Molecular Analysis of recA Gene in Rhodobacter sphaeroides

Madhusudan Choudhary (PI), Amber Neal and LeAnn Harrel
Department of Biological Sciences

Rhodobacter sphaeroides is a purple non-sulfur bacteria and its complex genome (two chromosomes and five endogenous plasmids) has been completely sequenced and annotated. The recA gene codes for the recombinase, which is responsible for homologous recombination and regulation of the bacterial SOS response under UV exposure. R. sphaeroides is a model organism to study the role of the RecA protein as it has an expanded range of metabolic capabilities. In addition, its completely sequenced genome reveals ~30% gene duplications, and these duplicate DNA sequences are necessary for the recombination mediated by RecA.

The following two hypotheses were tested in this study: (1) RecA is essential for the survival of R. sphaeroides. (2) RecA is essential for DNA repair after UV-induced DNA damage in R. sphaeroides

<Figure 1: Analysis of PCR Products

Methods: This study employs the construction of an in-frame deletion mutant (ΔrecA) and compares the phenotype and growth patterns between the wild-type and the ΔrecA mutant under different UV exposures. Results and Discussion: We have successfully constructed the ΔrecA strains, and confirmed the recA deletion by amplifying the specific deletion fragment from the mutant strain and subsequent sequencing the PCR product. All 8 ΔrecA strains showed the expected ~1.2 kb deletion fragment on 1% agarose gel (Fig. 1) and each fragment was then purified and subsequently sequenced (data not shown). The wild type strain showed the expected ~2.2 kb DNA fragment (Fig 1).

<Figure 2: Growth patterns of wild type and recA in aerobic condition

The wild type and eight ΔrecA strains exhibited similar growth patterns under aerobic growth condition (Fig 2). This result reveals that RecA protein is not essential for the cell survival of the R. sphaeroides. Result of aerobic growth curve characteristics of the wild type and the mutant strains, and their percent cell survivals under different UV exposures (0, 5, 10, 15, 20, 25, 50 mJ) is shown in Fig 3.

The mutant strain had ~30% less survival than the wild type at 5mJ UV exposure. Both strains had 0% survival at ≥20mJ UV exposure (data not shown). The result suggests that recA gene plays an important role when this bacterium grows under UV exposure. We expect that ΔrecA strains will have reduced expression of the downstream genes involved in the SOS Response.

<Figure 3: Percent cell survival under different UV exposures

Since the recA gene is conserved across all bacterial species, impairing the homologous recombination in R. sphaeroides would allow this bacterium to be used as a key biotechnological tool for cloning foreign genes in R. sphaeroides. Comparison of recA and rad51 (human homolog) functions will further provide a better understanding of the evolution of the SOS response in human cells, and allow DNA repair pathway may be further augmented, and such novel protein products will be used for medicinal applications to cure human skin cancer.