How to achieve cellular replication without fail: lessons from bacterial cells

Speaker:             Christine Jacobs-Wagner
Department:     Microbial sciences institute, dept. of molecular cellular and developmental biology
Subject:              How to achieve cellular replication without fail: lessons from bacterial cells
Location:            TU Delft, BN-seminar
Date:                    13-10-2017
Author:               Maricke Angenent

 A cell partitioning mechanism which does not involve any of the complicated processes such as cytoskeleton filament assembly, motor protein functioning and the participation of the actin proteins. That is a system we were introduced to by Christine Jacobs-Wagner, who is currently the principle investigator in the Jacobs-Wagner lab at the university of Yale. She started her seminar by giving a short outline on cell replication in general. Hereafter the presentation would consist of two distinct parts. Firstly the focus was on how to ensure that partition of cell compartments happens equally among daughter cells. The second part was supposed to be about how different processes are integrated, especially how the cell cycle function integrates cell metabolism. The latter topic would have been rather innovative as the cell cycle and metabolism are usually viewed separately. Unfortunately an hour proved too short to cover both of the subjects, so instead Christine set on answering some additional questions on the first subject.

 

Christine started by emphasizing the fact that replication is a truly remarkable process. As she said “the ability to replicate distinguishes the non-living organisms from the living”, which is the source of her fascination for the process. In her lab, bacterial replication in particular is analyzed, since replication is fast and it is an incredibly robust processes. Replication is a stochastic process, meaning that it is submissive to internal variations, yet the process seems to never fail. One of the proposed reasons for this observation is that various processes are highly integrated and often rely on intracellular organization. But how exactly is this spatial organization achieved?

 

Though diffusion is an effective means of molecular transport in bacteria, due to travel distances being small, this only leads to random distributions. This in  itself is not a problem, however when you have a low copy number, the probability of finding all copies of a cellular compartment on one side of the cell considerably increases. Consequently, the chance to obtain unequal daughter cells also rises. Diffusion in bacteria is thus ineffective for partitioning cellular compartments in low count. A different separation machinery must be present in bacteria to partition single components. As Christine explained, this mechanism makes use of stochastic interactions that are transformed into active transport, which results in the spatial patterning of low count cell compartments. Based on experiments, it was concluded that when you have more than one copy in the cell, they are equidistantly distributed among the axis of the cell ensuring their propagation to the daughter cells. This distribution was analyzed for the plasmid system, as this system is best understood compared to others.

In this experiment, the mechanism of just a single plasmid was taken into account rather than of a cluster of plasmids, which you would normally encounter in real situations. Two proteins are of central importance in the partitioning mechanism: parA and par B. The former mediates active transport and binds to the chromosomal DNA  as a dimer. The latter binds to a specific sequence of a plasmid, resulting in a plasmid rich region (or so called cargo). Bound parA interacts with the parB cargo, resulting in the oscillation of a single plasmid cluster over the nucleoid. What is interesting, is that parA does not form any cytoskeleton filaments, nor does it use any motor proteins, yet the oscillations point to a form of active transport. In modeling this process it is important to assume that chromosomal DNA is not static. Instead the DNA (and thus also when parA is bound) experiences intrinsic fluctuations, which is observed as “wiggling”. Now the plasmid cargo (=parB) diffuses until it encounters parA, where it activates hydrolyzation. Consequently the parA is released from the DNA and parB can go on to interact with a different parA-DNA complex. When plotting the movement of parB over time, you obtain evidence for oscillatory behavior, where the plasmid goes from one end of the nucleoid to the other end of the nucleoid. This movement usually follows the direction of the parA gradient as the chance for interactions is higher in an environment with a higher concentration of bound parA.

seminar report 2The above figure shows the interaction of two parB plasmids, resulting in an opposite spatial organization.

In practice not just one, but multiple plasmid cargo’s are present in the cell. All of them undergoing interactions with the DNA-bound parA. These cargo’s influence each other’s direction because as they get closer the surrounding will be depleted of parA making sure that the cargo’s will turn around to go to a more favorable surrounding (where more parA is present). This influence happens continuously and as a result a regular pattern arises in which the cargoes are aligned along the axis of the bacteria. The regular patterning leads to correct partitioning of low counts during replication.

 

So in a nutshell, Christine introduced us to a replication mechanism, simpler and easier to generate than the conventional system. A mechanism in which there is no need for cytoskeletal structures or motor proteins since the DNA fulfills a mechanical function instead. Only two factors are needed, factor A binding the DNA and factor B ensuring the lifetime of binding of A. This simple mechanism relies on random fluctuations in the chromosomal DNA and stochastic interactions between various plasmids, which leads to a perfectly organized, spatial pattern. Overall I thought the seminar was fascinating, especially because I had never heard of this alternative partitioning machinery before. In addition, Christine incorporated many expressions which we have just covered in our lectures on physical biology of the cell, showing me how the theoretical information is also really applied nowadays in the laboratories.

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How to achieve cellular replication without fail: lessons from bacterial cells.

Speaker: Christine Jacobs-Wagner
Department: Microbial Sciences Institute, Yale West Campus
Subject: How to achieve cellular replication without fail: lessons from bacterial cells.
Location: TU Delft (BN Seminar)
Date: 13-10-2017     

 Author: Nemo Andrea

 The topic of today’s talk was cellular replication, which, in Christine’s opinion, is the ability that separates the living from the non-living. In order to study this process, they study bacterial replication, as bacteria both divide rapidly and do so with high accuracy. The speaker stressed how, if one stops to think about it, achieving such short (20 minutes) division times in a varying environment with in the inherently highly stochastic environment of a living cell, is a truly remarkable feat. Thus, the robustness of bacterial replication and the relative simplicity of prokaryotic systems as compared to eukaryotic model systems make bacteria the candidate of choice for her research.

As there are many topics that can be explored within cellular replication, the speaker decided to focus on a single system that her group had recently done work on. Many bacteria have plasmids, which can contain vital functions for a bacterium, thus requiring efficient segregation of plasmids upon division. While plasmids diffuse throughout the cell and are thus, on average, equally divided among the two halves of a bacterium, suggesting that in the case of cell division no problem should arise, this is not the case. If a plasmid is present in high copy number, the chance of one daughter cell having significantly fewer copies of the plasmid is very small, but if a plasmid has low copy number the probability of one daughter cell ending up with no copies due to low number noise becomes significant. It is thus that bacteria have developed methods to effectively separate the plasmids that are present in low numbers. One could argue that it is not the bacterium driving the evolution of such a system but rather the plasmid itself, as plasmids that effectively do this ensure their survival, but that is really more a matter of perspective than anything else, and beside the point argued in this talk.

The plasmid that the group focused on displayed very curious behavior. The plasmids (in elongated bacteria) are distributed equidistantly along the long axis. The plasmids do diffuse, but stay in roughly the same area over time. It should be apparent that if such a distribution is maintained, the plasmids will be divided equally among daughter cells. As it turns out, this is achieved through a particular variant of the Par system. This variant uses just two proteins: PAR A/B, which are encoded on the plasmid itself. ParB binds to the plasmid, and does this by recognizing specific sequences on the plasmid, ensuring selective binding. ParA, on the other hand, binds to ParB, after which ParB will stimulate the ATPase activity of ParA, which will then unbind from the DNA. ParA also unspecifically binds to the DNA (of the bacterium). They observed (in case of a single plasmid) an oscillation of ParA from one side of the cell to the other, with the plasmid (with ParB) following the ParA signal. They then produced a first model, to test if simple Brownian dynamics of a diffusing plasmid could reproduce such behavior. They found that this was insufficient and concluded from this that a translocating force must be present to create this behavior.

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Figure taken from: DNA-relay mechanism is sufficient to explain ParA-dependent intracellular transport and patterning of single and multiple cargos. ; cropped

The key insight that was missing from the first simulation was the fact that DNA is not static, but moves around in the cell. They determined that the movement of a locus in the DNA can essentially be seen as an elastic harmonic potential, with a force of around 0.04pN. This is a really small force, even by cellular standards. They redid the simulations with this idea incorporated and found that they were able to reproduce the behavior observed in bacteria. The conceptual change is now as follows: The plasmid with ParB is pulled around through its multiple connections with multiple harmonic potentials through ParA. It will then unbind from the ParA in that area and move in a direction (slightly). In the new situation, the plasmid will ‘see’ more ParA in the direction of movement than behind it (which is the previous location, where it made ParA unbind) essentially creating a gradient of forces in the direction of movement. The speed of the plasmid seemed to correspond well with the force of the harmonic potentials. The initial symmetry breaking is caused by the stochastic nature of ATP hydrolysis.

The speaker mentioned another recent model that was developed that may explain similar phenomena, but pointed out that certain parts of this specific system are most likely not reconcilable with that model. The model presented today was also extremely elegant requiring just two components; one that binds to chromosomes, and another that modulates that first component’s lifetime. I quite enjoyed the talk, as Christine was an excellent presenter. While I am generally not easily convinced by simulations, this model seems to make sense both mechanistically and matches real world experiments. We also had a brief session afterwards to ask some questions, which was cut short due to unforeseen circumstances. I do appreciate her taking the time to sit down with us and answer some questions – even the ones not related to the talk.

How to achieve cellular replication without fail: Lessons from bacterial cells

Speaker:     Christine Jacobs-Wagner

Department: Molecular, Cellular and Developmental Biology, Yale University

Subject:       How to achieve cellular replication without fail: Lessons from bacterial cells

Location:    TU Delft (BN)

Date:           13-10-2017

Author: Antoine Rolland

This talk was given by Christine Jacobs-Wagner. She started her talk by saying that most of the work in her lab is based on different phases in the self-replication of the cell. The research is mostly done on bacterial cells, since these are for example simpler than eukaryotic cells. In this talk, she introduced a mechanism that made sure that some low copy number plasmids in bacterial cells are evenly distributed among the two daughter cells after replication.

This mechanism actually consists of a pretty simple biochemical cycle, where two molecules are the most important: parA and parB. Both of these factors are encoded by the plasmids themselves. ParB can bind to the plasmid and cover it almost completely and parA can, after it has bound an ATP,  bind to the chromosomal DNA, which is spread almost all around the bacterial cell. ParB has a high affinity for parA that is bound to the DNA, and in this way the plasmid, which is covered with parB, can be connected to a given loci of the chromosomal DNA where parA is bound. After a small time step, the parB unbinds and the parA loses its ATP, after which the cycle can be repeated. Experiments have shown that this cycle creates a near-perfect distribution of the plasmids  along the long axis of bacterial cells. In case all the plasmids are merged, so only one large plasmid is present, this plasmid oscillates along the long axis over time, following the gradient of the parA concentration. However, in a first model in which only this cycle was introduced, this behavior wasn’t observed. This was caused by the fact that it was assumed that the parA molecules, once they were bound to the chromosomal DNA, would be static. This is not the case, since loci oscillate from its equilibrium position in the cell, because of a certain spring force.

When this part was introduced to the model, exactly the same behavior was observed as in the in vitro and in vivo experiments. Also, changing some parameters, like the amount of parA or the cell shape or size, still produced a nice distribution of the plasmids. What I would be interested in, is which parameters would disturb this distribution, and how this is done.

model parA parB

One of the models of a bacterium cell which was used, with in green the parB covered plasmid and in red parA

What I conclude from this talk is that a simple system, like the biochemical cycle of parA and parB, can still create a very nice distribution of the plasmids before replication. Before this talk, I would think that some really complex processes with a lot of components would be needed to create such a distribution. Also, I think it is nice to see how the model first didn’t work at all, but after one small change, namely having the parA molecules experience a spring force, it made the model work perfectly.

Verrucomicrobia and the intestinal microbiome; the case of the Akkermansia muciniphila

Speaker:  Clara Belzer

Department: Laboratory of Microbiology, Wageningen University, The Netherlands

Subject: Verrucomicrobia and the intestinal microbiome; the case of the Akkermansia muciniphila

Location: BioTechnology department TU Delft

Date: 29-06-2017

Author: Mirte Golverdingen

To broaden my view of the studies done in the Applied Science building I visited a lunch lecture in the BioTechnology department. The microbiologist Clara Belzer from Wageningen University was the speaker and she spoke of the newly discovered Akkermansia muciniphila bacteria. Belzer and her group are interested in anaerobic micro-organisms that can degrade ligases and mucus in the gut. Verrucomicrobia is one of the phyla that lives in the gut, the bacteria Akkermansia muciniphila is one of the Verrucomicrobia and isolated by the group for a quest of new bacteria.

They discovered that the bacteria are very dominant along the mucus gut and an important player in human microbiota composition. The bacteria are a potential maker for healthy intestine and has an extraordinary mucus degrading capacity. The importance of the bacteria is shown in the fact that after a month after birth the bacteria can already be detected in the gut system. This is certainly not the case for all gut bacteria.

The human microbiota changes during life stages and perturbations, this is partly caused by the place of the bacteria on the body. However, over a life time the microbiota can also change due to physiological changes of the body by diet changes. Therefore, the change in microbiota can contribute to or starting diseases. Moreover, some microbiotia do not depend on food, they depend on mucus and mother milk.

Bacteria influence the host cell by a difference in glycal level. This has a microbial benefit; mucosal glycan foraging can solve the problem of the competition for glycans in the gut. For the host, the mucosal microbes can provide resistance against the colonization of pathogens. It directly inhibits the pathogens by production of antimicrobial compounds. Moreover, the depletion of nutrients modulates the immune response of the host.

In the gut, there are 5 major microbiological fila, some are mucus degraders and some are mucus binders. Mucus can be used as the source of growth because mucus glycans can be created by the bacteria. However, there are not only glucose molecules in these mucus glycans, there are also other more ‘awkward’ sugars which are hard to break down. These sugars need to be broken down by enzymes. The Akkermansia muciniphila is adapted to digest mucin, it contains a high number of genes that encodes for enzymes which can degrade mucin. None of all Verrucomicrobia have the same number of enzymes that are able to digest mucin as the A. muciniphila.  So, the A. muciniphila is really adapted to the work of mucin digestion.

So, to find out if A. muciniphila is also present in other animals, Belzer et al. went to the zoo to find the bacteria in mucus of mammals and non-mammals (see figure 1). The A. muciniphila found in these animals differ as the animals have different digestive physiologies, diets and mucus structure. Still, the bacteria were not very different, which is very remarkable. The only difference between the animals A. muciniphila was found in the antibiotic resistance and CRISPR Cas system, as the animals differ in defending to bacteria and viruses in their environment.

So, why are the bacteria so similar? This could be due to a spontaneous infection very recently, so the bacteria were not able to evolve since. The bacteria could also have a very stable genome that does not evolve. Or the bacteria are not specific on the type of mucus.  One genome, however, was different from the rest. The python strain of the A. muciniphila looks really different. Moreover, only a small number of all animals are tested on the A. muciniphila. Therefore, more different genomes could be discovered.

Belzer saw a similarity between organisms and the A. muciniphila system: As soon as the host was on a diet or starving, the A. muciniphila levels are going up. This is similar for all organisms and systems. This could indicate that A. muciniphila could be an obese related bacterium. Belzer moreover, showed that the A. muciniphila works as a growth support of beneficial microbes in the gut. Moreover, when the A. muciniphila levels rises in the gut, organisms were metabolic healthier.

It was very interesting to visit the BioTechonolgy department for this seminar. Some metabolic details were hard to follow for me; however, I was still able to follow her talk. I was surprised by the complex microbiota in the gut. The similarity of the A. muciniphila is very interesting and research for this bacteria in other animals can contribute to the understanding of this bacteria.

Honours11

Figure 1: Akkermansia muciniphila is universally distributed in intestinal tracts all over the animal kingdom. (a) Phylogenetic tree indicating the position of A. muciniphila among selected full-length 16S rRNA clones from mammalian gut samples. Red colored samples derive from human sources. Thermotoga thermarum is used as an outgroup. The tree was generated using the neighbor joining method. Full details and high-resolution information are provided in Supplementary Figure S1. (b) Schematic representation of the tree in (a) with the five different clades their position and similarity to A. muciniphila. (c) Taxonomic tree of mammals generated using iTol webtool from tree of life project using all available sequences from NCBI (Letunic and Bork). Animal silhouettes indicate single species as a representative of that order. When an animal species from the mammalian orders was positive for Akkermansia-like sequences the animal logo belonging to that order is colored red, when it was negative the animal logo is colored gray. No Akkermansia sequences have been reported yet in any of the animals belonging to the mammalian orders depicted in black.

Adapted from: Belzer, C., & De Vos, W. M. (2012). Microbes inside—from diversity to function: the case of Akkermansia. The ISME journal, 6(8), 1449-1458.