Lyndsey Schutte

Plasmid/all4312 experiment

Introduction:

Cyanobacteria are amongst the few bacteria that have cellular differentiation. They only have two main cell types as opposed to most eukaryotes who have thousands. Their simplicity allows them to be highly valuable organisms for the study the mechanisms of how cells differentiate. If people can learn how these bacteria differentiate, then they could apply it to human and other eukaryotes that are too complex to understand at this time.

The two cell types are the vegetative cells and the optional nitrogen fixing heterocysts. Photosynthesis takes place in the vegetative cells, producing sugar, which is distributed along all the neighboring cells. In the case of fixed nitrogen deprivation the bacteria will produce heterocysts at regular intervals, usually around ten cells apart, along the filament. These cells will be are able to fix nitrogen from N₂ and provide the other vegetative cells with needed nitrogenNH2. Heterocysts are physically distinguished by a cellular envelopes, a larger, somewhat rounder structure, and two polar endsplugs. Chemically, they contain a whole set of different enzymes leading to the fixation of nitrogen. The specialized envelope is necessary to keep out the oxygen produced by the vegetative cellπs photosynthesis, since nitrogenase, one of the main enzymes that fixes nitrogen, is inactivated by oxygen.

How do these cells know to differentiate when nitrogen levels drop to zero? NtcA is one of the main proteins that monitors the level of nitrogen. It is a transcription activator that induces genes that code for many of the enzymes that deal with the fixing of nitrogen. (Buikema and Haselkorn, 2001) It binds to the consensus sequence TGT-(N_9-10)-ACA. The NtcA-dependant promoters are active in nitrate-containing media and repressed in ammonia-containing media. (Buikema and Haselkorn, 2001)

HetR is the main gene that controls the heterocyst differentiation. Without it, there is no heterocyst formation, and over expression of hetR causes supernumerary heterocysts. (Buikema and Haselkorn 1990) HetR binds to one of its own three promoters, giving HetR positive feedback. If NtcA is mutated, then HetR is not expressed, so NtcA is somewhere upstream of HetR. However, the hetR promoter does not have the ntcA binding site. (Buikema and Haselkorn,1990)

What is the link between NtcA and HetR? In the simplest case, the gene should have an NtcA binding site and be a transcription factor in order to help activate HetR. A gene named all4312 has been found that meets those two requirements. It has a similar sequence to other genes that regulate transcription and it has what appears to be a NtcA binding site. When the all4312 gene was knocked out to see how or if it played a role in the cell differentiation, the mutation turned out to be a lethal and all the cells died.

In this lab, a conditional mutation will be created so the bacteria can stay alive long enough to be studied and so the effects of the gene can be determined at multiple periods along the life cycle. The lac promoter and repressor can make a very efficient and simple switch for turning a gene on and off, and so that is the one we have decided to use. It works by repressing DNA polymeraseπs ability to bind to the promoter between the two lac operators in the presence of lactose. When lactose is removed, the lac operon can no longer bind to its operators and so the DNA polymerase can bind to its promoter and transcribe the gene. (Muller et al., 1996) We will create a plasmid with the all4312 gene placed backwards behind the promoter. This way the RNA that is transcribed when lactose is present will base pair with the normal all4312 gene and prevent its translation.

Methods:

The plasmid VCU22 that will be placed inside the cyanobacteria is made of a combination of two different plasmids, VCU20 and VCU21. VCU20 comes from the plasmid PUR361, which is cut with KpnI and BamHI to allow Ptc, the sequence of DNA that contains the promoter and lac operons, to be cut with KpnI and BglII and inserted into PUR361. The VCU21 plasmid is a combination of PUR11, cut with ClaI and NsiI, and lacI, which is cut with AccI and PstI. The enzyme AgeI will be used to cut and piece together VCU20 and VCU21.

[insert pic of plasmid]

After VCU22 is created, all4312 will be inserted into the region after the promoter, and placed into Anabaena. The developing of the plasmids will take place in E. coli due to their significantly faster life cycle.

The bacteria will be grown on nitrogen-free medium, and lactose will be introduced to the bacteria before nitrogen deprivation, and at regular intervals after nitrogen deprivation begins. We will check for results both by looking for phenotypic changes and monitoring levels of HetR by northern blot.

Possible results and implications:

If no heterocysts are formed and HetR is not strongly expressed then this would be strong evidence that all4312 is in the pathway between NtcA and HetR. If no heterocysts are formed or if they are formed in a different pattern, but HetR is expressed normally, that implies that all4312 is in pathway for heterocyst differentiation, but it is after or unrelated to HetR, and assays can be performed to see what proteins have changed between the mutant and the normal to see what it is affecting.

If heterocyst development continues unaltered in any way, the mutation can still allow the research team to induce mutations earlier and earlier in development till it is understood where in development the protein is needed. If we induce the mutation and vegetative cells and heterocysts both die, the lactose can be given in lesser concentrations so that only some of the all4312 RNA is bound and the cell will have enough of the protein to survive but have enough of a lack that we can assess its role in the cell.

References:

Buikema, W. J., and Haselkorn, R. (2001). Expression of the Anabaena hetR gene from a copper-regulated promoter leads to heterocyst differentiation under repressing conditions. Proc. Natl. Acad. Sci. 98, 5, 2729-2734.

Buikema, W. J., and Haselkorn, R. (1990). Characterization of a gene controlling heterocyst differentiation in the cyanobacterium Anabaena 7120. Genes Dev. 5, 321-330.

Muller, J., Oehler, S., Muller-Hill, B. (1996). Repression of lac Promoter as a Function of Distance, Phase and Quality of an Auxiliary lac Operator. J. Mol. Biol. 257, 21-29.