Phenotypic Consequences of the Inactivation of a Urea Binding Protein in the Cyanobacterium Anabaena sp.

PCC 7120
Hector Emiko and Scott 1Pasadena City College, Pasadena, CA and 2University of Montana, Missoula, MT
1 Sandoval ,

2 Sano ,

2 Miller

Introduction
Understanding the functional consequences of genetic variation is a central but challenging goal of evolutionary biology. At White Creek, a geothermally-influenced stream in the Lower Geyser Basin of Yellowstone National Park, the thermophilic cyanobacterium Mastigocladus laminosus is a prominent component of the microbial mat community (right). We have recently identified an ~1 kbp deletion of the gene urtA that has attained >70% relative frequency in the White Creek M. laminosus population and therefore appears to be selectively favored (Fig 1A). urtA encodes the urea binding protein component of a high-affinity urea active transporter (ref. 1). By contrast with other nitrogen metabolism genes, however, urtA is strongly regulated under a number of environmental stresses, including phosphate limitation (ref. 2), high light (ref. 3) and low temperature (ref. 4). This suggests that this protein may have pleiotropic effects on other aspects of metabolism. The aim of this pilot project was to begin to develop an understanding of the possible functional consequences of the urtA deletion at White Creek using the genetic model system Anabaena PCC 7120. We compared the growth of wild type Anabaena with that of a mutant for which urtA has been insertionally inactivated (ref. 1; Fig. 1) under a variety of physical and chemical treatments to determine whether the mutant exhibited a growth phenotype under conditions unrelated to urea metabolism.
A
100 Klett Units (Log) 10

Results
B
100 Klett Units (Log) Anabaena sp. urtA Mutant 10 Anabaena sp. urtA Mutant

Conclusion
The urtA mutant appears to have higher fitness than wild type under high light in the presence of combined nitrogen (Fig 2c.; Fig. 3), indicated by a higher yield beginning around hour 89.Whether this is due to the light level per se or is a general effect of cell density remains to be determined. This result suggests that the urtA gene product may have another function(s) in addition to binding urea that is impacted by insertional inactivation. The urtA mutant generally exhibits shorter estimated generation times than wild type under a variety of growth conditions (Table 1), although these differences are not statistically significant. In future experiments, we will extend the duration of growth experiments to determine which phases of the growth cycle, if any, distinguish mutant and wild type performance. We will also sample more frequently to obtain more accurate estimates of exponential growth rate. Ultimately, revealing the phenotypic effects of urtA inactivation may lead to insights both on the mechanisms of UrtA function in Anabaena and the advantages of the urtA deletion in the White Creek population of M. laminosus.

1 -20 0 20 40 60 80 100 120 140 -20 Time (Hours) 100

1 0 20 40 60 80 100 120 140 Time (Hours)

Klett Units (Log)

-N Anabaena sp. 10 -N urtA Mutant +N Anabaena sp. +N urtA Mutant

1 0 20 40 60 Time 80 (Hours) 100 120 140 160

Figure 2. Representative Graph of PCC 7120 Anabaena sp. and urtA Mutant. Fig 2a growth conditions: BG-11 (+N) media. Fig 2b: BG-110 (-N) media. Fig 3c: High light +/- N

Mutant

Wild Type

Figure 3. Representative samples from high light intensity experiment.

References
1. Valladares et al., (2002). Molecular Microbiology 43(3), 703-715. 2. Suzuki et al., (2004). The Journal of Biological Chemistry. 279(13), 13234-13240. 3. Tu et al., 2004. J. Bacteriol. 186:3889. 4. Ehira et al., 2005. Plant Cell Physiol. 46:1237.

Figure 1A. The urt gene cluster found in Anabaena sp. PCC 7120 The C.S3 gene cassette was inserted into urtA to deactivate the expression of the urease transporter. Note M. laminous deletion 100 kb ladder Pes 66+67 Pes 67+urtA.R3 Figure 1B. C.S3 gene cassette amplified to confirm insertion in urtA mutants. Primers used are Pes 66 + 67 and Pes 66 + urtA.R3.

Table 1. Doubling times SE of exponentially growing wild type and mutant under various treatments.
Doubling Time (Hours)
Condition +N -N +N @ 22C +N #2 -N #2 +N #3 +NP -NHigh Light +NHigh Light

Anabaena sp.
20.0 5.07 43.0 11.67 21.5 2.00 16.9 1.50 21.5 6.76 22.2 2.60 19.02 1.08 28.16 0.32 23.82 1.8

urtA Mutant
18.8 2.70 30.1 1.50 18.8 2.94 16.7 1.48 17.7 3.64 17.36 0.90 17.43 0.77 25.2 2.56 25.29 1.46

Acknowledgments
I would like to the thank Miller lab for their support and guidance during this study. I would like to thank Enrique Flores and Ana Valladeres for providing Anabaena strains. Photo credit: Patrick Hutchins. This research was funded by National Science FoundationResearch Experience for Undergraduates Grant #DBI-1157101.