Bacterial utilization of creatine in seawater

Dissolved organic nitrogen (DON) is recognized as an important component of the marine carbon (C) and nitrogen (N) cycles. Creatine, a component of the dissolved free amino acid (DFAA) pool, is a known byproduct of metazoan metabolism, and genetic evidence suggests that some phytoplankton may also have the ability to produce creatine. We hypothesized that creatine utilization by marine bacteria is more widespread than commonly assumed. The diatom Thalassiosira pseudonana, for which genome analysis had indicated the potential for creatine synthesis, was used to verify the presence of creatine via liquid chromatography/mass spectrometry (LC/MS) analysis. The phylogenetic breadth of creatine-utilizing bacteria and protists was investigated via a bioinformatics approach. Uncharacterized creatinases found in the genomes of Roseobacter denitrificans Och114 and Roseobacter litoralis Och149 were sub-cloned, hexa-histidine tagged, and expressed in E. coli to confirm their functional annotation. Enzymatic activity assays indicated optima at pH 8.4 and 35 degrees C with Km values of 25 to 27 mM. A field experiment was conducted in the equatorial Pacific, where creatine concentrations were found to range between 19 and 171 nmol N l(-1), with higher concentrations at the surface than at the deep chlorophyll maxi mum (DCM). 15N-tracer techniques were used to measure creatine uptake rates, which were in the range of 0.08 to 0.66 nmol N l(-1) h(-1), and were higher in surface waters than at depth. Overall, these data support the idea that phytoplankton are a potential source of creatine to marine bacteria, and that creatine utilization by marine bacteria might account for a measurable fraction of DFAA turnover in the oceans.