Schuch also included earthworm guts in his search due to reading a biography about Louis Pasteur. The 19th-century microbiologist was the first to observe that areas with anthrax-infected carcasses were crawling with earthworms, although no one had actually proved that anthracis lived in these invertebrates. If earthworms do make a good habitat for anthracis, Schuch reasoned that they might also contain viruses that infect this and related Bacillus bacteria.
From their phage hunt, Schuch and Fischetti found eight new viruses, including two that they isolated from earthworm guts. When the researchers exposed a lab strain of anthracis to each of these phages separately, they found that, rather than bursting open and killing the bacteria, as lytic phages usually do, the viruses boosted the survival of their bacterial host: For the infected anthracis, the number of cells in test tube cultures of dirt and water remained constant for at least six months. In contrast, the population of phage-free anthracis dropped in half by 2.5 months, and was nonexistent after six months.
Although all of the phage types gave anthracis a survival advantage, they accomplished this effect through differing means. Three of the eight phages could speed the bacteria's transition from growing cells to spores when conditions were unfavorable for growth—low nutrient conditions or low temperatures (24 degrees Celsius instead of 30).
On the other hand, five of the phages triggered anthracis in nutrient-poor media to form biofilms, which are aggregates of vegetative cells encased in sugar-based matrices. Biofilms are the preferred state for other types of soil bacteria, helping them adhere to surfaces. Schuch and Fischetti found that these phages contain a gene whose protein activates the expression of a cluster of bacterial genes that are involved in growth and in sensing the environment. Unlike other bacteria in the Bacillus genus, however, whose genes get made into proteins, anthracis has a mutation that would normally prevent it from expressing this cluster. Anthracis is "just waiting for phages to turn on these genes," Fischetti says.
So far, the researchers have focused on the effect of one phage at a time—whether spore-promoting or growth- and biofilm-promoting—on laboratory strains of anthracis. But Fischetti says that more than one phage can infect anthracis simultaneously, and the researchers are planning to look at the interplay between different types of phages.
Worming out of detection
In addition to enhancing soil survival, Schuch and Fischetti found that the phages are critical for anthracis to colonize earthworms, which have been a suspected hideout since Pasteur's observation 130 years ago. Similar to how the viruses enhance the bacteria's survival in soil cultures, both spore- and biofilm-promoting phages kept the bacteria alive inside earthworms, without harming the invertebrates, for at least six months. But "if anthrax is in the earthworm and it loses the phage, it goes right out in the soil," where conditions are less favorable for the bacteria, Fischetti says.