Michal Koblížek

Rhizobiaceae serve as classical models for elucidating mutualistic plant-microbe interactions yet they represent a narrow phylogenetic subgroup of Alphaproteobacteria. Studying additional lineages of Rhizobiaceae, we observed broad associations with oxygenic phototrophs beyond land plants, including early branching clades of submerged plants, multicellular and unicellular algae, as well as cyanobacteria. In particular, bacteria of the genus Hoeflea were often affiliated with cyanobacteria or microbial algae, whereas Peteryoungia spp. colonized roots of submerged plants. While both genera were originally described as nonpigmented heterotrophs, our detailed genomic, biochemical and physiological analyses revealed that most strains actually contained genes for anoxygenic photosynthesis. Under oligotrophic, oxic growth conditions, each characterized representative expressed bacteriochlorophyll a-containing functional photosynthetic complexes. Photosynthesis genes shared the highest homology among phylogenetically closest relatives, displaying topologies congruent to cognate house-keeping gene phylogenies, and maintained highly conserved gene synteny across the chromosomes of different species. Together, this indicated a vertical inheritance and long ancestral history of aerobic anoxygenic photosynthesis in Rhizobiaceae rather than multiple recent horizontal transfers. Subsequent time-scale phylogenetic analysis suggested that the last common ancestor of Rhizobiaceae was an aquatic phototroph, with different lineages of Rhizobiaceae consecutively evolving in association with algae, land plants, then later legumes. While aquatic lineages maintained photosynthetic machinery till today, Rhizobia which developed symbioses with land plants either as mutualistic endosymbiosis within root nodules or as plant pathogens, concomitantly lost photosynthetic capability. Based on our results, multiple biotic interactions with diverse oxygenic phototrophs drove the early evolution of Rhizobiaceae.

A novel photoheterotrophic, beige-pigmented, bacteriochlorophyll a-containing strain KR11^T was isolated from Kaprový pond in Třeboň, Czechia. KR11^T cells were Gram-negative, rod-shaped, and motile. The isolated strain grew under photoheterotrophic conditions between 20 and 40 °C (optimum 22–25 °C), at pH ranges from 6.0 to 9.0 (optimum 7.0). It did not require NaCl for growth but tolerated NaCl concentrations up to 1.5% (w/v). No growth was observed under photoautotrophic conditions. Strain KR11^T showed the highest 16S rRNA gene sequence similarity to the type strains of Stagnihabitans tardus CYK-10^T (98.84%), Tabrizicola fusiformis SY72^T (95.95%), and Rhodobacter sediminis N1^T (95.37%). Phylogenetic analyses based on 16S rRNA gene sequences indicated that strain KR11^T clusters within the genus Stagnihabitans in the family Paracoccaceae of class Alphaproteobacteria. The whole-genome sequence of strain KR11^T comprises 4,085,976 bp with a 65 mol% G+C content. Phylogenomic analysis, including core-genome phylogeny, and the low genomic similarity (T at the species level. The distinctive phenotypic traits, chemotaxonomic studies, phylogenetic, and genomic analysis establish strain KR11^T as a novel species within the genus Stagnihabitans. Accordingly, we propose the name Stagnihabitans lacustris sp. nov. KR11^T (=CCUG 74777^T, LMG 31924^T), isolated from fresh water.

Phototrophic Gemmatimonadota represent a unique group of phototrophic bacteria that acquired a complete set of photosynthetic genes via horizontal gene transfer and later evolved independently. Gemmatimonas ( Gem. ) phototrophica contains photosynthetic complexes with two concentric light-harvesting antenna rings that absorb at 816 and 868 nm, allowing it to better exploit the light conditions found deeper in the water column. The closely related species Gem. groenlandica , with highly similar photosynthetic genes, harvests infrared light using a single 860 nm absorption band. The cryo-electron microscopy structure of the Gem. groenlandica photosynthetic complex reveals that the outer antenna lacks monomeric bacteriochlorophylls, resulting in a smaller optical antenna cross-section. The Gem. groenlandica spectrum is red-shifted relative to Gem. phototrophica due to the formation of a H-bond enabled by a different rotamer conformation of αTrp ³¹ in the outer ring. This H-bond forms with a neighboring bacteriochlorophyll and increases the intra-dimer exciton coupling, affecting the exciton localization probability within the rings and increasing exciton cooperativity between the complexes. The functional consequences of the spectral shift, caused solely by a subtle conformational change of a single residue, represent a novel mechanism in which phototrophic organisms adjust their antennae for particular light conditions and enable Gem. groenlandica to grow higher in the water column where more photons are available. IMPORTANCE: The photoheterotrophic species of the phylum Gemmatimonadota employ unique photosynthetic complexes with two concentric antenna rings around a central reaction center. In contrast to other phototrophic species, these organisms have not evolved any regulatory systems to control the expression of their photosynthetic apparatus under different light conditions. Despite the overall similarity, the complexes present in Gemmatimonas phototrophica and Gemmatimonas groenlandica have different absorption properties in the near-infrared region of the spectrum that make them more suitable for low or medium light, respectively. The main difference in absorption depends on the conformation of a single tryptophan residue that can form an H-bond with a neighboring bacteriochlorophyll. The presence or absence of this H-bond affects how the protein scaffold interacts with the bacteriochlorophylls, which in turn determines how light energy is transferred within and between the photosynthetic complexes.

A novel chlorophototrophic bacterium, strain BV2-C ^T , was isolated from a microbial mat (~40 °C) at Rupite hot springs, Bulgaria. Cells were rod-shaped, Gram-negative, with numerous fimbriae/pili, but non-motile. Optimal growth occurred under micro-oxic conditions in the light (20–50 µmol photons m ⁻² s ⁻¹ ) at 45 °C (range 35–50 °C) and pH 7.2 (range pH 5.5–9.0). Bacteriochlorophyll c , bacteriochlorophyll a _P , echinenone and γ -carotene were the major pigments in cells capable of producing chlorosomes and type-1 homodimeric reaction centres. Dense liquid culture appeared greenish-brown, with cells forming small clumps and aggregates. Strain BV2-C ^T grows photoheterotrophically using organic compounds as carbon source (i.e. a mixture of 20 proteinogenic amino acids or peptone) and thioglycolate as reduced sulphur source. Strain BV2-C ^T obligately required branched-chain amino acids ( l -isoleucine, l -leucine and l -valine) and l -lysine for growth. It is closely related to Chloracidobacterium thermophilum and ‘ Candidatus Chloracidobacterium aggregatum ’ with 16S rRNA gene sequence identities of 96.97 and 97.78%, respectively. The genome of strain BV2-C ^T comprises two circular chromosomes of 2,659,040 and 1,000,103 bp (total genome, 3.65 Mb) and G+C content of 59.9 mol%. Phenotypic, phylogenetic and genomic analyses demonstrated that Chloracidobacterium validum strain BV2-C ^T (=DSM 113832 ^T =JCM 39534 ^T ) represents a novel species of the genus ‘ Candidatus Chloracidobacterium’, which we hereby propose as Chloracidobacterium gen. nov. with Chloracidobacterium validum sp. nov. as the type species. Based on phylogenomic analyses, we also propose a new family, Chloroacidobacteriaceae fam. nov., and a new order, Chloracidobacteriales ord. nov., within the class Blastocatellia . We further propose the name ‘ Ca. C. aggregatum ’ for pro-valid publication with the genome sequence of strain E as nomenclatural type.

Intertidal biological mats are highly dynamic ecosystems typically dominated by filamentous cyanobacteria of the genus Coleofasciculus. These primary producers play important roles in primary production, biogeochemical cycling, and coastal protection. 16S rRNA gene profiling of non-axenic cultures has recently revealed an astonishing wealth of associated bacteria. We analyzed the microbiomes of 14 non-axenic Coleofasciculus cultures from nine globally distributed marine sampling sites, representing seven distinct phylogenomic lineages. Metagenome sequencing and binning resulted in 320 metagenome-assembled genomes (MAGs) representing a broad spectrum of “uncultivated” bacterial diversity mostly belonging to Pseudomonadota, Bacteroidota and Planctomycetota. Marinovum algicola, and Roseitalea porphyridii were found in 12 of the microbiomes studied, making them the most common housemates. The complex microbiome of Coleofasciculus sp. WW12 contained seven Planctomycetota MAGs from so far undescribed species, representing inter alia a new family in the order Phycisphaerales and an MAG from a deeply branching sister lineage of all cultivated planctomycetes. The discovery of 36 proteobacterial MAGs with photosynthesis gene clusters (PGCs) and 32 MAGs with proteorhodopsin or xanthorhodopsin operons documented the coexistence with many photoheterotrophic bacteria, indicating that the cyanosphere is a hotspot of phototrophic life. The presence of a PGC-containing Myxococcales MAG (Candidatus Photomyxococcus marinus) is of special interest because it paves the way to investigate photosynthesis in Deltaproteobacteria. In a Mediterranean Coleofasciculus culture, three alphaproteobacterial MAGs were found that have both a xanthorhodopsin operon and the PGC, suggesting that dual phototrophy is not restricted to alpine lakes or glaciers, and can also be found in marine habitats.

Bacterial contractile injection systems (CISs) are multiprotein complexes that facilitate the bacterial response to environmental factors or interactions with other organisms. Multiple novel CISs have been characterised in laboratory bacterial cultures recently; however, studying CISs in the context of the native microbial community remains challenging. Here, we present an approach to characterise a bioinformatically predicted CIS by directly analysing bacterial cells from their natural environment. Using cryo-focused ion beam milling and cryo-electron tomography (cryoET) imaging, guided by 16S rRNA gene amplicon sequencing, we discovered that thermophilic Chloroflexota bacteria produce intracellular CIS particles in a natural hot spring microbial mat. We then found a niche-specific production of CIS in the structured microbial community using an approach combining metagenomics, proteomics, and immunogold staining. Bioinformatic analysis and imaging revealed CISs in other extremophilic Chloroflexota and Deinococcota. This Chloroflexota/Deinococcota CIS lineage shows phylogenetic and structural similarity to previously described cytoplasmic CIS from Streptomyces and probably shares the same cytoplasmic mode of action. Our integrated environmental cryoET approach is suitable for discovering and characterising novel macromolecular complexes in environmental samples.

Bacterial contractile injection systems (CISs) are multiprotein complexes that facilitate the bacterial response to environmental factors or interactions with other organisms. Multiple novel CISs have been characterised in laboratory bacterial cultures recently; however, studying CISs in the context of the native microbial community remains challenging. Here, we present an approach to characterise a new, bioinformatically predicted CIS by directly analysing bacterial cells from their natural environment. Using cryo-focused ion beam milling and cryo-electron tomography (cryoET) imaging, guided by 16S amplicon sequencing, we discovered that thermophilic Chloroflexota bacteria produce intracellular CIS particles in a natural microbial hot spring mat. We then found a niche-specific production of CIS in the structured microbial community using an approach combining shotgun metagenomics, proteomics, and immunogold staining. Bioinformatic analysis and imaging revealed CISs in other extremophilic Chloroflexota and Deinococcota. This Chloroflexota/Deinococcota CIS lineage shows phylogenetic and structural similarity to previously described cytoplasmic CIS from Streptomyces and probably shares the same cytoplasmic mode of action. Our integrated environmental cryoET approach is suitable for discovering and characterising novel macromolecular complexes in environmental samples.

Aquatic bacterial rhodopsin proton pumps harvest light energy for photoheterotrophic growth and are known to contain hydroxylated carotenoids that expand the wavelengths of light utilized, but these have not been characterized in marine archaea. Here, by combining a marine chromophore extract with purified archaeal rhodopsins identified in marine metagenomes, we show light energy transfer from diverse hydroxylated carotenoids to heimdallarchaeial rhodopsins (HeimdallRs) from uncultured marine planktonic members of ‘Candidatus Kariarchaeaceae’ (‘Candidatus Asgardarchaeota’). These light-harvesting antennas absorb in the blue-light range and transfer energy to the green-light-absorbing retinal chromophore within HeimdallRs, enabling the use of light that is otherwise unavailable to the rhodopsin. Furthermore, we show elevated proton pumping by the antennas in HeimdallRs under white-light illumination, which better simulates the light conditions encountered by these archaea in their natural habitats. Our results indicate that light-harvesting antennas in microbial rhodopsins exist in families beyond xanthorhodopsins and proteorhodopsins and are present in both marine bacteria and archaea.

Background: Aerobic Anoxygenic Phototrophic (AAP) bacteria are an essential component of aquatic microbial communities and play an important role in carbon cycling due to their ability to supplement their chemoorganotrophic metabolism with light-derived energy. While most of the previous studies focused on abundance, species composition and seasonal changes of AAP bacteria, their affinity for the particle-attachment did not attract much attention. Similarly, it remains unclear whether the entire AAP community is phototrophically active. This study investigated the seasonal changes in the composition of free-living and particle-attached AAP bacteria in the central Adriatic Sea’s coastal waters using both DNA and RNA puf M amplicon gene sequencing in the particle-attached and the free-living fractions. Results: AAP bacterial abundance grew from 1.27 × 10 ⁴ cells mL ^-1 in winter to 8.30 × 10 ⁴ cells mL ^-1 in summer. The proportion of AAP bacteria was consistently higher in the particle-attached fraction, particularly in spring and summer. DNA and RNA puf M amplicon analyses revealed large differences in activity among the species forming the AAP communities. Additionally, DNA-based assessments underestimated the phototrophic activity of certain genera, demonstrating discrepancies between the gene presence and its functional activity. Conclusions: Our data demonstrated that the expression of phototrophic genes in AAP bacteria is not uniform and largely varies throughout seasons and fractions. The particle-attached fraction harboured more than twice as many active AAP bacteria as the free-living fraction, with seasonal shifts and lifestyle driving changes in the phototrophy gene expression. RNA and DNA libraries revealed discrepancies between total and active AAP bacterial communities, emphasizing the necessity of transcript-based approaches for accurately assessing photoheterotrophic activity in marine environments. The pronounced partitioning of AAP bacterial diversity and activity between free-living and particle-attached fractions indicated the ecological specialization of certain AAP lineages, which may have noteworthy implications for the consumption of particulate organic matter and, ultimately, carbon cycling in coastal waters.