11: CRISPRs, Enviropig, Brainbow, Lysozyme

CRISPR — a widespread system that provides acquired resistance against phages in bacteria and archaea
Sorek, et al. Nature Rev Microbiol 2008

CRISPRs are clustered, regularly interspaced short palindromic repeats found in bacterial genomes. Together, with a leader sequence and CRISPR-associated (CAS) genes, they believe that this system helps prevent phage infection....P.S. I didn't know phage were 5-10x more numerous than bacteria!

So, yea, they think this is a bacterial homolog (paralog?) of eukaryotic RNAi. They're still working out the details, but there are already patents on this for three applications, namely strain spoligotyping (spacer oligotyping), engineering phage resistance in industrial bacterial strains used to make dairy products, and engineering gene knockdown in bacteria. The cool thing is that because the spacers are numerous and repetitive, you could knockdown several genes with this machinery....but apparently, it's still not proven that it has RNAi function.

CRISPR Interference Limits Horizontal Gene Transfer in Staphylococci by Targeting DNA
Marraffini, et al. Science 2008

So, I think the title says it all, but to go on, they found a clinical strain of Staph. epidermis that has CRISPR loci with part of the nickase gene inside, a gene found in all staphylococal conjugative plasmids (recall conjugation is a form of either intra- or interspecies horizontal gene transfer).
Blah, blah, blah, complicated genetics that I don't really understand right now, and ...oh, what's this?

Apparently, they proved that CRISPR doesn't target RNA - it targets DNA! Also interesting is their concluding few lines:

"CRISPR function is not limited to phage defense, but instead encompasses a more general
role in the prevention of HGTand the maintenance of genetic identity, as with restriction-modification systems.A primary difference between restriction modification and CRISPR interference is that the latter can be programmed by a suitable effector crRNA. If CRISPR interference could be manipulated in a clinical setting, it would provide a means to impede the ever-worsening spread of antibiotic resistance genes and virulence factors in staphylococci and other bacterial pathogens."

Perhaps I'll follow up on this after a careful re-read and class discussion tomorrow morning.

Man, I just can't keep up on here with the amount of science that I take in everyday. I still haven't gotten to write entries about any of my lab work. And just yesterday, I was skimming an article (probably in Nature) about genetically modified animals and the legal implications of their potential approval. They talked about how there weren't too many companies working on it, but that they had developed the Enviropig, whose salivary glands secrete an enzyme that helps reduce the pig's phosphorus waste.(Picture from the UCSD news website)

And in today's Chemical Biology lecture, we discussed how GFP was developed (for which the 2008 Nobel Prize was awarded in Chemistry) as well as its colored variants. While I could go into detail on how it works, I'll just mention that the coolest application I thought was the "Brainbow", where this lab at Harvard used a Cre-recombinase system to label 1000s of murine neuronal cells with varying combinations of CFP, YFP, GFP, and RFP. They claim this will help them dissect the complex network of connections between cells in the brain. Certainly looks pretty : )


Disulfide Isomerization After Membrane Release of Its SAR Domain Activates P1 Lysozyme
Xu, et al. Science 2005

These authors argue about the activate-ability of lysozymes using cysteine-accessibility experiments.

10: Checkpoints, Flagella

Checkpoints: Controls That Ensure the Order of Cell Cycle Events
Hartwell & Weinert Science 1989

This is an older article, but it provides a good framework to think about control of the cell cycle. Here are two initial questions the authors posed:
"What principles does the cell use to establish an ordered pathway of events? Does the existence of such order imply the existence of control mechanisms that enforce order?"

They basically have two models they propose:
One is substrate-product assembly - basically, objects have inherent properties that cause them to come together in the proper way. They cite phage assembly as an example, where the proteins are created simultaneously but require the baseplate to come together first before the outer coating can assemble around it. In this example, the order of the process is controlled by the materials themselves

The other model is subtly different - phage DNA is processed in a matter that depends on a control mechanism. The DNA will not be processed by terminase unless proheads are in place and a special inhibitor is put in place to make sure the system abides by this principle.

So, in the early days of thinking about the cell cycle, I guess scientists were unsure which was true (although it seems so obvious now). But, they were able to use chemical treatment and special mutants to deduce things. They argue that, although you can't prove it for certain, if these types of treatments relieve the dependence relationship, then there's most likely a control mechanism in place. I wonder if they were the first people to coin "checkpoint". Anyway, they go through a few examples of checkpoints, namely how mitosis depends on DNA replication, how replicons depend on each other, how mitosis is dependent on cell growth, how spindle pole replication is dependent on DNA replication, etc. They also mention that they observe embryonic stem cells to be different from somatic cells in these areas of control, and speculate on why that would be - are they sacrificing fidelity for quicker division? They also mention the SOS response, which I will get to probably in the next entry.

So, after class discussion, I no longer felt that I understood the difference between the two models - I thought the presence of an extrinsic sensor/regulator was the key part - yet there are some systems in which the lines start to blur - for example, SlmA binds to nucleoid DNA and prevents FtsZ activity - it's both the sensor and the executioner.

Sensing Structural Intermediates in Bacterial Flagellar Assembly by Export of a Negative Regulator
Hughes, et al. Science 1993

Background: In this paper, they talk about how flagellar assembly is grouped into 3 classes of genes: the early and middle genes are responsible for assembling the basal body and hook, which are attached together and anchored in the membrane. These genes induce the expression of the late genes that build off of the hook by exporting flagellin, hook-associated proteins (HAPs) and the Cap protein. These middle genes affect expression of late genes through encoding an alternate sigma factor (fliA, sigma 28), but if there's a defect in assembly, flgM will negatively modulate this activity.

Results: They come up with a valid model, where if the base of the flagellum is correctly set up, it can export flgM, thus lifting the repressiong of fliA and corresponding late genes that can finish assembling the flagellum. What's interesting is that normally fliA and flgM exist in equilibrium when flagella are not being assembled, but if the flagellum gets sheared, it allows for flgM export and thus rebuilding. They came to this conclusion through a few experiments, namely analyzing spent media for exported flgM, analyzing motility, and determining whether fliC (flagellin) can get exported too. They use some mutants in these experiments, either defective in some of the flagellar components or with a lacZ fusion to the flgM that they hypothesized clogged up the export (though they don't really prove this is what's happening, this paper has yet to be disproven).

A Molecular Clutch Disables Flagella in the Bacillus subtilis Biofilm
Blair, et al. Science 2008

So, bacteria switch between being motile and burying themselves in biofilms, but how is this coordinated? I mean, you wouldn't want to just start lopping off your flagella when you're getting ready to settle down - they're expensive and take a lot of time/effort to make again.

So, these two processes are alternately controlled by the transcription factor SinR in B. subtilis, which represses the eps (Extracellular polysaccharide) operon. Mutants in sinR are nonmotile - but are the flagella just getting stuck in the sticky goo secreted?

No, sinR epsH mutants don't make EPS and are still stuck. But, after searching for suppressors of the nonmotile phenotype, they found mutants in EpsE, a family II glycosyltransferase. And when they continued to look for suppressors of suppressors, they found mutations in FliG, the protein that makes the C-ring at the base of the flagellum. This protein is responsible for transducing the rotational movement generated by MovA/MovB (yes, that's right, flagella rotate because of the proton motive force conducted through these channels, anchored to the peptidoglycan skeleton) to the flagellum. So, they wondered whether EpsE was acting either as a brake, to stop the flagellum from rotating, or if it was acting like a clutch and detaching the flagellum from the motion-generating apparatus. After immobilizing bacteria and inducing expression of EpsE, they measured that flagella were not rotating as normal, but they weren't locked in position - they still underwent Brownian motion. Thus, EpsE serves as a clutch!

Regulated Pole-to-Pole Oscillations of a Bacterial Gliding Motility Protein
Mignot, et al. Science 2005
So, there are all kinds of different ways that bacteria can move, some of which are still not understood. Some bacteria use pili-directed movements, where they shoot out a pilus kind of like a grapling hook, and then try to reel themselves in. One poorly understood way is through gliding.

In this paper, they used FRAP to show that they found another oscillatory protein that goes back and forth, from pole to pole (recall MinD in E. coli), called frzS (they were in Myxococcus xanthus).