Siderophores from neighboring organisms promote the growth of uncultured bacteria

The majority of bacterial cells in an environmental sample will not grow on synthetic media and are known as "unculturable". As high as 99.9% of total cells counted by microscopy will not form colonies on standard Petri dishes. This phenomenon is known as the Great Plate Count Anomaly and is a significant unsolved problem in microbiology. The data presented in this study tests the hypothesis that some unculturable bacteria fail to grow on synthetic media because they are lacking specific growth factors from neighboring species, which signal a suitable environment to grow. In order to test this hypothesis, environmental bacteria were isolated from intertidal sediment and examined for helper-dependent relationships where an unculturable bacteria would only grow in the presence of a culturable helper species. Several such pairs were identified and a model unculturable, M. polysiphoniae KLE1104, was chosen for further study with model helper strain, M. luteus KLE1011, isolated from the same environment. Filtered spent supernatant of M. luteus KLE1011 as well as E. coli was capable of inducing growth of M. polysiphoniae KLE1104. E. coli knockout strains deficient in production of the iron chelating siderophore, enterobactin, were unable to induce growth. Testing purified enterobactin confirmed the compound was necessary and sufficient to induce growth of macrocolonies of M. polysiphoniae KLE1104. Five siderophores were purified from M. luteus KLE1011, and each was capable of inducing growth of the unculturable. Structure elucidation of the siderophores revealed that they were novel acyl-desferrioxamines with variable terminal modifications to increase hydrophobicity. Several species were then isolated from the same environment, which were dependent on M. luteus KLE1011. Six of these isolates along with two others previously isolated from the same environment were tested for growth induction by a panel of 16 commercial siderophores and the five M. luteus KLE1011 siderophores. Each unculturable isolate was helped by a different set of siderophores, ranging from 6 to all 21 siderophores tested. This growth dependence on a varying set of siderophores suggests a strategy of only growing in the presence of a suitable environment, which is signalled by the presence of siderophores from appropriate neighbors.