Isolation of a Heme-dependent Uncultured Marine Bacterium

Abstract

It has long been observed that most bacterial species do not grow in the laboratory. We previously showed that siderophores can induce the growth of a subset of uncultured bacteria. We reasoned that there are other growth-promoting factors required to cultivate additional unculturable bacteria. Using co-culture techniques on media supplemented with soluble iron to bypass siderophore dependency, we cultivated an isolate from marine sand biofilm which would grow only in the presence of a helper. While Escherichia coli also acts as a helper, neither purified enterobactin (a siderophore produced by E. coli) nor soluble iron induced growth, as expected. We hypothesized that heme, another iron-binding compound from E. coli, may be inducing growth. Purified heme spotted on petri plates spread with the unculturable isolate induced the growth of colonies, while no growth was visible on the control plates. Experimental results suggest that the iron in heme is not responsible for this effect. Heme-dependent bacteria have been identified, such as the human pathogens Haemophilus influenzae and Haemophilus ducreyi. Heme growth dependence has not, however, been reported for marine bacteria. This discovery suggests a surprising growth factor for bacteria from the marine environment and provides a new tool to access microbial diversity.