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Reading for this lecture:

• Viollier PH, Thanbichler M, McGrath PT, West L, Meewan M, McAdams HH & Shapiro L. 2004 Rapid and sequential movement of individual chromosomal loci to specific subcellular locations. Proc. Natl. Acad. Sci. USA 101:9257-9262

REQUIRED READING FOR THIS LECTURE:

• Thanbichler M & Shapiro L 2008 Getting organized - how bacterial cells move proteins and DNA. Nat. Rev Microbiol. 6:28-40

Bacterial Cell Biology

The physical organization of bacterial chromosomes and bacterial mitosis

Most textbooks, when they talk about replication and cell division in Bacteria, show a picture of a cell containing a circle inside representing the circular genome, attached at one point to the cell membrane. This circle is replicated, then the two circles are pulled to opposite ends of the cell by movement of the membrane attachment points to the poles of the cell. Then cytokinesis can take place, dividing the cell in two between the two DNA circles:

The problem with this view is one of scale - E. coli cells are 2-3 microns in length, but the DNA circle, at 4 million basepairs, is about a millimeter long. The nucleoid is a mass of DNA strands, and no amount of the movement of the membrane attachment points will detangle or separate the 2 copies of the chromosome. How do Bacteria deal with this physical problem? This isn't well understood, but what's becoming clear is that Bacteria do deal with this problem in many ways like eukaryotes do, with organized chromosomes and some kinds of physical mechanism for organizing and partitioning chromosomes before division. In eukaryotes, we call this mitosis; do Bacteria have some form of mitosis as well? The answer seems to be "Yes"..

The paper today deals with two aspects of this issue: how genes are arranged in the nucleoid, and how they reorganize during DNA replication. The first, the physical arrangement of genes in the nucleoid, had already been looked at a little in E. coli and B. subtilis, but with conflicting results and not at all to this scale.

Caulobacter, however, offers some unique properties that allow much better examination of gene localization in the nucloid:

1. It's easy to isolate swarmer cells from a growing culture, and these cells will grow synchronously when put back into warm culture medium. It other words, you can grow cultures in which all of the cells are at about the same phase of the life cycle.
2. There is a distinct pause (interphase) between rounds of DNA replication in Caulobacter. In other words, each round of replication is distinct. This isn't true in E. coli or B. subtilis, in which new rounds of replication can begin well before the previous round is completed. (This allows E. coli to divide every 30 minutes even though it takes about 50 minutes for the genome to replicate.)
3. Both the stalked and swarmer cells are polarized - the stalk or flagellum (respectively) mark the "old" end of the cell. In addition, the old end of swarmer cells contain the receptors for chemotaxis, so flourescent antibodies against these can be used to label this end of living cells (the flagella can't be seen by light microscopy).

Remember the Caulobacter life cycle:

In addition, Caulobacter can be manipulated genetically almost as easily as E. coli, the complete genome sequence is available, and it's easy to grow.

Viollier PH, Thanbichler M, McGrath PT, West L, Meewan M, McAdams HH & Shapiro L. 2004 Rapid and sequential movement of individual chromosomal loci to specific subcellular locations. Proc. Natl. Acad. Sci. USA 101:9257-9262

Question : How are genes localized in the cell during replication?

This paper really addresses two issues: are genes in Caulobacter (and generally in Bacteria, by extension) just wherever they happen to end up in the nucleoid or are they organized and placed in specific positions, and what happens to the localization of genes during DNA replication (in preparation for division).

The authors use two methods to pinpoint the location of specific genes or chromosomal locations. The standard method is fluorescent in situ hybridization (FISH), but this has limited sensitivity and can only be done on fixed (i.e. dead) cells. The authors only use this method to validate their better approach, the fluorescent repressor/operator system (FROS). This method involves using genetics to put large blocks (arrays) of lac or tet repressor binding sites (operators) randomly in the genome. The location of the these blocks of operators (there's only one block per chromosome) is mapped in each strain. Then a fluorescent protein-tagged repressor is expressed in the cell from a plasmid (these autofluorescent proteins originated in jellyfish, and are common genetic tools these days). The fluorescent proteins bind the operators, and there's so many of them there that they light up as a spot in the cell. By using different versions of the fluorescent protein that glow with different colors (cyan-tetR vs yellow-lacR), they can simultaneously label two genes, usually the origin and one other place.

The authors start out by showing that they can get the same result using either method to pinpoint the locations of 4 genes in the cell. They use swarmer cells, because they're not replicating their DNA. They start out using FISH to show (Fig 1) that ori is always found near the flagellated end of the cell (which they identify using fluoresescent McpA chemoreceptor antibodies) , and 3 other genes, pilA (10 o'clock), pleC (8 o'clock) and podU (7 o'clock) are found sequentially arranged along the axis of the cell. They get the same result with FROS, although here they use the ori (also tagged by FROS) to mark the flagellated end.

Having shown that FROS works, they go on to put FROS tags at 112 different sites around the chromosome, and map the location of the resulting spot relative to the ori end of the cell. Amazingly, the genes form a nice orderly arrangement, with the ori and one end, the ter at the other, and all of the genes arranged along the axis of the cell according to position along the chromosome. The chromosome is not a pile of spaghetti, it's organized just like a eukaryotic chromosome!

But they could have used traditional FISH for that experiment. The real power of FROS is that genes can be watched by video during the life cycle of the cell. It this case (Fig 3) they watch the labeled ori during the first round of replication as the cell transitions from a swarmer to a stalked cell. What they see is that when the ori is replicated, one copy stays put at the original end of the cell, but the other moves quickly (at about the speed of DNA replication) to the other end of the cell.

They go on to show the same thing for a variety of other genes in the chromosome - that they start out in their place along the cell axis, but at the time the replication fork should be passing through, the spot splits into two and they each move to their appropriate location for the two daughter cells. In other words, the closer to the ori the gene is, the closer to opposite ends of the cell the two results spots go.

Notice, however, that they only look at genes/locations in the chromosome on the ori side of things - they say the results from tags on the ter side were difficult to interpret, but this would be interesting.

Recent work shows pretty well that in E. coli, unlike Caulobacter, both the ori and the ter sequence are in the middle of the cell and the right and left arms (hemispheres) of the chromosomes are on opposite sides of the cells. Genes halfway down each arm are at polar opposites of the cell. When the chromosome replicates, each chromosome sits side-by-side in the same (not opposite, as you might think) orientation.

So, there is a lot of similarity between bacterial and eukaryotic chromosome structure and segregation. But there is one big difference; in eukaryotes, DNA replication (S phase) and chromosone segregation (mitosis) are unlinked, but in Bacteria, they seem to be concurrent.

The organization of the bacterial cell

This paper is a great recent review about how bacterial cells are organized and how things get moved around:

Thanbichler M & Shapiro L 2008 Getting organized - how bacterial cells move proteins and DNA. Nat. Rev Microbiol. 6:28-40

Two other things are also peripherally discussed that also also very interesting: the basic structure of the bacterial cytoskeleton (and how it's related to cytokenesis), and cell cycling and it's regulation.

Bacteria do have a cytoskeleton, made up (at least in part) of homologs of the same proteins that make up the eukaryotic cytoskeleton: mreB (actin), ftsZ (tubulin), and crescentin (as an example; intermediate filaments). Both mreB and ftsZ (at least in some Bacteria) form coiled spring-like cytoskeletal structure underneath the cell envelop ; at least in the case of mreB this is a closed loop of filaments:

When the cell is preparing to divide, the ftsZ coil closed up to form a band around the middle of the cell, along with a number of other proteins, to form the division machinery that constricts to divide the cell. Another protein, mreC, forms a similar helical structure (but not in phase with the mreB filaments) in the periplasm that is associated with the cell wall synthesis machinery.

The intermediate filament homologs (e.g. crescentin) seem to be related to directing cell morphology. In Caulobacter, crescentin is concentrated in the inner surface of the curved cells. The filaments relieve tension on the cell wall, and as a result, the cell wall synthesis machinery (which is regulated by tension on the cell wall strands), is inhibited and so the cells grow into a curved shape.

In addition, there are filaments of various kinds that run from one end of the cell to the other, that seem to be involved in trafficking material around and organizing various organellar structures (gas vacuoles, magnetosomes, plasmids, strome, &c).

Questions for thought:

• Do you know of any other developmental cycles in bacteria?
  
  
• Where would you predict markers near ter (or ter itself) to end up after replication in Fig 4? Near the middle, or also at the ends?
  
  
• How would you visualize the process of chromosome segregation taking place in rapidly growing E. coli where replication starts again before the previous round is finished?
  
  
• How would you imagine the chromosomes might be arranged in an organism like Deinococcus or Azotobacter, in which each cell typically contains several copies of the chromosome? What about organisms like Methanococcus that has 3 different chromosomes? How about filamentous organisms like Streptomyces with multiple nucleoids? How would to test your ideas?
  
  
• The motor driving cytokenesis is the ftsZ ring, which it inside the cell and anchored to the cell membrane. Do you suppose this constriction also drives pinching-off of more external structures - the cell wall and outer membrane? How? If not, how might this work?
  
  
• How do you suppose the helical cytoskeletal structures (mreB, mreC) are "divided" during cytokinesis? How about in Bacteria that divide by budding instead of fission?
  
  
• What do you suppose goes on in the skeleton and chromosomes of organism that divide in different orientations, e.g. to produce tetrads?
  
  
• Oh no! What about Gemmata, with its nuclear envelop and divides by budding?
  
  
• How do you suppose chromosomes are organized in the cell in case (very common) of species that have more than one chromosome? How would you test your hypothesis?