Master’s Project

December 25, 2011

In the lab of Prof. A.J.M. Driessen (Molecular Microbiology) I am currently working on the first of my two master’s projects. This project has an approximate duration of six months and is centered on some molecular aspects of bacterial protein secretion across the cytoplasmic membrane. In order to give you the chance of learning something about what I do,  I summarized my work-plan and some of the theory behind it.  If you have questions, do not hesitate to contact me!

Background

Cells are no closed and just internally highly complex entities that are sealed from the outside world. In fact in one way or the other every cell directly or indirectly communicates with its environment through processes which are mediated by channels or receptors. For multicellular eukaryotic organisms it is evident and well-known that cells secrete substances which serve as substrates for a wide array of physiological functions. These substances are ranging from simple ions for neuronal signaling purposes or pH homeostasis to more complex peptide hormones and other proteins. A general issue which was overlooked by science for decades is the fact that secretion and communication also appears in and among prokaryotic cells. Since these organisms most of the time consist of single cells, systemically relevant excretion of compounds which are synthesized within the cell is occurring in a different manner and on a different scale. Bacteria, representing one of the two prokaryotic domains, indeed possess highly complex systems which enable interaction with the environment in the form of other cells or an adherence substrate.

A key role for a bacterial cells ability to survive and to secrete molecules into the environment plays the bacterial secretory (Sec) system. Genetic, biochemical, biophysical, and structural approaches during the last two decades have shaped a detailed understanding of the functional elements and dynamics of this system. The so-called translocase system encompasses an array of proteins which are functionally centered around the translocon channel which mediates the export of proteins across the bacterial cytoplasmic membrane and the insertion of membrane proteins into it. The focus of this work lies on the study the SecA motor proteins that occur in bacteria that next to the general Sec system also possess a so-called accessory Sec system which is responsible for the export of a subset of proteins. A significant number of pathogenic bacteria make use of this subsystem to export protein virulence factors. SecA is a motor protein and ATPase which is thought to be mainly responsible for the movement of proteins across the membrane via the SecYEG translocon channel. How the two different SecA motor proteins that occur in the general and accessory Sec systems interact and what their exact functional relationship is, is currently not understood. By making use of genetic, biochemical, and fluorescence microscopic techniques the focus of the present study is to elucidate a possible interaction between Staphylococcus aureus’ general Sec systems SecA1 and its accessory Sec systems counterpart SecA2. Parts of the bacterial Sec system are conserved in all three kingdoms of life and among others also function in the eukaryotic endoplasmic reticulum in order to secrete proteins into the cytoplasm. Embedding the performed research into its context is essential and therefore brief introductions to bacterial cell structure and the two Sec systems follow with a focus on SecA structure and function.

It does, however, not lie within the scope of this work to formulate a wholesome review on the translocase system. A number of reviews have already been published on this topic, which offer an in-depth overview and cover this subject more explicitly (1, 2, 3).

The Sec translocase system

Since the SecA proteins of S. aureus, which stand central in my research project, are part of the functional complex Sec translocase system, here a brief overview is given about this system. Fig. 1 summarizes the most important aspects of this kind of bacterial protein translocation across the cytoplasmic membrane.

Fig. 1: Schematic overview of the bacterial protein translocation system termed Sec translocase. Proteins which are synthesized within the ribosome are exported from the cytoplasm over the cytoplasmic membrane into the periplasm by the proton motive force (PMF) and the ATPase SecA via the heterotrimeric membrane channel SecYEC. Two major ways of protein translocation exist: posttranslational secretion and cotranslational insertion into the cytoplasmic membrane. Secretory proteins are either directly targeted to SecA by means of their N-terminal signal sequence or are bound by the chaperone SecBfirst and translocate later via SecA and SecYEG. Membrane proteins are translocated cotranslational. Their C-terminal signal sequence is bound by the signal recognition particle (SRP) and targeted to the SRP receptor (FtsY). Consequently SecAtranslocates these proteins via a lateral gate in the SecYEG channel into the membrane. SecDF(yajC) is an accessory factor which seems to improve preprotein translocation. YidC associates with the translocon during protein insertion into the membrane (figure derived from (1)).

Some bacteria posses two different versions of SecA

In bacteria SecA universally functions as the ATPase which delivers the chemical energy to physically translocate proteins across the cytoplasmic membrane in a post-translational manner. However, some bacteria posses two homologues of this protein termed SecA1 and SecA2. SecA1 functions as the essential housekeeping protein for translocation, while SecA2 seems to be especially important for a subset of proteins. Studies on bacteria which are expressing  SecA2 have shown, that this subset of proteins often includes virulence factors, as for example adhesion molecules (4). In addition to SecA1/A2 some of these bacteria also contain a homologue monomer of the heterotrimeric translocon channel SecYEG (Fig. 1) termed SecY2.  Summarizing, bacteria containing either only SecA1/A2 or SecA1/A2 + SecY2 are said to express an accessory Sec system next to the general Sec system which is depicted in Fig. 1.

The question

The intriguing research questions of my project therefore is: Do SecA1 and SecA2 dimerize with each other or which other combinations are required to be functional? This is an important question, since SecA1/A2 might function as a link between the general and accessory Sec systems. As the accessory Sec system is important for virulence in some bacteria it is essential to learn more about its protein molecular basis. Fig. 2 summarizes the hypothetical dimers which SecA1/A2 might form in bacteria with or without an additional SecY2 homologue. Currently I am investigating the SecA proteins of S. aureus which is not expressing an additional SecY2. Some of the combinations depicted below (Fig. 2, (D) through (F)) therefore are not predicted to apply to the current state of my project.

Fig. 2: Schematic overview of the hypothetical combinations of two SecA motor proteins in S. aureus with the two SecYEG homologues when assuming a functional SecAdimer. Dimers (A) through (C) might function with the canonical SecY1EG translocation channel, while dimers (D) through (F) might also interact with the accessory SecY2EG translocation channel which is present in S. aureus. In addition it is also theoretically possible that dimer (A) binds to SecY2EG and dimer (D) to SecY1EG (created by myself, based on earlier work of Irfan Prabudiansyah).

How to get there

Here I will present a very brief work-plan, just to give you an idea about which techniques and experimental approaches my project entails in order to investigate the properties of SecA1/A2 and their functional relation to each other in S. aureus. The aim is to use molecular genetics to overexpress both proteins, which is followed by using knowledge of biochemistry to purify and fluorescently label the proteins. The final step is to use confocal microscopy (cross-correlation) and FRET microscopy to examine the potential interaction between both proteins.

Genetics

The molecular cloning of of both secA1 and secA2 gene sequences from S. aureus into plasmid vectors involves some bioinformatics work and the designing of the needed primers. In addition DNA sequencing is performed to confirm correct sequence insertion. The Isopropyl β-D-1-thiogalactopyranoside (IPTG) inducible plasmids are isolated and heat-shock transformed into E. coli cells. These cells containing either secA1 or secA2 are consequently grown in large amounts at 25°C to 30°C to reduce inclusion body formation after overexpression induction by the addition of IPTG into the growth medium. After cell harvesting and cell membrane disruption by french pressing or sonication, the overexpressed protein-containing cytoplasmic fraction is isolated by ultracentrifugation. Results are confirmed by SDS-PAGE and Western blots.

Biochemistry

Overexpressed SecA1/A2 is purified by HPLC mediated cation exchange and size-exclusion chromatography. After desalting on a size-exclusion column both proteins are labeled with two fluorophores (Cy5 & fluoresceine) via a maleimide group to their external cysteines (50% of purified SecA1 is labeled with first fluorophore, other half with second, same procedure for SecA2). Labeling efficiency is checked by photometry at different wavelengths specific to excitations spectra of fluorophores. Removal of excess fluorohore molecules is essential for avoiding too strong background noise during microscope steps. This is achieved by HPLC cation exchange at protein specific salt concentration. Results are checked on SDS-PAGE under fluorescence detection conditions.

Microscopy

Analysis of dimer formation is first achieved by an array of different concentrations of SecA1 and SecA2 combined to each other. Secondly, the potential dimers are analyzed with the help of a confocal microscope observing a volume of approximately 90 femtoliters. Fluorescence cross-correlation spectroscopy is applied to this volume of the solution in order to determine the diffusion coefficient of the fluorescently labeled particles (5). Potential dimer formation can then be described by altered dual color absorption/emission spectra of the proteins (FRET), as well as their unchanged or slower diffusion time through the observed volume in the case of dimer formation.

Final remarks

If you also consider the approach of my work as interesting as I do, you can always contact me back via mail. In the future I would like to continue my work and extend my knowledge into the direction of biophysics. Describing the properties of proteins by interdisciplinary means has definitively caught my eye.

(1) Du Plessis, Nouwen, Driessen, The Sec translocase, Biochimica et BiophysicaActa (2010).

(2) Driessen & Nouwen, Protein Translocation Accross the Bacterial Cytoplsmic Membrane. Annual Reviews of Biochemistry (2008).

(3) Papanikou, Karamanou, Economou, Bacterial protein secretion through the translocase nanomachine, Nature Reviews Microbiology (2007).

(4) Rigel & Braunstein, A new twist on an old pathway – accessory Sec systems, Molecular Microbiology (2008).

(5) Schwille, Mayer-Almes, Rigler, Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution, Biophysical Journal (1997)

The Experiment

December 19, 2011

“[…] One reaction tube contains much more bacteria than the other one and they want to know why. In their own discussion the students conclude that something went wrong during the procedure. The experiment failed. […] But there is no course with the title “How to do proper Science”. […] Daring to make your own conclusions […]. […] Realizing what you have learned over the years […]. […] It sounds so simple, but it is a giant leap. […] Tomorrow we will do a new experiment. An experiment with an unknown result. Educational objective: There will come a day on which you can beg for an explanation of your results, but your teacher will also not know the correct answer. There will come a day on which you will have to figure it out yourself.”

by Rosanne Hertzberger (excerpt from her column in the Dutch newspaper NRC next, December 19th 2011)

The biology of light

December 16, 2011

If you want to know something about the connection of biomolecules and light, then this might be of interest to you. I also thought it is pretty interesting, so I wrote this piece on proteins, linker-molecules, emission, and fluorophores. First of all because I wanted to understand the principles of fluorescence better since it is important for my project (FRET and confocal microscopy), but secondly also because I like the interplay between biology and physics. For my work on the hypothetical interaction of Staphylococcus areus SecA1 and SecA2 proteins I use two molecules that have fluorophore properties. Fig.1 depicts these molecules called fluorescein-5-maleimide (A) and Cy5-maleimide (B). Obviously both names are not the IUPAC nomenclature names for chemical compounds, however in everyday lab-life, these two names seemed more practical.

 

Fig. 1: Chemical structure of two fluorophores that can be used to label proteins via sulfur containing thiol groups on external cysteines. Left fluorescein-malamide is depicted and on the right side Cyanine (Cy5) maleimide. The maleimide group is the lowest identical portion of both molecules.  It functions as a linker between the thiol group and the actual fluorophores.

To be fluorescent actually describes a property that some molecules have. This includes the absorption of light at a specific wavelength (relatively high energy) and the subsequent emission of light with a longer wavelength and lower energy per photon. The relation between the energy of a photon and its wavelength is important when talking about fluorophores (Fig. 2). The frequency of a photon (≈ internal energy) is inversely proportional to its wavelength, thus photons with a short wavelength contain more energy than photons with longer wavelength.

 Fig. 2: The relation of a photon energy with its wavelength. Long wavelength means lower energy, because the frequency of the photon decreases (not shown in this formula) (1).

If one concentrates on the chemical structures depicted in Fig. 1 it becomes apparent that they both contain a large number of double bond containing cyclic carbon rings. And this is also the case for most fluorophores in nature. These structures are one secret of fluorescent properties.

Light low wavelength transfers energy on electrons in double bond which becomes excited and “jumps up” a band, thereby stabilizing the energy which has just been absorbed. This excited state of the electron does not last forever and it will fall back to its original and more stable state sooner or later. However, thermodynamics tells us that energy does not just disappear so the energy which is lost (because the electron has fallen back) must be somewhere. Indeed the energy is somewhere. The fluorescence effect that we see is the electromagnetic radiation in the form of photons that is emitted from the falling electron. Important to notice is, however, that not all energy is emitted as visible electromagnetic radiation. The physical turnover processes also create other emissions that cannot simply be seen by eye and in addition energy is “lost” through collisions of our fluorophores with other molecules. Since this is the case, the emitted energy is somewhat lower than the absorbed energy and therefore also the wavelength of the emitted photons becomes longer, as Fig. 2 tells us. As light color depends on wavelength, the emitted color that can be observed is different from the previously absorbed one (Fig. 3).

 Fig. 3: Emission spectra of green fluorescent protein (GFP). The wavelength of the emitted light is longer than the wavelength of the absorbed light. Therefore also the color of the light appears different (2).

Fluorescent properties of molecules enable biologists to mark proteins or other biomolecules in order to make them easily detectable by eye or with the help of photomultipliers. Two or more fluorescent markers can even be combined and might transfer energy to each other when their carrier molecules make contact. Changes emission spectra can consequently be observed. This is the basis of FRET microscopy and allows me to study the potential dimerization of proteins that are thought to function in the same protein translocation pathway.

(1)http://astronomyonline.org/Science/Frequency.asp

(2)http://www.invitrogen.com/site/us/en/home/support/Product-Technical-Resources/Product-Spectra.EGFPpH7.html

Lamarck’s revenge

December 7, 2011

Lamarck himself of course did not know about molecular genetics, but in biology it is becoming more and more clear that inheritance of acquired traits might also be possible. However, most of these traits are transmitted via epigenetics (methylation, acetylation, etc.) which is essentially chromatin and thus DNA based. A mechanism which enables the “inheritance” of acquired traits and in which DNA is not the central element, has now been published in the Cell journal (Transgenerational Inheritance of an Acquired Small RNA-Based Antiviral Response in C. elegans).   The group of  Oliver Hobert from the Department of Biochemistry at Columbia University in New York has reported that upon viral infection of their model organism Caenorhabditis elegans, the worm activates its defense mechanisms in such a way that it is also transferred to later generations which did not have contact with the virus at all (1). In this case the immunological response of C. elegansis is based on the so-called RNA interference (RNAi) mechanism. RNAi is generally known to silence genes in the cadre of genetic regulation and works by the binding of specially synthesized small RNA molecules to regular mRNA which is subsequently up- or downregulated thereby influencing protein production. For quite some years it has also been known that RNAi is used in viral defense by utilizing the double stranded RNA (dsRNA) of the virus as a template for the production small RNAs (viRNA) that subsequently lead to the breakdown of the viral genes and an effective protection. Also in the present study this behavior of the RNAi system is important.

By saying “gene-silencing effects evoked by exogenously added dsRNA can be observed not only in the treated animals, but […] also in the progeny of the treated worms” the authors summarize the findings of other scientists, who had found in previous studies that upon RNAi system stimulation by foreign dsRNA (for example from a virus) the viRNA parts of this defense mechanisms could also be traced back in generations that never encountered the dsRNA (i. e. virus). Therefore some kind of “inheritance” must have taken place. In this paper Hobert and coworkers tried to identify whether this “inheritance”  is rather traditional and therefore DNA or chromatin mediated (for example epigenetic) or whether it involves some kind of novel mechanism that transgenerationally “directly” transmits the RNA .

In order to test this the scientists introduced dsRNA containing the FR1gfp transgene into the C. elegans worms. This dsRNA was responsive to a heat-shock signal due to a promoter which was able to sense abrupt increase of temperature. By means of raising the temperature the virus could thus be induced. Under conditions where the worm RNAi machinery is functional the replication and distribution of the foreign viral RNA is of course inhibited. The worms keep their regular phenotype. When, however, the RNAi machinery is mutated the worm cannot defend itself against the replication of the foreign RNA. In this case also the FR1 gene which is fused to a green fluorescent protein (gfp) gene is expressed. The worms appear greenish after excitation with light of the fitting wavelength. Mutations where induced in two genes called rde-1 and rde-4. These two genes are normally responsible for the expression of dsRNA binding proteins that induce the RNAi response. Fig. 1 (left) depicts the general experimental setup in which immunodeficient mutant worms are crossed with immunocompetent worms (parental generation) what results in heterozygotic (for this allele) worms in the F1 generation (kids). When these worms are crossed with each other a Mendl-like inheritance pattern results in the F2 generation (grandchildren). The essential question is: Are the homozygotic mutants of the F2 generation resistant to the virus dsRNA even though they have never encountered the virus before and do not possess the machinery to combat the dsRNA? In case they are not resistant the researchers would observe a green fluorescence signal due to the replicating RNA (see also above).

Fig. 1: The genetic principle behind this study (left) and some results showing the generational transmittance of viRNA into mutant worms what consequently renders them resistant to the virus (no green fluorescent signal) (right). The transmission effect eventually “wears off” after 4-5 generations.

After performing the above described heat-shock induction of the F2 generation worms, the hypothesis derived results could indeed be observed (Fig. 1 (right)). Even viRNA transmission up to the 5th generation was present. The authors excluded the possibility that the resistance of the homozygous mutants is based exclusively on maternal deposition by the heterozygous parents since it has been demonstrated before that none of the gene activity is transmitted between generations (2). In addition the necessary viRNA is transmitted over several generations also if no viral stimulus is added. However the virus resistance “wears off” after 4-5 generations.

That viRNA is really transmitted among generations is proven by a physical RNA detection assay in which the 20–30 nt viRNAs are quantified in accordance to their alignment on the viral RNA and qualified based on their strandedness.

All together these results indicate that classical genetics based on DNA and even relatively new elements of it such as epigenetics are not the whole picture of biological adaption and evolution. However, important molecular components of this viRNA transmittance system remain unidentified. Why does the protective effect “wear off”? How are the viRNAs transmitted from generation to generation if not by maternal deposition? What is the code if no viral dsRNA template is present and nothing is deposited?

In their conclusion the authors stress the point that the above described mechanism can have a strong evolutionary significance for organisms since it renders it possible to rapidly transmit the mechanisms of an adaptive response to the following generations. Short-term environmental stress can thus be effectively counter acted.

Even though many important questions, especially concerning the molecular basis, remain unanswered, this study shows that biology is far from being elucidated completely. In the search for answers it can thus sometimes be helpful to look into a direction that seems the least probable. Lamarck might agree.

This blog-article is especially based on the two articles named below:

(1) O. Rechavi, G. Minevich, O. Hobert, Transgenerational Inheritance of an Acquired Small RNA-Based Antiviral Response in C. elegans. Cell147 (2011). (online publication)

(2) D. Blanchard, P. Parameswaran, J. Lopez-Molina, J. Gent, J.F. Saynuk and A. Fire, On the nature of in vivo requirements for rde-4 in RNAi and developmental pathways in C. elegans. RNA Biol.,  8  (2011).

A brief intro

December 1, 2011

This is what I do during my Master project number one (of two). As you can see, the Sec systemsSecA motor protein is the center of my work. It is the molecular motor which transports proteins which are made within the (bacterial) cell via the membrane to the outside.  Wordle created this nice overview for me by copy/pasting the introduction of my report into it. I actually borrowed the idea to this from Stefan Kowalczyk who is working at Bionanoscience Department of TU Delft in the Netherlands.