A Promiscuous Phage and Its Illiterate Transcriptase
Bacteroides thetaiotaomicron coats its cell surface with sugar. These complex sugars form a capsule, which interacts with the environment and neighboring microbes. Many bacteria make capsules and often switch between different capsule types. Each capsule type is like a different disguise and like a con artist changes disguises, switching between capsules enables the bacteria to evade host immune systems or avoid infection by bacteriophages. B. thetaiotaomicron genomes can encode 8 different capsule polysaccharides from 182 genes.
But it can take a lot of precious genetic space to encode 180, or even 8, versions of a gene or operon with a single function. So how do phages, with the smaller genomes limited by their capsid size, adapt? Are they left at the mercy of random mutation or is there another way?
In 2004, Jeff F. Miller’s research group published a story in which they stumbled across one answer to this very question. While investigating Bordetella-specific phage, Liu et al. noticed that a small number of phage could always infect Bordetella, even when the target binding molecule, in this case pertactin, was missing. In other words, a consistent but small portion of the phage population could use different molecules on the cell surface to initiate infection.
Figure 1. Negative-stain transmission electron micrographs of the Bordetella phage BPP-1 A) intact virus with the genome containing capsid, tail, and tail fibers with globular ends B) tail and tail fibers alone E) Schematic of the phage. Source.
Curious about why there were always phage capable of infecting Bordetella, regardless of their surfaces, the authors sequenced the DNA of phage targeting three different Bordetella surface molecules. Comparing the 42.5-kilobase genomes against each other, they realized that 90% of the genetic mutations occurred in a span of DNA comprising less than 0.5% of the total genome. This 134-base-pair stretch of DNA was at the end of the gene encoding the major tropism determinant, or mtd.
The protein encoded by mtd is responsible for recognizing and binding the target on bacterial surfaces that the phage use to dock and initiate infection. These proteins determine which bacterial species a phage can infect—in a word, its tropism. While most of the protein is responsible for maintaining structure and interacting with other viral proteins like the tail fiber (Fig.1), the highly variable region (termed VR1) identified by Liu et al. is the portion that interacts with bacterial target proteins such as pertactin (the globular ends of the tail fibers, Fig.1). By changing the DNA in the region of the gene responsible for docking to the bacterial surface, the phage can adapt to infect bacteria without the initial target protein.
But how can a phage restrict such high genetic variability to a segment of DNA? How could a phage initiate and direct mutagenesis to enhance its infectivity?
To answer this question, the authors turned to a couple of other features nearby. Located about 100 bp away from mtd and VR1 was another gene encoding the accessory protein avd, a copy of VR1 whose sequence rarely changed, and a gene that coded for a reverse transcriptase. Reverse transcriptase is an enzyme that builds DNA from an RNA template; this is the opposite of what typically happens in a cell, where RNA is transcribed from a DNA template like the genome.
Figure 2. A schematic of the phage DNA encoding mtd and its downstream diversity generating features. Source.
If researchers deleted either the nonvariant copy of VR1 (termed the template region, TR) or the reverse transcriptase from the phage DNA, the phage lost the ability to mutate the VR1. These phage could no longer infect Bordetella that did not display pertactin. Continuing their studies, the researchers found that the phage reverse transcriptase was using an RNA copy of the TR to generate the DNA in the VR1 region. Peculiarly, all the mutations in the VR1 mapped back to adenine bases in the TR, suggesting that the reverse transcriptase couldn’t read adenine bases and would randomly fill in the blanks with other bases.
This means that when the reverse transcriptase was active, it could use the TR region to replace certain base pairs in the VR1 in a process facilitated by an Avd pentamer. This site-directed mutagenesis often changed the amino acids coded for in those sections of DNA, thereby affecting what proteins or surface molecules the virus could bind. In turn, this altered the range of bacteria it could infect, thus changing the virus tropism without expanding the genome.
This Bordetella bacteriophage isn’t the only microbe to employ a VR1, avd, TR, reverse transcriptase system to introduce site-directed mutagenesis. When Miller’s group examined bacteria and archaea with limited genomes, they found identical systems associated with cell attachment proteins. Other phage and even B. thetaiotaomicron have similar versions of diversity generating systems. This protein diversification tool allows microbes with limited genomes to rapidly adapt to their environments. The adenine-illiterate reverse transcriptase and the resulting promiscuous phage are just another example of Dr. Ian Malcolm’s observation in Jurassic park that “life, uh, finds a way.”