Tucker Matthews, Elizabeth Hussa , and Heidi Goodrich-Blair

1. -280 -221 lrp gene Lrp promoter lacZgene • lacZgene • lacZgene • lacZgene • lacZgene • lacZgene Transcriptional regulation of Leucine-responsive regulatory protein (Lrp) in Xenorhabdusnematophila Tucker Matthews, Elizabeth Hussa, and Heidi Goodrich-Blair Department of Bacteriology, University of Wisconsin-Madison Conclusions Abstract Background -The 50 base pair region of DNA between the -647 and -597 sites is necessary for Lrp-independent negative regulation (Fig. 4 and Fig. 5). -There is also a lesser amount of Lrp-dependent positive regulation in this region (Fig. 6, promoter construct -647). -The 101 base pair region of DNA between the -597 and -446 sites is necessary for Lrp-independent positive regulation (Fig. 4 and Fig. 5). -The 62 base pair region of DNA between the -397 and -335 sites is necessary for Lrp-independent negative regulation (Fig. 4 and Fig. 5). -The region of DNA downstream of the -335 site is necessary for Lrp-dependent positive regulation (Fig. 6, promoter construct -335). Leucine responsive regulatory protein (Lrp) is a transcriptional regulatory protein found in all bacteria, including Xenorhabdusnematophila (1). In X. nematophila, Lrp is a global regulatory protein that has been shown to be necessary in both the mutualism and pathogenesis segments of the symbiotic life cycle (3) (see Fig. 3). In other bacterial systems, Lrp is known to be negatively autoregulated, so we wanted to test this hypothesis (4, 5). Lrp mutant strains of X. nematophila have shown many defects, including the following: -attenuated colonization of IJ nematodes (2), and diminished outgrowth within nematode host (3) -S. carpocapsae nematodes reared on Lrp mutant lawns produced fewer offspring (3) -delayed growth when shifted from rich to poor nutrient sources (2) -attenuated virulence (2) -defective suppression of insect immune response (3) The bacterium Xenorhabdusnematophila exists in a non-obligate symbiotic relationship with the nematode Steinernemacarpocapsae(see Fig. 2). During colonization, S. carpocapsae in the infective juvenile (IJ) developmental stage are colonized by X. nematophila cells in a region inside of the nematode, known as the receptacle. The IJ nematodes then serve as a vector for the bacteria by entering an insect host and releasing the X. nematophila. X. nematophila then serves as a pathogen in the insect host, by suppressing the host insect’s immune system and releasing toxins, which act to kill the insect. The resulting insect cadaver provides both the bacteria and the nematode with a supply of nutrients for reproduction and growth. Once either space or nutrient availability becomes scarce, this reproduction portion of the symbiotic life cycle terminates, and the bacteria re-associate with the nematodes, leave the cadaver, and set off to find another insect host (1). Leucine-responsive regulatory protein (Lrp) is a global transcriptional regulatory protein that is necessary for both the colonization and pathogenesis segments of Xenorhabdusnematophila’s symbiotic life cycle with the nematode, Steinernemacarpocapsae. This study was done to characterize the regulatory mechanisms behind the expression of Lrp, in X. nematophila. Promoter constructs containing varying lengths of the intergenic region of DNA upstream from the Lrp promoter were made and fused to a lac-Z reporter gene, to determine the effects of the different sections of DNA on Lrp promoter activity. This was done in both a wild type and Lrp mutant background, to determine whether or not the regulation is Lrp-dependent. Our results show areas of both Lrp-dependent and Lrp-independent regulation—both positive and negative—in this regulatory region. Discussion and Future Directions Methods The most notable, and statistically significant, regulation in our target regulatory region occurs in the 50 base pair region between the -647 and -597 sites. This Lrp-independent negative regulation is drastic and undeniable. Our other conclusions are less assured due to certain possible causes of error. First, due to time constraints, each construct strain was assayed in triplicate in two independent experiments, generating only three data points. While this may allow for obvious trends to be inferred, a higher number of replicates would allow for greater exactness in our data. This has the possibility of either reaffirming—with more confidence—or disproving our observed conclusions. Therefore, further experiments, with a higher number of replicates, will be necessary to confirm our less statistically significant conclusions. Another potential source of error stems from the fact that the samples were measured during X. nematophila’s stationary phase. Further studies should be conducted in which the bacteria are in other stages of growth, such as log phase growth. A seventh promoter construct at the -495 site is in the process of being constructed, which will help further characterize the 101 base pair region in between the -547 and -446 base pairs. The regions of DNA that our data indicate are involved in Lrp regulation can be assessed based on known regulatory binding sequences in other bacterial regulatory systems to try and better determine which exact sequences are involved in the binding. This can be performed for both Lrp-independent, as well as Lrp-dependent regulatory mechanisms. Due to the potential for rapid changes in X. nematophila’s environment—such as between colonization and pathogenesis—studying the regulatory systems behind such global regulatory proteins as Lrp can provide critical information into understanding the ability of X. nematophila—and other species of bacteria—to thrive. Adapted from reference 1 The purpose of our experiment was to characterize the transcriptional regulation of Lrp. The region of DNA directly upstream from the Lrp promoter sequence is likely involved in regulation of Lrp expression, so our goal was to get a better idea of both how this occurs (Lrp-dependent or Lrp-independent regulation) and of what the roles are for specific regions of DNA in this upstream area (i.e. a more localized description). To do this, we fused promoter constructs of varying lengths to lacZ, and performed beta-galactosidase assays. Figure 3. The role of Lrp as a global regulator. Through both inhibitory and activating mechanisms, Lrp has been shown to regulate levels of a number of other factors in X. nematophila. This, in turn, results in Lrp being necessary in both the mutualism and pathogenesis stages of the symbiotic life cycle. Adapted from reference 1 Figure 2. Life cycle of Xenorhabdusnematophila. Steinernemacarpocapsae nematodes and X. nematophila have a mutualistic relationship in which they infect and kill an insect host. Results -647 Experiments done on overnight cultures performed in triplicate. This is one representative experiment of two independent experiments. -597 Figure 4. Lrp promoter activity among varying promoter construct lengths in an Lrp mutant background. Using two-tailed t-tests, there were statistically significant differences in Lrp promoter activity between the -647 construct and all of the other 5 constructs (all 5 P values<0.0001). There were also statistically significant differences between the -597 construct and the -446 and -397 constructs (P values of 0.0110 and 0.0017, respectively). Finally, there were statistically significant differences between the -397 construct and the -547 and -335 constructs (P values of 0.0370 and 0.0298, respectively). -547 -446 Literature cited -397 • 1. Herbert, E. E., Goodrich-Blair, H. (2007). Friend and foe: the two faces of Xenorhabdusnematophila. Nature, 5, 634-636. doi: 10.1038/nrmicro1706 • 2. Richards, G. R., Goodrich-Blair, H. (2009). Masters of conquest and pillage: Xenorhabdusnematophila global regulators control transitions from virulence to nutrient acquisition. Cellular Microbiology, 11(7), 1025-1033. doi: 10.1111/j.1462-5822.2009.01322.x • 3. Cowles, K. N., Cowles, C. E., Richards, G. R., Martens, E. C., Goodrich-Blair, H. (2007). The global regulator Lrp contributes to mutualism, pathogenesis and phenotypic variation in the bacterium Xenorhabdusnematophila. Cellular Microbiology, 9(5), 1311-1323. doi: 10.1111/j.1462-5822.2006.00873.x • 4. Wang, Q., Wu, J., Friedberg, D., Plakto, J., Calvo, J.M. (1994). Regulation of the Escherichia coli lrp Gene. Journal of Bacteriology, 176(7), 1831-1839. doi: 0021-9193/94/$04.00+0 • 5. McFarland, K. A., Dorman, C. J. (2008). Autoregulated expression of the gene coding for the leucine-responsive protein, Lrp, a global regulator in Salmonella entericaserovarTyphimurium. Microbiology, 154, 2008-2016. doi: 10.1099/mic.0.2008/018358-0 -335 Figure 1. Schematic of the 6 variable promoter constructs. The promoter for the gene encoding Lrp is 60 base pairs in length, and sits 221 base pairs upstream of the Lrp start codon. 6 constructs were made containing decreasing lengths of the intergenic region upstream of the promoter region in which regulation of promoter binding is believed to occur. All values on the schematic are upstream relative to the gene’s start codon. Note: -647 and -335 constructs are courtesy of Elizabeth Hussa Figure 5. Lrp promoter activity among the varying promoter construct lengths in a wild type background. Using two-tailed t-tests, there were statistically significant differences in Lrp promoter activity between the -647 construct and all of the other 5 constructs (all P values<0.0001). There were also statistically significant differences between the -335 construct and the -547, -446, and-397 constructs (P values of 0.0399, 0.0079, 0.0242, respectively). Figure 6. Comparison of Lrp promoter activity in Lrp mutants versus wild type for each promoter construct. Using two-tailed t-tests, there was a statistically significant difference in Lrp promoter activity between the wild type background versus the Lrp mutant background for the -647 constructs (P=0.0018) and the -335 contructs (P=0.0280). The remaining four constructs did not yield statistically significant differences (-597, P=0.4881; -547, P=0.4007; -446, P=0.2768; -397, P=0.0871)