Growing unculturable bacteria

Abstract

The bacteria that can be grown in the laboratory are only a small fraction of the total diversity that exists in nature. At all levels of bacterial phylogeny, uncultured clades that do not grow on standard media are playing critical roles in cycling carbon, nitrogen, and other elements, synthesizing novel natural products, and impacting the surrounding organisms and environment. While molecular techniques, such as metagenomic sequencing, can provide some information independent of our ability to culture these organisms, it is essentially impossible to learn new gene and pathway functions from pure sequence data. A true understanding of the physiology of these bacteria and their roles in ecology, host health, and natural product production requires their cultivation in the laboratory. Recent advances in growing these species include coculture with other bacteria, recreating the environment in the laboratory, and combining these approaches with microcultivation technology to increase throughput and access rare species. These studies are unraveling the molecular mechanisms of unculturability and are identifying growth factors that promote the growth of previously unculturable organisms. This minireview summarizes the recent discoveries in this area and discusses the potential future of the field.

Fig 1

Bringing the environment into the laboratory. Image of a 76-l (20-gallon) aquarium for the incubation of marine sand biofilm bacteria in a simulated environment. The aquarium contains natural seawater, beach sand, flora, and invertebrate fauna from Canoe Beach, Nahant, MA. Operated in the laboratory of Kim Lewis at Northeastern University, the simulated environment is maintained with a filter system, a protein skimmer, a circulator, and regular exchanges of water freshly collected from the beach. This system can be used as an incubation environment, and the sand sediment can be used as a source of environmental bacteria.

Fig 2

Coculture-dependent growth of an unculturable isolate. This petri plate shows a helper strain inducing the growth of a previously unculturable bacterium. A freshwater sediment isolate (a relative of Bacillus marisflavi that was originally isolated from Punderson Lake State Park, Newbury, OH) was diluted and spread evenly over the entire petri plate (R2A medium), and a culture of a helper (a relative of Bacillus megaterium that was isolated from the same environment) was spotted on the plate. The unculturable isolate grows only close to the helper, where a growth factor has diffused into the medium of the plate. Preliminary results indicate that the growth factor is a siderophore (A. D'Onofrio, J. M. Crawford, E. J. Stewart, K. Witt, E. Gavrish, S. Epstein, J. Clardy, and K. Lewis, unpublished data).

Fig 3

Model of mechanisms of coculture helping. Unculturable bacteria are schematically represented in the center of the figure, while known and potential helpers are arrayed around the periphery. Arrows indicate positive growth effects; stopped lines indicate inhibitory growth effects. Dashed arrows and inhibition lines indicate effects caused by as-yet-unidentified factors, while solid symbols indicate known factors. (Top center) In aerobic environments, molecular iron is completely oxidized (Fe³⁺) and is unavailable to cells without specific systems for acquiring it (gray arrow with prohibition symbol). Siderophores from neighboring bacteria bind and solubilize Fe³⁺, making it available to bacteria that cannot grow without this help. (Top right) Hydrogen peroxide (and possibly other forms of reactive oxygen species) can prevent the growth of sensitive bacteria. Helper organisms can protect against this effect by removing the oxidative stress, allowing the growth of the sensitive bacteria. (Bottom right) Helper bacteria can provide amino acids, vitamins, carbon sources, and other common nutrients that are often included in rich laboratory medium. (Bottom left and top left) Depictions of growth factors and stress-relieving effects yet to be discovered.