# Presentation: 184924

## SENSING THE ENVIRONMENT:

- Deciphering the roles of two-component systems in *Desulfovibrio vulgaris* (Hildenborough)

- _Alyssa Redding_,  Aindrila Mukhopadhyay, Vincent Martin, Jay D. Keasling
- Department of Chemical Engineering, University of California, Berkeley

**Stress Response in Bacteria**
- In bacteria, two-component systems are comprised of Histidine Sensor Kinases (HSKs) and Response Regulators (RRs). Such systems serve as the primary means for response to changes in the environment.1
- The genome of *D. vulgaris *contains several Open Reading Frames (ORFs) which have been annotated as HSKs.2 This set of proteins provides an ideal system to start examining stress response.
- To enable evaluation of stress response in *D. vulgaris*,* *a library of HSK knock out mutants would be very valuable. A high throughput method has been designed to generate such a library.

- The gene of interest is amplified using PCR primers that generate 15 base pair overhangs.3
- Complimentary overhangs are created in a vector suitable for knockout mutagenesis.
- The gene of interest can now be cloned independent of restriction endonuclease sites or enzymatic ligation.
- A second round of PCR and cloning is used to replace an internal sequence of the gene with an antibiotic resistance cassette.
- Gene disruption is coupled with the introduction of unique UP and DOWN tags selected for each gene.4
- Using the resulting suicide vectors for homologous recombination, a library of tagged knock-out mutants will be generated for *D. vulgaris*.

- Several candidate HSKs have been selected to develop the methods and evaluate the efficiency of the high throughput strategy.
- For example the TIGR ORF05659, a HSK, is annotated as LytS due to its similarity to LytS found in several other bacteria. In *S.aureus* the knock out mutant of LytS showed the striking phenotype of altered cell structure, which was clearly visualized via microscopy*.*5

- Surveying the HSK proteins in *D. vulgaris *has provided a starting point for experimental evaluation.
- Scrutinizing the conserved domains, comparisons with similar HSKs in other bacteria, and information from operon predictions will also provide valuable insights into two-component signal transduction in *D.vulgaris.*

- The *D.vulgaris* genome has 74 ORFs annotated as histidine kinases. Shown above are 13 examples of HSKs that contain the expected conserved motifs. Of the HSKs shown, 10 are hybrid HSKs, containing a C-terminal receiver domain in addition to the HSK domain. Most HSKs present in *D. vulgaris* appear to be hybrid HSKs.

- Acknowledgements
- The list of ORFs annotated as HSKs was obtained from Eric Alm, LBNL.
*D.vulgaris* genomic DNA used in the PCR experiments to date was a gift from Swapnil Chabra, SNL.
- Professor Judy Wall, for all her advice and providing strains and plasmids for use in our strategy.
- DOE for funding*.*

- References
- Hoch, J. A. and Silhavy, T. J. 1995. Two-Component Signal Transduction. American Society for Microbiology, Washington D.C.
*D. vulgaris* annotations posted on Virtual Institute for Microbial Stress and Survial (VIMSS) website. http://vimss.lbl.gov.
- Tillett, D. and Neilan, B. A. (1999) Enzyme-free cloning: a rapid method to clone PCR products independent of vector restriction enzyme sites. Nucleic Acids Res. **27:**e26.
- Winzeler, E. A. *et al.* (1999) Functional Characterization of the *S. cerevisiae* Genome by Gene Deletion and Parallel Analysis. Science. **285:**901-906.
- Brunskill, E. W. and Bayles, K. W. (1996) Identification and Molecular Characterization of a Putative Regulatory Locus That Affects Autolysis in *Staphylococcus aureus*.  J. Bacteriol. **178:**611-618.

**A High Throughput Knock Out Method**

**Candidate HSKs**

- Using high thoughput assays, the mutant library can be used to understand stress response in *D. vulgaris*:
- The unique UP and DOWN tags incorporated into each mutant will allow microchip technology to be utilized. The mutants can then be analysed separately, in parallel, or even in head-to-head competitions.
- Proteomic research, using techniques such as the Isotope-Coded Affinity Tag (ICAT), will allow comparison of stressed and unstressed samples, or between a mutant and the wild type.