Bacteriophages — or “phages” for short — are viruses that infect bacteria. Phages are a high priority for exploration at Arcadia. They are extremely under-studied relative to the great richness of biological novelty that they represent, are highly abundant in most ecosystems, and serve as excellent test cases for the development of novel computational and experimental approaches to explore non-model biology.
Phages’ pervasive use of non-canonical nucleic acid chemistries is especially interesting to us. DNA is often represented as a simple four-letter alphabet. However, there are actually way more than four types of DNA nucleotides out there [1]. And while use of non-standard or modified nucleotides is seen across the tree of life, phages in particular display a striking diversity of non-canonical DNA chemistries [2][3][4][5].
Why? Diverse bacterial immune systems have evolved to recognize and destroy phage genomes. This puts extraordinary evolutionary pressure on phages to subvert these defenses. By changing the chemical nature of their DNA through genome modification or use of non-standard nucleotides, phages fortify their genome against bacterial attack [6][7][8][9][10].
Our overarching goal is to discover new and exciting nucleic acid chemistries from bacteriophages in natural ecosystems.
Phages protect their genomes with chemical modifications.
Bacteriophages are vulnerable to a wide variety of bacterial immune systems. Some immune systems work by chewing up the bacterial genome. In response, phages have evolved special, protective nucleic acid modifications that bacterial enzymes can't destroy. We suspect there is a rich diversity of nucleic acid chemistry waiting to be uncovered.
Key questions
What omics tools can we leverage or adapt to learn more about the chemistry of phage nucleic acids in natural ecosystems?
How do we get more phages from the environment into the lab, where their nucleic acids can undergo detailed study?
And at the big-picture level, how can we bridge the gap between laboratory studies of isolated phages and omics-based studies of environmental phages?
Progress
We have started simple, getting our molecular biology and analytical techniques up and running on a couple laboratory-culturable phages with well-characterized DNA modifications. For our first pub, we are sharing some of the techniques we have been using, with the hope that it may be useful to others tackling similar problems.
Isolating and analyzing phage nucleic acids
We are using phage T4 and phage SPO1 as our model non-canonical phage genomes. Phage T4 infects E. coli, and has unusual chemistry at its cytosines, where each cytosine is modified with a glucosyl-methyl group [11][12]. Phage SPO1 infects B. subtilis and has completely replaced thymine with hydroxy-methylated uracil [13][14].
This short pub details phage amplification and concentration, high molecular weight DNA extraction, and HPLC and MS analysis of phage nucleosides:
This pub details a process for phage amplification and concentration, DNA extraction, and HPLC and MS analysis of phage nucleosides. We optimized the approach with model phages known to use non-canonical nucleosides in their DNA, but plan to apply it for other phages.
When we tried applying a similar approach to isolating phage RNA, we hit a few stumbling blocks. We describe these challenges and offer a possible path forward here:
We struggled to isolate enough phage mRNA for HPLC as we searched for new nucleoside chemistries. Ribodepleting the abundant extracted rRNA introduced contaminating DNA, and we were still left with more bacterial mRNA than phage transcripts. We suggest an alternative approach.
We are building out a phage culture collection, by isolating new bacteriophages from cheese microbial communities. We will chemically analyze each of these phage genomes using HPLC and MS to test for nucleic acid chemistries of interest. In parallel, we are using LC-MS/MS to analyze the chemical content of DNA extracted from whole microbial communities and community viromes.
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