Jacob Erickson and Dr. Joel Griffitts, Microbiology and Molecular Biology

Bacterial infection has been fought against successfully since the discovery of penicillin, and since then other antibiotics. Despite this, bacterial strains continue to mutate and find resistance to commonly used antibiotics. Gaining an understanding the genetic process of infection within bacteria will aid in the fight of harmful bacteria and the use of symbiotic bacteria.

The bacteria used in the this study was Sinorhizobium meliloti. In order for bacteria to go into the infection mode it must first receive a stimulus from the outside environment. This stimulus then causes a chain of events to happen within the cell on a molecular level.

The model used for this experiment involves three separate proteins within the Sinorhizobium meliloti bacteria.
1. FeuQ: believed to be a sensor kinase.
2. FeuP: believed to be a transcription factor.
3. NdvA: proven to be required for bacterial infection.

It has been proposed that the protein FeuQ is a sensor kinase that is positioned within the inner membrane of the cell. This sensor kinase receives a phosphate group and then transfers this phosphate group onto a response regulator protein called FeuP. This FeuP protein in turn binds to the promoter region of ndvA which causes transcription (see Figure 1).

This two component system is believed to be part of a symbiotic relationship that Sinorhizobium meliloti (Rm1021) and alfalfa have with each other. The bacteria infect the roots of the plant causing pink nodules to form, and in return the plant received needed nitrogen from the bacteria. Infection is measured by placing the bacteria on alfalfa plants and monitoring if pink nodules form on the roots. If pink nodulation occurs then successful infection has taken place. If the nodules are not pink, or if no nodules grow at all then the organisms did not reach symbiotic behavior, despite the fact that some infection may have occurred.

Through experimentation FeuP was found to be required for proper infection and symbiosis.
In order to verify that FeuQ was necessary for symbiosis the feuQ gene sequence was removed from the DNA sequence. When the mutated form of Rm1021 was placed on the alfalfa roots normal pink nodulation occurred. This showed that FeuQ was not necessary for healthy symbiosis. However this did not disprove that FeuP may still be acting as a response regulator for FeuP.

Efforts were shifted from the relationship between FeuQ and FeuP to FeuP and NdvA. The FeuP protein was purified through a series of steps. The feuP genetic sequence was copied and placed on a plasmid directly downstream of a sequence containing six histidines and three glycines, also known as a 6-His tag (see Figure 2). These histidines are used as a purifying agent when the proteins containing the 6-His tag are flushed through a column containing nickel. The 6-His tag binds to the nickel, allowing the other protein’s and substances to pass through. After washing the column to remove any residual material, the 6-His tag is competed off the nickel. The resulting flow through is a purified protein. Because the 6-His tag is so small, the protein is left with minimal change to it. The glycines within the 6-His tag are used to allow the 6-His tag flexibility to find and bind to the nickel.

For the experiment, the cells containing the new 6-His and FeuP protein were grown up to saturation overnight. The saturated culture was then diluted down ten-fold and allowed to grow for four additional hours. This four hour culture was then pelleted down for future use. The pelleted cells were then lysed open using lysing buffer as well as sonication. The lysed cells were then pelleted down once again and the resulting supernatant was separated from the pellet. This supernatant was used for the protein purification.

The supernatant was washed through a nickel column as was mentioned before. Currently, efforts must still be made to ensure the removal of small amounts of protein contamination as can be seen (see Figure 3).

Future plans to map this molecular model will include verifying where FeuP binds to the DNA. FeuP is thought to bind to the promoter region of ndvA and is thought to be a response regulator for the NdvA protein. This protein has been found to be necessary for cell motility and cell health and growth.