**In-Situ Metatranscriptomic Analyses Reveal the Metabolic Flexibility of the Thermophilic Anoxygenic Photosynthetic Bacterium** *Chloroflexus aggregans* **in a Hot Spring Cyanobacteria-Dominated Microbial Mat**

**Shigeru Kawai 1,2,\*, Joval N. Martinez 1,3, Mads Lichtenberg 4, Erik Trampe 4, Michael Kühl 4, Marcus Tank 1,5, Shin Haruta 1, Arisa Nishihara 1,6, Satoshi Hanada 1 and Vera Thiel 1,5,\***


**Abstract:** *Chloroflexus aggregans* is a metabolically versatile, thermophilic, anoxygenic phototrophic member of the phylum *Chloroflexota* (formerly *Chloroflexi*), which can grow photoheterotrophically, photoautotrophically, chemoheterotrophically, and chemoautotrophically. In hot spring-associated microbial mats, *C. aggregans* co-exists with oxygenic cyanobacteria under dynamic micro-environmental conditions. To elucidate the predominant growth modes of *C. aggregans*, relative transcription levels of energy metabolism- and CO2 fixation-related genes were studied in Nakabusa Hot Springs microbial mats over a diel cycle and correlated with microscale in situ measurements of O2 and light. Metatranscriptomic analyses indicated two periods with different modes of energy metabolism of *C. aggregans*: (1) phototrophy around midday and (2) chemotrophy in the early morning hours. During midday, *C. aggregans* mainly employed photoheterotrophy when the microbial mats were hyperoxic (400–800 μmol L−<sup>1</sup> O2). In the early morning hours, relative transcription peaks of genes encoding uptake hydrogenase, key enzymes for carbon fixation, respiratory complexes as well as enzymes for TCA cycle and acetate uptake sugges<sup>t</sup> an aerobic chemomixotrophic lifestyle. This is the first in situ study of the versatile energy metabolism of *C. aggregans* based on gene transcription patterns. The results provide novel insights into the metabolic flexibility of these filamentous anoxygenic phototrophs that thrive under dynamic environmental conditions.

**Keywords:** filamentous anoxygenic phototroph; microbial mats; hot springs; metatranscriptomics; energy metabolism; carbon fixation
