Utilization of Heme as an Iron Source by Marine Alphaproteobacteria in the Roseobacter Clade

Abstract

The bioavailability and utilization of porphyrin-bound iron, specifically heme, by marine microorganisms have rarely been examined. This study used Ruegeria sp. strain TrichCH4B as a model organism to study heme acquisition by a member of the Roseobacter clade. Analogs of known heme transporter proteins were found within the Ruegeria sp. TrichCH4B genome. The identified heme uptake and utilization system appears to be functional, as the heme genes were upregulated under iron stress, the bacterium could grow on ferricporphyrin complexes as the sole iron source, and internalization of 55 Fe from ferric protoporphyrin IX was observed. The potential ability to utilize heme in the Roseobacter clade appears to be common, as half of the isolates in the RoseoBase database were found to have a complete heme uptake system. A degenerate primer set was designed and successfully used to identify the putative heme oxygenase gene (hmus) in the roseobacter heme uptake system from diverse nonenriched marine environments. This study found that members of the Roseobacter clade are capable of utilizing heme as an iron source and that this capability may be present in all types of marine environments. The results of this study add a new perspective to the current picture of iron cycling in marine systems, whereby relatively refractory intracellular pools of heme-bound iron may be taken up quickly and directly reincorporated into living bacteria without previous degradation or the necessity of a siderophore intermediate.

Direct Heme Uptake by Phytoplankton-Associated Roseobacter Bacteria

“…Previous work suggested that heme uptake is a mechanism by which heterotrophic bacteria remineralize particulate organic iron in the marine environment (17, 21). Phytoplankton cells contain relatively high levels of hemoproteins that are mostly associated with the photosynthetic apparatus (40), and lysed or decaying phytoplankton cells could be a significant source of heme or hemoproteins for nearby bacteria.…”

“…Kanamycin (120 μg/ml) and chloramphenicol (10 μg/ml) were used for selection in plates and liquid cultures to ensure selection of insertion mutants. Iron-limited cultures were grown in a modified PC medium (21) here termed PC+ medium. Briefly, the medium consisted of 1 liter of 0.2 μM filtered north Pacific seawater collected from the Scripps Institution of Oceanography pier, 1 g bacteriological peptone, 1 g casein, 10 mM glucose, 4.7 mM NH 4 Cl, 600 μM KH 2 PO 4 , 50 μM Na 2 -EDTA, 40 nM ZnSO 4 , 230 nM MnCl 2 , 25 nM CoCl 2 , 10 nM CuSO 4 , 100 nM Na 2 MoO 4 , and 10 nM Na 2 SeO 3 .…”

“…Because of its prevalence in marine phytoplankton, heme may be a potentially abundant local iron source for bacteria living in close proximity to phytoplankton. Indeed, one prior study identified a Roseobacter strain associated with the cyanobacterium Trichodesmium that could grow on heme and further identified 19 other Roseobacter genomes with putative heme transport gene clusters (21). Due to the lack of a knockout system for the particular strain, that study was not able to conclusively link growth on heme to a specific set of genes or completely distinguish direct heme transport from extracellular heme processing with subsequent free iron transport.…”

Ecotype diversification of an abundant Roseobacter lineage

“…Whereas a functional heme uptake and utilization system was characterized in Ruegeria sp. TrichCH4B and predicted to exist in 40% of surveyed roseobacters (Roe et al, 2013), it was not found in any DC5-80-3 genomes. Another potentially important adaptive mechanism for microbes inhabiting low iron marine environments is to eliminate ironcontaining proteins, which has been shown in a Prochlorococcus lineage inhabiting iron-limited highnutrient, low chlorophyll (HNLC) ocean regions (Rusch et al, 2010;Thompson et al, 2011).…”

Molecular determination of marine iron ligands by mass spectrometry

“…Some bacteria, such as Roseobacter possess heme uptake systems similar to those used for siderophores, which allows them to directly access heme-bound Fe released from dead cells [Roe et al, 2013]. This strategy is likely important for recycling Fe in the open ocean.…”