Structural colors (SC), generated by light interacting with nano-structured materials, are responsible for the brightest and most vivid coloration in nature. Despite being widespread within the tree of life, there is little knowledge of the genes involved. Partial exceptions are some Flavobacteriia in which genes involved in a number of pathways, including gliding motility and polysaccharide metabolism, have been linked to SC. A previous genomic analysis of SC and non-SC bacteria suggested that the pterin pathway is involved in the organization of bacteria to form SC. Here, we focus on moeA, a molybdopterin molybdenum transferase. When this gene was deleted from Flavobacterium IR1, the knock-out mutant showed a strong blue shift in SC of the colony compared to the wild-type. The moeA mutant showed a particularly strong blue shift when grown on kappa-carrageenan and was upregulated for starch degradation. To further analyze the molecular changes, proteomic analysis was performed, showing the upregulation of various polysaccharide utilization loci, which supported the link between moeA and polysaccharide metabolism in SC. Overall, we demonstrated that a targeted approach, modifying a single gene identified by genomics, could change the optical properties of bacteria.

Interspecies interactions shape microbial communities; this is central for microbial ecology. Cellulophaga lytica PlyA2 is a marine flavobacterium, which glides over surfaces and forms ordered, structurally coloured colonies, which display angle-dependent reflection of light. Pseudosulfitobacter pseudonitzschiae SW is an apparently nonmotile, nonstructurally coloured marine bacterium. Here, we aim to understand the interaction of both strains at cellular, genomic, optical, and proteomic levels. Cocultivation on agar showed that P. pseudonitzschiae uses gliding C. lytica to spread by microbial hitchhiking in which Pseudosulfitobacter appears to "surf" on basal layers of motile Cellulophaga. This dispersal mechanism was found to be often beneficial for P. pseudonitzschiae, which could maximally expand its population up to 350-fold relative to monoculture. Coculture was often of limited benefit for C. lytica, only in extended cultivation on rich medium was the presence of P. pseudonitzschiae detrimental to its viability. The proteome of P. pseudonitzschiae was strongly impacted by the association with C. lytica. Quorum-sensing signalling, potential exchange of amino acids, vitamins, and other metabolites are likely mediating this hitchhiking interaction. In contrast, C. lytica made minimal adjustments to its proteome composition in coculture. Supported by optical analysis, P. pseudonitzschiae patterned C. lytica by changing how groups of the latter organised to reflect light. Our results underscore the unusual, dynamic interplay between two bacterial species and provide insights on the mechanisms underlying this relationship.

Structural color is an optical phenomenon resulting from light interacting with nanostructured materials. Although structural color (SC) is widespread in the tree of life, the underlying genetics and genomics are not well understood. Here, we collected and sequenced a set of 87 structurally colored bacterial isolates and 30 related strains lacking SC. Optical analysis of colonies indicated that diverse bacteria from at least two different phyla ( Bacteroidetes and Proteobacteria ) can create two-dimensional packing of cells capable of producing SC. A pan-genome-wide association approach was used to identify genes associated with SC. The biosynthesis of uroporphyrin and pterins, as well as carbohydrate utilization and metabolism, was found to be involved. Using this information, we constructed a classifier to predict SC directly from bacterial genome sequences and validated it by cultivating and scoring 100 strains that were not part of the training set. We predicted that SCr is widely distributed within gram-negative bacteria. Analysis of over 13,000 assembled metagenomes suggested that SC is nearly absent from most habitats associated with multicellular organisms except macroalgae and is abundant in marine waters and surface/air interfaces. This work provides a large-scale ecogenomics view of SC in bacteria and identifies microbial pathways and evolutionary relationships that underlie this optical phenomenon.

Members of the candidate phylum Dadabacteria, recently reassigned to the phylum Candidatus Desulfobacterota, are cosmopolitan in the marine environment found both free-living and associated with hosts that are mainly marine sponges. Yet, these microorganisms are poorly characterized, with no cultured representatives and an ambiguous phylogenetic position in the tree of life. Here, we performed genome-centric metagenomics to elucidate their phylogenomic placement and predict the metabolism of the sponge-associated members of this lineage. Rank-based phylogenomics revealed several new species and a novel family (Candidatus Spongomicrobiaceae) within a sponge-specific order, named here Candidatus Nemesobacterales. Metabolic reconstruction suggests that Ca. Nemesobacterales are aerobic heterotrophs, capable of synthesizing most amino acids, vitamins and cofactors and degrading complex carbohydrates. We also report functional divergence between sponge- and seawater-associated metagenome-assembled genomes. Niche-specific adaptations to the sponge holobiont were evident from significantly enriched genes involved in defense mechanisms against foreign DNA and environmental stressors, host-symbiont interactions and secondary metabolite production. Fluorescence in situ hybridization gave a first glimpse of the morphology and lifestyle of a member of Ca. Desulfobacterota. Candidatus Nemesobacterales spp. were found both inside sponge cells centred around sponge nuclei and in the mesohyl of the sponge Geodia barretti. This study sheds light on the enigmatic group Ca. Nemesobacterales and their functional characteristics that reflect a symbiotic lifestyle.

Flavobacteria, which can be found in marine environments, are able to grow in highly organized colonies producing vivid iridescent colorations. While much is known about the biology of these organisms, their design potential as responsive media in user interfaces has not been explored. Our paper aims at bridging this gap by providing insights into the type, degree, and duration of change in Flavobacteria’s expression, i.e., their living aesthetics. We present a tool to capture and characterize these changes concerning form, texture and iridescent color. To support the long-term study of their living aesthetics, we designed Flavorium. This bio-digital artifact provides the necessary habitat conditions for Flavobacteria to thrive for a month. Granting insights into the responsive behavior of this organism, this work presents a design space, vocabulary, and application concepts to inspire HCI and design scholars to investigate the complex temporal qualities of living media for future user interfaces.

The brightest colours in nature often originate from the interaction of light with materials structured at the nanoscale. Different organisms produce such coloration with a wide variety of materials and architectures. In the case of bacterial colonies, structural colours stem for the periodic organization of the cells within the colony, and while considerable efforts have been spent on elucidating the mechanisms responsible for such coloration, the biochemical processes determining the development of this effect have not been explored. Here, we study the influence of nutrients on the organization of cells from the structurally coloured bacteria Flavobacterium strain IR1. By analysing the optical properties of the colonies grown with and without specific polysaccharides, we found that the highly ordered organization of the cells can be altered by the presence of fucoidans. Additionally, by comparing the organization of the wild-type strain with mutants grown in different nutrient conditions, we deduced that this regulation of cell ordering is linked to a specific region of the IR1 chromosome. This region encodes a mechanism for the uptake and metabolism of polysaccharides, including a polysaccharide utilization locus (PUL operon) that appears specific to fucoidan, providing new insight into the biochemical pathways regulating structural colour in bacteria.

ABSTRACT CRISPR-Cas is a powerful tool for genome editing in bacteria. However, its efficacy is dependent on host factors (such as DNA repair pathways) and/or exogenous expression of recombinases. In this study, we mitigated these constraints by developing a simple and universal genome engineering tool for bacteria which we termed SIBR-Cas (Self-splicing Intron-Based Riboswitch-Cas). SIBR-Cas was generated from a mutant library of the theophylline-dependent self-splicing T4 td intron that allows for universal and inducible control over CRISPR-Cas counterselection. This control delays CRISPR-Cas counterselection, granting more time for the editing event (e.g., by homologous recombination) to occur. Without the use of exogenous recombinases, SIBR-Cas was successfully applied to knock-out several genes in three bacteria with poor homologous recombination systems. Compared to other genome engineering tools, SIBR-Cas is simple, tightly regulated and widely applicable for most (non-model) bacteria. Furthermore, we propose that SIBR can have a wider application as a universal gene expression and gene regulation control mechanism for any gene or RNA of interest in bacteria.

Structural color occurs by the interaction of light with regular structures and so generates colors by completely different optical mechanisms to dyes and pigments. Structural color is found throughout the tree of life but has not, to date, been reported in the fungi. Here we give an overview of structural color across the tree of life and provide a brief guide aimed at stimulating the search for this phenomenon in fungi.

Marine sponges harbor diverse microbial communities that represent a significant source of natural products. In the present study, extracts of 21 sponge-associated bacteria were screened for their antimicrobial and anticancer activity, and their genomes were mined for secondary metabolite biosynthetic gene clusters (BGCs). Phylogenetic analysis assigned the strains to four major phyla in the sponge microbiome, namely Proteobacteria, Actinobacteria, Bacteroidetes, and Firmicutes. Bioassays identified one extract with anti-methicillin-resistant Staphylococcus aureus (MRSA) activity, and more than 70% of the total extracts had a moderate to high cytotoxicity. The most active extracts were derived from the Proteobacteria and Actinobacteria, prominent for producing bioactive substances. The strong bioactivity potential of the aforementioned strains was also evident in the abundance of BGCs, which encoded mainly beta-lactones, bacteriocins, non-ribosomal peptide synthetases (NRPS), terpenes, and siderophores. Gene-trait matching was performed for the most active strains, aiming at linking their biosynthetic potential with the experimental results. Genetic associations were established for the anti-MRSA and cytotoxic phenotypes based on the similarity of the detected BGCs with BGCs encoding natural products with known bioactivity. Overall, our study highlights the significance of combining in vitro and in silico approaches in the search of novel natural products of pharmaceutical interest.

Vivid colours found in living organisms are often the result of scattering from hierarchical nanostructures, where the interplay between order and disorder in their packing defines visual appearance. In the case of Flavobacterium IR1, the complex arrangement of the cells in polycrystalline three-dimensional lattices is found to be a distinctive fingerprint of colony organization. By combining analytical analysis of the angle-resolved scattering response of in vivo bacterial colonies with numerical modelling, we show that we can assess the inter-cell distance and cell diameter with a resolution below 10 nm, far better than what can be achieved with conventional electron microscopy, suffering from preparation artefacts. Retrieving the role of disorder at different length scales from the salient features in the scattering response enables a precise understanding of the structural organization of the bacteria.

CRISPR-Cas is a powerful tool for genome editing in bacteria. However, its efficacy is dependent on host factors (such as DNA repair pathways) and/or exogenous expression of recombinases. In this study, we mitigated these constraints by developing a simple and widely applicable genome engineering tool for bacteria which we termed SIBR-Cas (Self-splicing Intron-Based Riboswitch-Cas). SIBR-Cas was generated from a mutant library of the theophylline-dependent self-splicing T4 td intron that allows for tight and inducible control over CRISPR-Cas counter-selection. This control delays CRISPR-Cas counter-selection, granting more time for the editing event (e.g. by homologous recombination) to occur. Without the use of exogenous recombinases, SIBR-Cas was successfully applied to knock-out several genes in three wild-type bacteria species (Escherichia coli MG1655, Pseudomonas putida KT2440 and Flavobacterium IR1) with poor homologous recombination systems. Compared to other genome engineering tools, SIBR-Cas is simple, tightly regulated and widely applicable for most (non-model) bacteria. Furthermore, we propose that SIBR can have a wider application as a simple gene expression and gene regulation control mechanism for any gene or RNA of interest in bacteria.

FlavobacteriumIR1 is a gliding bacterium with a high degree of colonial organization as a 2D photonic crystal, resulting in vivid structural coloration when illuminated.Enterobacter cloacaeB12, an unrelated bacterium, was isolated from the brown macroalgaFucus vesiculosusfrom the same location as IR1. IR1 was found to be a predator of B12. A process of surrounding, infiltration, undercutting and killing of B12 supported improved growth of IR1. A combination of motility and capillarity facilitated the engulfment of B12 colonies by IR1. Predation was independent of illumination. Mutants of IR1 that formed photonic crystals less effectively than the wild type were reduced in predation. Conversely, formation of a photonic crystal was not advantageous in resisting predation byRhodococcusspp. PIR4. These observations suggest that the organization required to create structural colour has a biological function (facilitating predation) but one that is not directly related to the photonic properties of the colony. This work is the first experimental evidence supporting a role for this widespread type of cell organization in theFlavobacteriia.

BACKGROUND:Members of the bacterial family Flavobacteriaceae are widely distributed in the marine environment and often found associated with algae, fish, detritus or marine invertebrates. Yet, little is known about the characteristics that drive their ubiquity in diverse ecological niches. Here, we provide an overview of functional traits common to taxonomically diverse members of the family Flavobacteriaceae from different environmental sources, with a focus on the Marine clade. We include seven newly sequenced marine sponge-derived strains that were also tested for gliding motility and antimicrobial activity.RESULTS:Comparative genomics revealed that genome similarities appeared to be correlated to 16S rRNA gene- and genome-based phylogeny, while differences were mostly associated with nutrient acquisition, such as carbohydrate metabolism and gliding motility. The high frequency and diversity of genes encoding polymer-degrading enzymes, often arranged in polysaccharide utilization loci (PULs), support the capacity of marine Flavobacteriaceae to utilize diverse carbon sources. Homologs of gliding proteins were widespread among all studied Flavobacteriaceae in contrast to members of other phyla, highlighting the particular presence of this feature within the Bacteroidetes. Notably, not all bacteria predicted to glide formed spreading colonies. Genome mining uncovered a diverse secondary metabolite biosynthesis arsenal of Flavobacteriaceae with high prevalence of gene clusters encoding pathways for the production of antimicrobial, antioxidant and cytotoxic compounds. Antimicrobial activity tests showed, however, that the phenotype differed from the genome-derived predictions for the seven tested strains.CONCLUSIONS:Our study elucidates the functional repertoire of marine Flavobacteriaceae and highlights the need to combine genomic and experimental data while using the appropriate stimuli to unlock their uncharted metabolic potential.

Naturally occurring photonic structures are responsible for the bright and vivid coloration in a large variety of living organisms. Despite efforts to understand their biological functions, development, and complex optical response, little is known of the underlying genes involved in the development of these nanostructures in any domain of life. Here, we used Flavobacterium colonies as a model system to demonstrate that genes responsible for gliding motility, cell shape, the stringent response, and tRNA modification contribute to the optical appearance of the colony. By structural and optical analysis, we obtained a detailed correlation of how genetic modifications alter structural color in bacterial colonies. Understanding of genotype and phenotype relations in this system opens the way to genetic engineering of on-demand living optical materials, for use as paints and living sensors.

Our understanding of microbial natural environments combines in situ experimentation with studies of specific interactions in laboratory-based setups. The purpose of this work was to develop, build and demonstrate the use of a microbial culture chamber enabling both in situ and laboratory-based studies. The design uses an enclosed chamber surrounded by two porous membranes that enables the comparison of growth of two separate microbial populations but allowing free exchange of small molecules. Initially, we tested if the presence of the macroalga Fucus vesiculosus inside the chamber affected colonization of the outer membranes by marine bacteria. The alga did indeed enrich the total population of colonizing bacteria by more than a factor of four. These findings lead us to investigate the effect of the presence of the coccolithophoric alga Emiliania huxleyi on attachment and biofilm formation of the marine bacterium Phaeobacter inhibens DSM17395. These organisms co-exist in the marine environment and have a well-characterized interdependence on secondary metabolites. P. inhibens attached in significantly higher numbers when having access to E. huxleyi as compared to when exposed to sterile media. The experiment was carried out using a wild type (wt) strain as well as a TDA-deficient strain of P. inhibens. The ability of the bacterium to produce the antibacterial compound, tropodithietic acid (TDA) influenced its attachment since the P. inhibens DSM17395 wt strain attached in higher numbers to a surface within the first 48 h of incubation with E. huxleyi as compared to a TDA-negative mutant. Whilst the attachment of the bacterium to a surface was facilitated by presence of the alga, however, we cannot conclude if this was directly affected by the algae or whether biofilm formation was dependent on the production of TDA by P. inhibens, which has been implied by previous studies. In the light of these results, other applications of immersed culture chambers are suggested.

*. py files are executed using Python 2.7. x with numpy/matplotlib (pylab)/scipy installed. It has been tested under OS X. With a few modifications (ie change print statements) it should be able to run under Python 3. x with similar modules installed. ImageJ (https://imagej. nih. gov/ij/download. html) was used for measuring sizes for the size histograms of the SEMs. The file" how_to_measure. txt" explains how to do this in more detail. The raw goniometer data (*. mat) is in HDF5 format and saved from matlab (mathworks. com), but the actual post processing is in our case done in Python. Any HDF5 viewer should be able to read the data. We recommend VLC for playing back the movie (www. videolan. org).

aUniversity of Cambridge, Department of Chemistry, Lensfield Road, Cambridge, CB2 1EW, United Kingdom; bHoekmine BV, Room 1.091 (iLab), Kenniscentrum Technologie en Innovatie, Hogeschool Utrecht, Heidelberglaan 7, 3584 CS, Utrecht, The Netherlands; cWageningen Food & Biobased Research, Bornse Weilanden 9, 6708 WG, Wageningen, The Netherlands; dDepartment of Physics, University of Cambridge, JJ Thomson Avenue, CB3 0HE, Cambridge, United Kingdom; eAdolphe Merkle Institute, University of Fribourg, Chemin des Verdiers 4, CH-1700 Fribourg, Switzerland; fDepartment of Biotechnology and Biomedicine – Infection Microbiology, Technical University of Denmark, Anker Engelunds Vej, Building 301, 2800 Kgs. Lyngby, Denmark