Uncultured microorganisms from marine biofilms can be coaxed to grow when they are exposed to natural marine sediment or other bacterial species from the same environment. The research team suspected that some type of natural diffusible molecule stimulates this syntrophy among marine microbes.
The researchers inoculated the uncultured isolate Marinbacter polysiphoniae KLE1104 with strains of Escherichia coli containing deletions for various growth factors to identify metabolites that promote growth. From among three likely candidates- the siderophore enterobactin, the universal quorum sensing factor AI-2, and an autoinducer indole-only the strain missing enterobactin fails to induce growth. "We had no idea what the growth factor would be, and we got lucky using our knockout collection of E. coli mutants," says study leader Kim Lewis, director of Northeastern's Antimicrobial Discovery Center.
Lewis' team also discovered that another strain, Micrococcus luteus KLE1011, is a potent natural helper that promotes the growth of M. polysiphoniae KLE1104 and other uncultured strains in the laboratory. They purified five novel siderophores in media collected from cultures of M. luteus KLE1011. These newly identified siderophores belong to the desferrioxamine class of iron-binding agents, and they all have flanking acyl side chains that make them very hydrophobic. Their unusual chemical structure "probably helps them stay within the biofilm, and they do not easily leak out into the open ocean," Lewis says. Moreover, the five new siderophores individually induce the growth of uncultured M. polpolysiphoniae KLE1104, confirming their role as factors that aid the growth of uncultured bacteria.
The importance of siderophores was further explored by screening uncultured bacterial strains induced by M. luteus KLE1011 in marine environmental biofilms. Six isolates were selected that prove particularly dependent on M. luteus KLE1101 for growth. The six isolates and M. polysiphoniae KLE1104 were treated with 20 commercial siderophores, representing different chemical classes, such as phenols/catechols and oxazolines/ thiazolines. The different uncultured bacterial strains, including Cyclobacterium, Sulfitobacter, and Bacteroidetes, show distinctly different patterns of siderophore dependence. Some siderophores universally induce growth, whereas others act more specifically. The results suggest that siderophores show wide variations in their preference for aiding growth of uncultured isolates.
The multitude of uncultured microbes likely will require other yetundiscovered factors to stimulate growth. Nonetheless, siderophores are a first important step towards solving the 100-year-old mystery of the great plate count anomaly. "It gives us a tool to access biodiversity that has been hidden from us," says Lewis, thus allowing exploration of unknown bacterial metabolites, such as much-needed novel antibiotics.
"Improving our ability to culture difficult micoorganisms promises to increase our understanding of them and verify that bacteria detected indirectly using gene sequencing techniques are truly present in particular environments," says Garth James, medical projects manager at the Center for Biofilm Engineering at Montana State University, Bozeman. For example, 16S rRNA data indicate that chronic wounds contain diverse bacteria that cannot be cultured. "It would not surprise me," says Garth, "to find siderophore- based syntrophy in wounds and across the human microbiome."
Carol Potera is a freelance writer in Great Falls, Mont.