steps in substrate utilization, uptake and phosphorylation, have been largely neglected. However, glucose transporters are known to play key roles in regulation and metabolism(1). In L. lactis, glucose is taken up and phosphorylated via the specific mannose/glucose (EIIMan/Glc) and/or cellobiose (EIICel) PEP-dependent phosphotransferase systems, or is imported via a non-PTS permease and phophorylated by glucokinase(2).

Protein 1. The cellobiose-PTS protein complex (EIIBA, soluble components, EIIC, membrane-bound components). The cellobiose-PTS (ptcBAC) shows homology to proteins in the PTS lactose-N,N'-diacetylchitobiose-β-glucoside (Lac) family, which are known to take up lactose, aromatic β-glucosides, cellobiose, N,N-diacetylchitobiose and lichenan oligosaccharides(3). Therefore, L. lactis PTSCel is the first transporter of this family with proven affinity for glucose(2). A dramatic effect on glucose metabolism (growth rate and glucose consumption rate negatively affected) was observed in mutants with disrupted EIIBACel (soluble components), but not EIICCel (membrane-bound)(4). These data implies a role of EIIBACel also in the regulation of sugar uptake, most likely through phosphorylation of other proteins(1). A mutant where glucose is preferentially taken up by the PTSCel (glucokinase and PTSman disrupted) shows a clear preference for the -glucose, indicating this anomer as the substrate for PTSCel. Kinetic parameters for this transporter will be determined in the near future. To further understand the features of this protein-complex a more detailed structure-function study is required.

Protein 2. The non-PTS glucose transporter GlcU. The non-PTS glucose transporter, glcU, has recently been identified in L. lactis(5). Deletion of this gene in a background where PEP:PTS transport was blocked resulted in a strain unable to grow on glucose as sole carbon source. The kinetic parameters of this newly discovered transporter were determined by measuring the uptake of 14C-glucose by whole cells. When cells were energized by supplying arginine a 5-fold increase in Vmax was observed, indicating that the transport via GlcU is closely dependent on the cell energy status. GlcU has homology with porters in the drug/metabolite transporter (DMT) superfamily, more specifically with the glucose uptake permease of Staphylococcus xylulosus (2.A.7.5. the glucose/ribose porter (GRP) family, 6). To the best of our knowledge, structure-function information on this subclass of proteins is still missing, which would provide important insights into the sugar transport mechanisms.

The groups headed by Ana Rute Neves and Helena Santos are interested in the study of the mechanism involved in the transport of sugar compounds and on their regulatory pathways. The group headed by Margarida Archer aims at the determination of the three-dimensional structure of these enzymes by X-ray Crystallography (11). The first step involves the crystallization of these membrane-bound proteins using detergents. Crystal growth in lipidic mesophases (in meso) can also be tried.

So far, most structural and functional approaches have been limited by the immense difficulties in the production of sufficient amounts of protein samples in conventional expression systems based on living cells. One goal of the current project is to use the cell-free expression system for the production of the sugar transporters. This work will be performed under the supervision of Frank Bernhard. The open nature of cell-free systems allows the addition of detergents in order to provide an artificial hydrophobic environment for the reaction. This strategy defines a completely new technique for the production of membrane proteins that can directly associate with detergent micelles upon translation. This strategy has already been successfully applied, namely to produce high levels of different G-protein coupled receptors (GPCRs) in an individual cell-free expression system based on Escherichia coli extracts(10).

Reference List:

  1. J. Deutscher, C. Francke, P. W. Postma.
    Microbiol. Mol. Biol. Rev., 70, 939-1031, (2006).
  2. J. Abranches, M. M. Candella, Z. T Wen, H. V. Baker, R. A. J. Burne.
    J. Bacteriol. 188, 3748-3756, (2006).
  3. G. E. Kaufman, J. Yother.
    J. Bacteriol., 189, 5183- 5192, (2007).
  4. D. B. Diep, M. Skaugen, Z. Salehian, H. Holo, I. F. Nes
    Proc. Natl. Acad. Sci. U. S. A, 104, 2384-2389, (2007).
  5. W. A. Pool, A. R. Neves, J. Kok, H. Santos, O. P. Kuipers,
    Metab. Eng., 8, 456-464, (2006).
  6. R. D. Barabote, M. H. Saier, Jr.
    Microbiol. Mol. Biol. Rev., 69, 608-634, (2005).
  7. A. R. Neves, W. A. Pool, R. Castro, J. Kok, H. Santos, O. P. Kuipers.
    Manuscript in preparation.
  8. R. Castro, A. R. Neves, J. Kok, O. P. Kuipers, H. Santos.
    Unpublished results.
  9. Fiegler, H., J. Bassias, I. Jankovic, R. Brückner.
    J. Bacteriol., 181, 4929-4936, (1999).
  10. C. Klammt, D. Schwarz, N. Eifler, A. Engel, J. Piehler, W. Haase, S. Hahn, V. Dötsch, F. Bernhard.
    J Struct Biol., 159, 194-205, (2007).
  11. Hickman A.B & Davies D.R.
    Principles of Macromolecular X-ray Crystallography. In Current Protocols in Protein Science (ed.) pp 17.3.1-15, John Wiley&Sons, Inc., (1997)