We are interested in understanding how bacteria duplicate and repair their DNA. To examine these processes, we study the model bacterium Bacillus subtilis. B. subtilis is a common Gram-positive soil bacterium that is well developed for genetics, biochemistry, genomics and cell biological approaches. B. subtilis DNA repair and DNA replication pathways show a high degree of conservation with the DNA repair pathways of eukaryotes, including humans. Thus, B. subtilis provides an ideal platform for understanding conserved mechanisms shared across biology using the numerous experimental tools that are available for study in this organism. Our lab is currently studying ribonucleotide excision repair, helicase loading, N6 methyladenosine (m6A) and we are discovering uncharacterized genes that contribute to DNA repair or the DNA damage checkpoint.
Ribonucleotide excision repair
Our work has shown that high levels of ribonucleotides are incorporated into bacterial DNA. To better understand how bacteria cope with and correct ribonucleotides incorporated into chromosomal DNA, we have biochemically characterized the RNase H enzymes capable of removing RNA from the myriad of RNA-DNA hybrids that form in vivo. To understand the contribution of uncorrected ribonucleotides to genome integrity, we have combined mutation accumulation lines followed by whole-genome resequencing experiments to determine the mutational signature in RNase HII defective cells. We found that increased ribonucleotides cause an increase in mutagenesis in a specific sequence context. Our work has identified the DNA resynthesis step that results in mutagenesis. Our multidisciplinary experimental approach provides insight into the mechanisms of ribonucleotide removal from bacterial chromosomes. Our work coupled with a genome-wide mutational analysis resulting from defects in ribonucleotide excision repair has provided insight into how mutations can result in antibiotic resistance.
Randall, J.R., Nye, T.M., Wozniak, K.J. and L.A. Simmons (2019) Okazaki fragment maturation in Bacillus subtilis. Journal of Bacteriology pii: JB.00686-18. doi: 10.1128/JB.00686-18
Randall, J.R., Hirst, W.G. and L.A. Simmons (2018) Substrate specificity for bacterial RNase HII and HIII is influenced by metal availability. Journal of Bacteriology JB.00401-17.
Schroeder,J.W., Randall, J.R., Hirst, W.G. O’Donnell, M.E. and L.A. Simmons (2017) Mutagenic cost of ribonucleotides in bacterial DNA. Proc. Natl. Acad. Sci. USA. Oct 31;114(44):11733-11738. doi: 10.1073/pnas.1710995114.
Discovery of gene function
Understanding the pathways that regulate cell growth and genome integrity remains an important challenge in microbiology. This difficulty is underscored by our recent deep sequencing and genome annotation, which indicates that 46.2% of the B. subtilis genes have no demonstrated experimental function. Identifying the contribution of genes of unknown function, also called y-genes, to processes such as cell growth, DNA replication and repair represents an important challenge. To gain a genome-wide understanding of uncharacterized genes that are important for cell growth and surviving DNA damage we designed a transposon-insertion mutagenesis followed by deep sequencing (Tn-seq) experiment. We used the Tn-seq library to screen for B. subtilis genes that are important for surviving DNA damage induced by three different types of DNA damaging agents. Our screen yielded many uncharacterized genes that showed strong decreases in fitness when carrying transposon insertions. Our results strongly illustrate the power of our ability to find genes of unknown function that contribute to surviving DNA damage and other antibiotic treatments. Three recent papers from our efforts in discovering gene function are listed below.
Burby, P.E. and L.A. Simmons (2019) A bacterial DNA repair pathway specific to a natural antibiotic(in press) Molecular Microbiology111(2):338-353. doi: 10.1111/mmi.14158
Burby, P.E., Simmons, Z.W., and L.A. Simmons (2019) DdcA antagonizes a bacterial DNA damage checkpoint. Molecular Microbiology. Jan;111(1):237-253. doi: 10.1111/mmi.14151
Burby, P.E. Simmons, Z.W., Schroeder J.W., and L.A. Simmons 2018) Discovery of a dual protease mechanism that promotes DNA damage checkpoint recovery PLoS Genetics Jul 6;14(7):e1007512. doi: 10.1371/journal.pgen.1007512 (‡corresponding author)
The regulation of gene expression by N6 methyladenosine
More recently we have become interested in understanding how DNA methylation in the form of N6 methyladenosine (m6A) contributes to gene regulation in bacteria. We have used Pacific Biosciences single-molecule real time (SMRT) sequencing to identify sites of m6A in bacterial genomes. We have completed the analysis with the model bacterium Bacillus subtilis and the important human pathogen Streptococcus pyogenes. In the case of S. pyogenes we found that m6A from a Type I restriction-modification system regulates the expression of a master virulence regulator Mga. In the absence of m6A the expression of the mga gene is turned off causing the Mga regulon to remain in the off state. Mga controls the expression of several genes important for binding human cells and evading the host immune response. S. pyogenes cells lacking m6A fail to adhere to human cells and cause a more robust immune response in animal models. Our current work uses genome-wide protein occupancy experiments and biochemical approaches to determine how m6A causes changes in RNA polymerase association with promoter regions in bacterial cells. A recent paper is listed below.
Nye, T. M., K. M. Jacob, S. R. Dawid, L. A. Simmons, and M. E. Watson, Jr. (2019) DNA methylation from a type I restriction modification system influences gene expression and virulence in Streptococcus pyogenes. PLoS Pathogens17;15(6):e1007841. doi: 10.1371/journal.ppat.1007841.