Temporal metatranscriptomic patterning in phototrophic Chloroflexi inhabiting a microbial mat in a geothermal spring

Abstract

Filamentous anoxygenic phototrophs (FAPs) are abundant members of microbial mat communities inhabiting neutral and alkaline geothermal springs. Natural populations of FAPs related to Chloroflexus spp. and Roseiflexus spp. have been well characterized in Mushroom Spring, where they occur with unicellular cyanobacteria related to Synechococcus spp. strains A and B'. Metatranscriptomic sequencing was applied to the microbial community to determine how FAPs regulate their gene expression in response to fluctuating environmental conditions and resource availability over a diel period. Transcripts for genes involved in the biosynthesis of bacteriochlorophylls (BChls) and photosynthetic reaction centers were much more abundant at night. Both Roseiflexus spp. and Chloroflexus spp. expressed key genes involved in the 3-hydroxypropionate (3-OHP) carbon dioxide fixation bi-cycle during the day, when these FAPs have been thought to perform primarily photoheterotrophic and/or aerobic chemoorganotrophic metabolism. The expression of genes for the synthesis and degradation of storage polymers, including glycogen, polyhydroxyalkanoates and wax esters, suggests that FAPs produce and utilize these compounds at different times during the diel cycle. We summarize these results in a proposed conceptual model for temporal changes in central carbon metabolism and energy production of FAPs living in a natural environment. The model proposes that, at night, Chloroflexus spp. and Roseiflexus spp. synthesize BChl, components of the photosynthetic apparatus, polyhydroxyalkanoates and wax esters in concert with fermentation of glycogen. It further proposes that, in daytime, polyhydroxyalkanoates and wax esters are degraded and used as carbon and electron reserves to support photomixotrophy via the 3-OHP bi-cycle.

Figure 1

Expression of FAP phototrophy genes over a diel cycle. The mean relative expression level (±s.e.) is displayed for photosynthetic reaction center genes pufLMC (dark) and BChl biosynthesis genes (light) for Roseiflexus spp. (red), Chloroflexus spp. (green) and Anaerolineae-like (orange) Chloroflexi. BChl biosynthesis gene expression was the mean expression level of all BChl biosynthesis genes known in Roseiflexus and Chloroflexus spp. genomes, while for Anaerolineae-like Chloroflexi, the mean expression was taken from bchH, bchX, bchY and bchZ identified in previous metagenomic analyses.

Figure 2

Model for integrated TCA and 3-OHP pathways for FAP mixotrophic metabolism. The TCA cycle (blue) operates in the oxidative direction, while the 3-OHP cycle (red) reduces inorganic carbon. Shared steps are in purple, and the glyoxylate bypass is indicated in green. Metabolites indicated in light blue are substrates that can be obtained from outside the cell. G, glycogen; PHA, polyhydroxyalkanoic acids; WE, wax esters.

Figure 3

Models for diurnal (a) and nocturnal (b) central carbon metabolism, hydrogenase and putative nitrogenase activity in Roseiflexus spp. based on transcriptional results. The top panel displays a simplified diagram of Figure 3, where bold arrows indicate the predicted dominant flow of carbon through the 3-OHP/TCA cycles and related pathways. The coloring is consistent with that in Figure 2. The bottom panel shows transcription patterns for relevant genes for these pathways. (a) Transcripts for genes with diurnal transcription patterns, such as malonyl-CoA reductase (mcr) and propionyl-CoA synthase (pcs), were averaged for the 3-OHP bi-cycle (red). Malonyl-CoA mutase and malonyl-CoA epimerase were averaged to indicate the expression of shared components of the TCA and 3-OHP pathways (purple). The remaining genes of the TCA cycle were separately averaged (blue). The mean relative transcript levels for genes involved in the β-oxidation of fatty acids are shown in black. (b) Nocturnally expressed genes are shown as the mean expression values of those encoding subunits of hydrogenase (hydABCD) and the putative nitrogenase (nifHBDK). The mean expression values for genes involved in poly-hydroxybutyrate synthesis/degradation (including multiple paralogs of β-ketothiolase and acetoacetyl-CoA reductase) are represented by 3-hydroxybutanoyl-CoA synthesis. The expression values are also displayed for the single genes wax ester (WE) synthase and polyhydroxyalkanoic acid (PHA) synthase. G, glycogen.