Mid-day photomixotrophy by Roseiflexus spp. and implications for the ¹³ C content of hot spring cyanobacterial mats

Microbial mats inhabiting extreme environments have been studied as modern analogs of stromatolites. Mats in Octopus Spring and Mushroom Spring, Yellowstone National Park, are predominated by unicellular photoautotrophic cyanobacteria ( Synechococcus spp.), which are thought to cross-feed filamentous photoheterotrophic bacteria (mainly Roseiflexus spp.), except under early morning anoxic conditions when Roseiflexus spp. have been shown to fix dissolved inorganic carbon (DIC). Transcription patterns, however, suggest that Roseiflexus spp. may perform photomixotrophy, in which DIC is incorporated together with organic compounds during the daytime. We investigated the roles played by Synechococcus spp. and Roseiflexus spp. in DIC and organic matter uptake in mid-day light and oxic mats. Mass spectrometry was used to show that 13 C-bicarbonate uptake under infrared (IR) light (utilized by anoxygenic phototrophs) or visible-minus-blue light (V-B) (used by cyanobacteria) was about two-thirds and one-third, respectively, of that incorporated in full light. Laser-ablation mass spectrometry analysis demonstrated that 13 C incorporation under V-B light was restricted to the uppermost portion of the mat, whereas 13 C incorporation under IR light was maximal in deeper mat layers. 13 C-acetate, -propionate, -lactate, and -glycolate were incorporated to an equal or greater extent under IR and full light. Incorporation of 13 C into peptides showed that both Synechococcus spp. and Roseiflexus spp. were active in DIC uptake, whereas Roseiflexus spp. exhibited greater uptake of 13 C-organic acids, especially glycolate and lactate, into peptides. Peptides of proteins of the 3-hydroxypropionate pathway were labeled. Thus, Roseiflexus spp. appears to exhibit photomixotrophy throughout the day. IMPORTANCE In these mats, Synechococcus spp. fix dissolved inorganic carbon (DIC) via photoautotrophy using the Calvin-Benson-Bassham cycle, which imparts a 13 C isotopic fractionation (ε = δ 13 C REACTANT - δ 13 C PRODUCT ) of ~20‰ between DIC and photosynthate. However, the mat and DIC carbon isotope ratios suggest a much lower fractionation of ~6.4‰ to 10‰. We previously showed that Roseiflexus spp. fix DIC during the early morning, contributing isotopically heavier organic carbon by using the 3-hydroxypropionate pathway, which has a lower fractionation of ~13.7‰. The results of this study suggest that Roseiflexus spp. incorporates DIC during the day, most likely due to photomixotrophy, thus contributing to an isotopic signature heavier than expected for the Calvin-Benson-Bassham pathway. This likely also applies to other well-studied cyanobacterial mats, where it may cause the isotopic composition of mats containing cyanobacteria to approach that of mats formed exclusively by anoxygenic phototrophs, making it more difficult to distinguish these types of mats in stromatolitic organic carbon.