Lars Behrendt

Endolithic chlorophyll d-containing phototrophs

Cyanobacteria in the genus Acaryochloris are the only known oxyphototrophs that have exchanged chlorophyll a (Chl a) with Chl d as their primary photopigment, facilitating oxygenic photosynthesis with near infrared (NIR) light. Yet their ecology and natural habitats are largely unknown. We used hyperspectral and variable chlorophyll fluorescence imaging, scanning electron microscopy, photopigment analysis and DNA sequencing to show that Acaryochloris-like cyanobacteria thrive underneath crustose coralline algae in a widespread endolithic habitat on coral reefs. This finding suggests an important role of Chl d-containing cyanobacteria in a range of hitherto unexplored endolithic habitats, where NIR light-driven oxygenic photosynthesis may be significant.

Microbial diversity of biofilm communities in microniches associated with the didemnid ascidian Lissoclinum patella

We assessed the microbial diversity and microenvironmental niche characteristics in the didemnid ascidian Lissoclinum patella using 16S rRNA gene sequencing, microsensor and imaging techniques. L. patella harbors three distinct microbial communities spatially separated by few millimeters of tunic tissue: (i) a biofilm on its upper surface exposed to high irradiance and O2 levels, (ii) a cloacal cavity dominated by the prochlorophyte Prochloron spp. characterized by strong depletion of visible light and a dynamic chemical microenvironment ranging from hyperoxia in light to anoxia in darkness and (iii) a biofilm covering the underside of the animal, where light is depleted of visible wavelengths and enriched in near-infrared radiation (NIR). Variable chlorophyll fluorescence imaging demonstrated photosynthetic activity, and hyperspectral imaging revealed a diversity of photopigments in all microhabitats. Amplicon sequencing revealed the dominance of cyanobacteria in all three layers. Sequences representing the chlorophyll d containing cyanobacterium Acaryochloris marina and anoxygenic phototrophs were abundant on the underside of the ascidian in shallow waters but declined in deeper waters. This depth dependency was supported by a negative correlation between A. marina abundance and collection depth, explained by the increased attenuation of NIR as a function of water depth. The combination of microenvironmental analysis and fine-scale sampling techniques used in this investigation gives valuable first insights into the distribution, abundance and diversity of bacterial communities associated with tropical ascidians. In particular, we show that microenvironments and microbial diversity can vary significantly over scales of a few millimeters in such habitats; which is information easily lost by bulk sampling.

Chlorophyll f -driven photosynthesis in a cavernous cyanobacterium

Chlorophyll (Chl) f is the most recently discovered chlorophyll and has only been found in cyanobacteria from wet environments. Although its structure and biophysical properties are resolved, the importance of Chl f as an accessory pigment in photosynthesis remains unresolved. We found Chl f in a cyanobacterium enriched from a cavernous environment and report the first example of Chl f-supported oxygenic photosynthesis in cyanobacteria from such habitats. Pigment extraction, hyperspectral microscopy and transmission electron microscopy demonstrated the presence of Chl a and f in unicellular cyanobacteria found in enrichment cultures. Amplicon sequencing indicated that all oxygenic phototrophs were related to KC1, a Chl f-containing cyanobacterium previously isolated from an aquatic environment. Microsensor measurements on aggregates demonstrated oxygenic photosynthesis at 742 nm and less efficient photosynthesis under 768- and 777-nm light probably because of diminished overlap with the absorption spectrum of Chl f and other far-red absorbing pigments. Our findings suggest the importance of Chl f-containing cyanobacteria in terrestrial habitats.

Biofilm Growth and Near-Infrared Radiation-Driven Photosynthesis of the Chlorophyll d-Containing Cyanobacterium Acaryochloris marina

ABSTRACT The cyanobacterium Acaryochloris marina is the only known phototroph harboring chlorophyll (Chl) d. It is easy to cultivate it in a planktonic growth mode, and A. marina cultures have been subject to detailed biochemical and biophysical characterization. In natural situations, A. marina is mainly found associated with surfaces, but this growth mode has not been studied yet. Here, we show that the A. marina type strain MBIC11017 inoculated into alginate beads forms dense biofilm-like cell clusters, as in natural A. marina biofilms, characterized by strong O2 concentration gradients that change with irradiance. Biofilm growth under both visible radiation (VIS, 400 to 700 nm) and near-infrared radiation (NIR, ∼700 to 730 nm) yielded maximal cell-specific growth rates of 0.38 per day and 0.64 per day, respectively. The population doubling times were 1.09 and 1.82 days for NIR and visible light, respectively. The photosynthesis versus irradiance curves showed saturation at a photon irradiance of E k (saturating irradiance) >250 μmol photons m−2 s−1 for blue light but no clear saturation at 365 μmol photons m−2 s−1 for NIR. The maximal gross photosynthesis rates in the aggregates were ∼1,272 μmol O2 mg Chl d −1 h−1 (NIR) and ∼1,128 μmol O2 mg Chl d −1 h−1 (VIS). The photosynthetic efficiency (α) values were higher in NIR-irradiated cells [(268 ± 0.29) × 10−6 m2 mg Chl d −1 (mean ± standard deviation)] than under blue light [(231 ± 0.22) × 10−6 m2 mg Chl d −1]. A. marina is well adapted to a biofilm growth mode under both visible and NIR irradiance and under O2 conditions ranging from anoxia to hyperoxia, explaining its presence in natural niches with similar environmental conditions.

Microenvironmental Ecology of the Chlorophyll b-Containing Symbiotic Cyanobacterium Prochloron in the Didemnid Ascidian Lissoclinum patella

The discovery of the cyanobacterium Prochloron was the first finding of a bacterial oxyphototroph with chlorophyll (Chl) b, in addition to Chl a. It was first described as Prochloron didemni but a number of clades have since been described. Prochloron is a conspicuously large (7–25 μm) unicellular cyanobacterium living in a symbiotic relationship, primarily with (sub-) tropical didemnid ascidians; it has resisted numerous cultivation attempts and appears truly obligatory symbiotic. Recently, a Prochloron draft genome was published, revealing no lack of metabolic genes that could explain the apparent inability to reproduce and sustain photosynthesis in a free-living stage. Possibly, the unsuccessful cultivation is partly due to a lack of knowledge about the microenvironmental conditions and ecophysiology of Prochloron in its natural habitat. We used microsensors, variable chlorophyll fluorescence imaging and imaging of O2 and pH to obtain a detailed insight to the microenvironmental ecology and photobiology of Prochloron in hospite in the didemnid ascidian Lissoclinum patella. The microenvironment within ascidians is characterized by steep gradients of light and chemical parameters that change rapidly with varying irradiances. The interior zone of the ascidians harboring Prochloron thus became anoxic and acidic within a few minutes of darkness, while the same zone exhibited O2 super-saturation and strongly alkaline pH after a few minutes of illumination. Photosynthesis showed lack of photoinhibition even at high irradiances equivalent to full sunlight, and photosynthesis recovered rapidly after periods of anoxia. We discuss these new insights on the ecological niche of Prochloron and possible interactions with its host and other microbes in light of its recently published genome and a recent study of the overall microbial diversity and metagenome of L. patella.

Reactive oxygen production induced by near-infrared radiation in three strains of the Chl d-containing cyanobacterium Acaryochloris marina.

Cyanobacteria in the genus Acaryochloris have largely exchanged Chl a with Chl d, enabling them to harvest near-infrared-radiation (NIR) for oxygenic photosynthesis, a biochemical pathway prone to generate reactive oxygen species (ROS). In this study, ROS production under different light conditions was quantified in three Acaryochloris strains (MBIC11017, HICR111A and the novel strain CRS) using a real-time ethylene detector in conjunction with addition of 2-keto-4-thiomethylbutyric acid, a substrate that is converted to ethylene when reacting with certain types of ROS. In all strains, NIR was found to generate less ROS than visible light (VIS). More ROS was generated if strains MBIC11017 and HICR111A were adapted to NIR and then exposed to VIS, while strain CRS demonstrated the opposite behavior. This is the very first study of ROS generation and suggests that Acaryochloris can avoid a considerable amount of light-induced stress by using NIR instead of VIS for its photosynthesis, adding further evolutionary arguments to their widespread appearance.

Rapid TaqMan-Based Quantification of Chlorophyll d-Containing Cyanobacteria in the Genus Acaryochloris

ABSTRACT Reports of the chlorophyll (Chl) d-containing cyanobacterium Acaryochloris have accumulated since its initial discovery in 1996. The majority of this evidence is based on amplification of the gene coding for the 16S rRNA, and due to the wide geographical distribution of these sequences, a global distribution of Acaryochloris species was suggested. Here, we present a rapid, reliable, and cost-effective TaqMan-based quantitative PCR (qPCR) assay that was developed for the specific detection of Acaryochloris species in complex environmental samples. The TaqMan probe showed detection limits of ∼10 16S rRNA gene copy numbers based on standard curves consisting of plasmid inserts. DNA from five Acaryochloris strains, i.e., MBIC11017, CCMEE5410, HICR111A, CRS, and Awaji-1, exhibited amplification efficiencies of >94% when tested in the TaqMan assay. When used on complex natural communities, the TaqMan assay detected the presence of Acaryochloris species in four out of eight samples of crustose coralline algae (CCA), collected from temperate and tropical regions. In three out of these TaqMan-positive samples, the presence of Chl d was confirmed via high-performance liquid chromatography (HPLC), and corresponding cell estimates of Acaryochloris species amounted to 7.6 × 101 to 3.0 × 103 per mg of CCA. These numbers indicate a substantial contribution of Chl d-containing cyanobacteria to primary productivity in endolithic niches. The new TaqMan assay allows quick and easy screening of environmental samples for the presence of Acaryochloris species and is an important tool to further resolve the global distribution and significance of this unique oxyphototroph.

In situ metabolomic- and transcriptomic-profiling of the host-associated cyanobacteria Prochloron and Acaryochloris marina

The tropical ascidian Lissoclinum patella hosts two enigmatic cyanobacteria: (1) the photoendosymbiont Prochloron spp., a producer of valuable bioactive compounds and (2) the chlorophyll-d containing Acaryochloris spp., residing in the near-infrared enriched underside of the animal. Despite numerous efforts, Prochloron remains uncultivable, restricting the investigation of its biochemical potential to cultivation-independent techniques. Likewise, in both cyanobacteria, universally important parameters on light-niche adaptation and in situ photosynthetic regulation are unknown. Here we used genome sequencing, transcriptomics and metabolomics to investigate the symbiotic linkage between host and photoendosymbiont and simultaneously probed the transcriptional response of Acaryochloris in situ. During high light, both cyanobacteria downregulate CO2 fixing pathways, likely a result of O2 photorespiration on the functioning of RuBisCO, and employ a variety of stress-quenching mechanisms, even under less stressful far-red light (Acaryochloris). Metabolomics reveals a distinct biochemical modulation between Prochloron and L. patella, including noon/midnight-dependent signatures of amino acids, nitrogenous waste products and primary photosynthates. Surprisingly, Prochloron constitutively expressed genes coding for patellamides, that is, cyclic peptides of great pharmaceutical value, with yet unknown ecological significance. Together these findings shed further light on far-red-driven photosynthesis in natural consortia, the interplay of Prochloron and its ascidian partner in a model chordate photosymbiosis and the uncultivability of Prochloron.

Deep mutational scanning by FACS-sorting of encapsulated E. coli micro-colonies

We present a method for high-throughput screening of protein variants where the signal is enhanced by micro-encapsulation of single cells into 20-30 μm agarose beads. Cells inside beads are propagated using standard agitation in liquid media and grow clonally into micro-colonies harboring several hundred bacteria. We have, as a proof-of-concept, analyzed random amino acid substitutions in the five C-terminal β-strands of the Green Fluorescent Protein (GFP). Starting from libraries of variants, each bead represents a clonal line of cells that can be separated by Fluorescence Activated Cell Sorting (FACS). Pools representing collections of individual variants with desired properties are subsequently analyzed by deep sequencing. Notably, the encapsulation approach described holds the potential for high-throughput analysis of systems where the fluorescence signal from a single cell is insufficient for detection. Fusion to GFP, or use of fluorogenic substrates, allows coupling protein levels or activity to sequence for a wide range of proteins. Here we analyzed more than 10,000 individual variants to gauge the effect of mutations on GFP-fluorescence. In the mutated region, we observed virtually all amino acid substitutions that are accessible by single nucleotide exchange. Lastly, we assessed the performance of biophysical protein stability predictors, FoldX and Rosetta, in predicting the outcome of the experiment. Both tools display good performance on average, suggesting that loss of thermodynamic stability is a key mechanism for the observed variation of the mutants. This, in turn, suggests that deep mutational scanning datasets may be used to more efficiently fine-tune such predictors, especially for mutations poorly covered by current biophysical data.