Streamlined CRISPR genome engineering in wild-type bacteria using SIBR-Cas.

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.

Genetic manipulation of structural color in bacterial colonies.

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.

Comparative genomic analysis of Flavobacteriaceae: insights into carbohydrate metabolism, gliding motility and secondary metabolite biosynthesis.

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.

Mutually facilitated dispersal between the nonmotile fungus Aspergillus fumigatus and the swarming bacterium Paenibacillus vortex.

In the heterogeneous environment surrounding plant roots (the rhizosphere), microorganisms both compete and cooperate. Here, we show that two very different inhabitants of the rhizosphere, the nonmotile fungus Aspergillus fumigatus and the swarming bacterium Paenibacillus vortex, can facilitate each other's dispersal. A. fumigatus conidia (nonmotile asexual fungal spores) can be transported by P. vortex swarms over distances of at least 30 cm and at rates of up to 10.8 mm h(-1). Moreover, conidia can be rescued and transported by P. vortex from niches of adverse growth conditions. Potential benefit to the bacteria may be in crossing otherwise impenetrable barriers in the soil: fungal mycelia seem to act as bridges to allow P. vortex to cross air gaps in agar plates. Transport of conidia was inhibited by proteolytic treatment of conidia or the addition of purified P. vortex flagella, suggesting specific contacts between flagella and proteins on the conidial surface. Conidia were transported by P. vortex into locations where antibiotics inhibited bacteria growth, and therefore, growth and sporulation of A. fumigatus were not limited by bacterial competition. Conidia from other fungi, similar in size to those fungi from A. fumigatus, were not transported as efficiently by P. vortex. Conidia from a range of fungi were not transported by another closely related rhizosphere bacterium, Paenibacillus polymyxa, or the more distantly related Proteus mirabilis, despite both being efficient swarmers.

Candidatus Nemesobacterales is a sponge-specific clade of the candidate phylum Desulfobacterota adapted to a symbiotic lifestyle.

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.

Polysaccharide metabolism regulates structural colour in bacterial colonies.

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.

Organic modification and subsequent biofunctionalization of porous anodic alumina using terminal alkynes.

Porous anodic alumina (PAA) is a well-defined material that has found many applications. The range of applications toward sensing and recognition can be greatly expanded if the alumina surface is covalently modified with an organic monolayer. Here, we present a new method for the organic modification of PAA based on the reaction of terminal alkynes with the alumina surface. The reaction results in the the formation of a monolayer within several hours at 80 °C and is dependent on both oxygen and light. Characterization with X-ray photoelectron spectroscopy and infrared spectroscopy indicates formation of a well-defined monolayer in which the adsorbed species is an oxidation product of the 1-alkyne, namely, its α-hydroxy carboxylate. The obtained monolayers are fairly stable in water and at elevated temperatures, as was shown by monitoring the water contact angle. Modification with 1,15-hexadecadiyne resulted in a surface that has alkyne end groups available for further reaction, as was demonstrated by the subsequent reaction of N-(11-azido-3,6,9-trioxaundecyl)trifluoroacetamide with the modified surface. Biofunctionalization was explored by coupling 11-azidoundecyl lactoside to the surface and studying the subsequent adsorption of the lectin peanut agglutinin (PNA) and the yeast Candida albicans, respectively. Selective and reversible binding of PNA to the lactosylated surfaces was demonstrated. Moreover, PNA adsorption was higher on surfaces that exposed the ß-lactoside than on those that displayed the α anomer, which was attributed to surface-associated steric hindrance. Likewise, the lactosylated surfaces showed increased colonization of C. albicans compared to unmodified surfaces, presumably due to interactions involving the cell wall ß-glucan. Thus, this study provides a new modification method for PAA surfaces and shows that it can be used to induce selective adsorption of proteins and microorganisms.

Does Structural Color Exist in True Fungi?

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.

Mixed-culture transcriptome analysis reveals the molecular basis of mixed-culture growth in Streptococcus thermophilus and Lactobacillus bulgaricus.

Many food fermentations are performed using mixed cultures of lactic acid bacteria. Interactions between strains are of key importance for the performance of these fermentations. Yogurt fermentation by Streptococcus thermophilus and Lactobacillus bulgaricus (basonym, Lactobacillus delbrueckii subsp. bulgaricus) is one of the best-described mixed-culture fermentations. These species are believed to stimulate each other's growth by the exchange of metabolites such as folic acid and carbon dioxide. Recently, postgenomic studies revealed that an upregulation of biosynthesis pathways for nucleotides and sulfur-containing amino acids is part of the global physiological response to mixed-culture growth in S. thermophilus, but an in-depth molecular analysis of mixed-culture growth of both strains remains to be established. We report here the application of mixed-culture transcriptome profiling and a systematic analysis of the effect of interaction-related compounds on growth, which allowed us to unravel the molecular responses associated with batch mixed-culture growth in milk of S. thermophilus CNRZ1066 and L. bulgaricus ATCC BAA-365. The results indicate that interactions between these bacteria are primarily related to purine, amino acid, and long-chain fatty acid metabolism. The results support a model in which formic acid, folic acid, and fatty acids are provided by S. thermophilus. Proteolysis by L. bulgaricus supplies both strains with amino acids but is insufficient to meet the biosynthetic demands for sulfur and branched-chain amino acids, as becomes clear from the upregulation of genes associated with these amino acids in mixed culture. Moreover, genes involved in iron uptake in S. thermophilus are affected by mixed-culture growth, and genes coding for exopolysaccharide production were upregulated in both organisms in mixed culture compared to monocultures. The confirmation of previously identified responses in S. thermophilus using a different strain combination demonstrates their generic value. In addition, the postgenomic analysis of the responses of L. bulgaricus to mixed-culture growth allows a deeper understanding of the ecology and interactions of this important industrial food fermentation process.

The micro-Petri dish, a million-well growth chip for the culture and high-throughput screening of microorganisms.

A miniaturized, disposable microbial culture chip has been fabricated by microengineering a highly porous ceramic sheet with up to one million growth compartments. This versatile culture format, with discrete compartments as small as 7 x 7 mum, allowed the growth of segregated microbial samples at an unprecedented density. The chip has been used for four complementary applications in microbiology. (i) As a fast viable counting system that showed a dynamic range of over 10,000, a low degree of bias, and a high culturing efficiency. (ii) In high-throughput screening, with the recovery of 1 fluorescent microcolony in 10,000. (iii) In screening for an enzyme-based, nondominant phenotype by the targeted recovery of Escherichia coli transformed with the plasmid pUC18, based on expression of the lacZ reporter gene without antibiotic-resistance selection. The ease of rapid, successive changes in the environment of the organisms on the chip, needed for detection of beta-galactosidase activity, highlights an advantageous feature that was also used to screen a metagenomic library for the same activity. (iv) In high-throughput screening of >200,000 isolates from Rhine water based on metabolism of a fluorogenic organophosphate compound, resulting in the recovery of 22 microcolonies with the desired phenotype. These isolates were predicted, on the basis of rRNA sequence, to include six new species. These four applications suggest that the potential for such simple, readily manufactured chips to impact microbial culture is extensive and may facilitate the full automation and multiplexing of microbial culturing, screening, counting, and selection.

The cell organization underlying structural colour is involved in Flavobacterium IR1 predation.

Flavobacterium IR1 is a gliding bacterium with a high degree of colonial organization as a 2D photonic crystal, resulting in vivid structural coloration when illuminated. Enterobacter cloacae B12, an unrelated bacterium, was isolated from the brown macroalga Fucus vesiculosus from 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 by Rhodococcus spp. 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 the Flavobacteriia.

Complex photonic response reveals three-dimensional self-organization of structural coloured bacterial colonies.

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.

MEMS and the microbe.

In recent years, relatively simple MEMS fabrications have helped accelerate our knowledge of the microbial cell. Current progress and challenges in the application of lab-on-a-chip devices to the viable microbe are reviewed. Furthermore, the degree to which microbiologists are becoming the engineers and are tailoring microbial cells and protocells as potential components for bioMEMS devices is highlighted. We conclude this is a highly productive time for microbiologists and microengineers to unite their shared interest in the micron scale world.

Population heterogeneity of Lactobacillus plantarum WCFS1 microcolonies in response to and recovery from acid stress.

Within an isogenic microbial population in a homogenous environment, individual bacteria can still exhibit differences in phenotype. Phenotypic heterogeneity can facilitate the survival of subpopulations under stress. As the gram-positive bacterium Lactobacillus plantarum grows, it acidifies the growth medium to a low pH. We have examined the growth of L. plantarum microcolonies after rapid pH downshift (pH 2 to 4), which prevents growth in liquid culture. This acidification was achieved by transferring cells from liquid broth onto a porous ceramic support, placed on a base of low-pH MRS medium solidified using Gelrite. We found a subpopulation of cells that displayed phenotypic heterogeneity and continued to grow at pH 3, which resulted in microcolonies dominated by viable but elongated (filamentous) cells lacking septation, as determined by scanning electron microscopy and staining cell membranes with the lipophilic dye FM4-64. Recovery of pH-stressed cells from these colonies was studied by inoculation onto MRS-Gelrite-covered slides at pH 6.5, and outgrowth was monitored by microscopy. The heterogeneity of the population, calculated from the microcolony areas, decreased with recovery from pH 3 over a period of a few hours. Filamentous cells did not have an advantage in outgrowth during recovery. Specific regions within single filamentous cells were more able to form rapidly dividing cells, i.e., there was heterogeneity even within single recovering cells.

Swarming and complex pattern formation in Paenibacillus vortex studied by imaging and tracking cells.

BACKGROUND: Swarming motility allows microorganisms to move rapidly over surfaces. The Gram-positive bacterium Paenibacillus vortex exhibits advanced cooperative motility on agar plates resulting in intricate colonial patterns with geometries that are highly sensitive to the environment. The cellular mechanisms that underpin the complex multicellular organization of such a simple organism are not well understood. RESULTS: Swarming by P. vortex was studied by real-time light microscopy, by in situ scanning electron microscopy and by tracking the spread of antibiotic-resistant cells within antibiotic-sensitive colonies. When swarming, P. vortex was found to be peritrichously flagellated. Swarming by the curved cells of P. vortex occurred on an extremely wide range of media and agar concentrations (0.3 to 2.2% w/v). At high agar concentrations (> 1% w/v) rotating colonies formed that could be detached from the main mass of cells by withdrawal of cells into the latter. On lower percentage agars, cells moved in an extended network composed of interconnected "snakes" with short-term collision avoidance and sensitivity to extracts from swarming cells. P. vortex formed single Petri dish-wide "supercolonies" with a colony-wide exchange of motile cells. Swarming cells were coupled by rapidly forming, reversible and non-rigid connections to form a loose raft, apparently connected via flagella. Inhibitors of swarming (p-Nitrophenylglycerol and Congo Red) were identified. Mitomycin C was used to trigger filamentation without inhibiting growth or swarming; this facilitated dissection of the detail of swarming. Mitomycin C treatment resulted in malcoordinated swarming and abortive side branch formation and a strong tendency by a subpopulation of the cells to form minimal rotating aggregates of only a few cells. CONCLUSION: P. vortex creates complex macroscopic colonies within which there is considerable reflux and movement and interaction of cells. Cell shape, flagellation, the aversion of cell masses to fuse and temporary connections between proximate cells to form rafts were all features of the swarming and rotation of cell aggregates. Vigorous vortex formation was social, i.e. required > 1 cell. This is the first detailed examination of the swarming behaviour of this bacterium at the cellular level.

Isolation by Miniaturized Culture Chip of an Antarctic bacterium Aequorivita sp. with antimicrobial and anthelmintic activity.

Microbes are prolific sources of bioactive molecules; however, the cultivability issue has severely hampered access to microbial diversity. Novel secondary metabolites from as-yet-unknown or atypical microorganisms from extreme environments have realistic potential to lead to new drugs with benefits for human health. Here, we used a novel approach that mimics the natural environment by using a Miniaturized Culture Chip allowing the isolation of several bacterial strains from Antarctic shallow water sediments under near natural conditions. A Gram-negative Antarctic bacterium belonging to the genus Aequorivita was subjected to further analyses. The Aequorivita sp. genome was sequenced and a bioinformatic approach was applied to identify biosynthetic gene clusters. The extract of the Aequorivita sp. showed antimicrobial and anthelmintic activity towards Multidrug resistant bacteria and the nematode Caenorhabditis elegans. This is the first multi-approach study exploring the genomics and biotechnological potential of the genus Aequorivita that is a promising candidate for pharmaceutical applications.

Physically Triggered Morphology Changes in a Novel Acremonium Isolate Cultivated in Precisely Engineered Microfabricated Environments.

Fungi are strongly affected by their physical environment. Microfabrication offers the possibility of creating new culture environments and ecosystems with defined characteristics. Here, we report the isolation of a novel member of the fungal genus Acremonium using a microengineered cultivation chip. This isolate was unusual in that it organizes into macroscopic structures when initially cultivated within microwells with a porous aluminum oxide (PAO) base. These "templated mycelial bundles" (TMB) were formed from masses of parallel hyphae with side branching suppressed. TMB were highly hydrated, facilitating the passive movement of solutes along the bundle. By using a range of culture chips, it was deduced that the critical factors in triggering the TMB were growth in microwells from 50 to 300 µm in diameter with a PAO base. Cultivation experiments, using spores and pigments as tracking agents, indicate that bulk growth of the TMB occurs at the base. TMB morphology is highly coherent and is maintained after growing out of the microwells. TMB can explore their environment by developing unbundled lateral hyphae; TMB only followed if nutrients were available. Because of the ease of fabricating numerous microstructures, we suggest this is a productive approach for exploring morphology and growth in multicellular microorganisms and microbial communities.

Rapid antibiotic sensitivity testing and trimethoprim-mediated filamentation of clinical isolates of the Enterobacteriaceae assayed on a novel porous culture support.

A porous inorganic material (Anopore) was employed as a microbial culture and microcolony imaging support. Rapid Anopore-based antibiotic sensitivity testing (AST) methods were developed to assess the growth of clinical isolates, with the primary focus on testing the response of the Enterobacteriaceae to trimethoprim, but with the method supporting a wider applicability in terms of strains and antibiotics. It was possible to detect the growth of Enterobacter aerogenes after 25 min culture and to distinguish a trimethoprim-sensitive from a trimethoprim-resistant strain with 40 min incubation. MIC(90) determinations were made on Anopore; these were in good agreement with the results from the Vitek 2 and E-test methods. The Anopore method correctly identified sensitive (40/40) and resistant (17/17) strains of the Enterobacteriaceae and other Gram-negative rods within only 2-3 h culture. Additionally, a trimethoprim-resistant subpopulation (10 % of population) could be detected by microcolony formation within 2 h, and a smaller subpopulation (1 %) after 3.5 h. These results suggest that this is a viable approach for the rapid AST of purified strains, and that it may be able to deal with mixed populations. The microscopic examination of microcolonies during AST is an advantage of this method which revealed additional information. Filamentation triggered by trimethoprim was discovered in many species of the Enterobacteriaceae for which this phenomenon has not previously been reported. Filamentation was characterized by heterogeneity in terms of cell length, and also uneven nucleic acid distribution and flattening of damaged cells. The development and application of Anopore-based AST within clinical diagnostics is discussed.

EstDZ3: A New Esterolytic Enzyme Exhibiting Remarkable Thermostability.

Lipolytic enzymes that retain high levels of catalytic activity when exposed to a variety of denaturing conditions are of high importance for a number of biotechnological applications. In this study, we aimed to identify new lipolytic enzymes, which are highly resistant to prolonged exposure to elevated temperatures. To achieve this, we searched for genes encoding for such proteins in the genomes of a microbial consortium residing in a hot spring located in China. After performing functional genomic screening on a bacterium of the genus Dictyoglomus, which was isolated from this hot spring following in situ enrichment, we identified a new esterolytic enzyme, termed EstDZ3. Detailed biochemical characterization of the recombinant enzyme, revealed that it constitutes a slightly alkalophilic and highly active esterase against esters of fatty acids with short to medium chain lengths. Importantly, EstDZ3 exhibits remarkable thermostability, as it retains high levels of catalytic activity after exposure to temperatures as high as 95°C for several hours. Furthermore, it exhibits very good stability against exposure to high concentrations of a variety of organic solvents. Interestingly, EstDZ3 was found to have very little similarity to previously characterized esterolytic enzymes. Computational modeling of the three-dimensional structure of this new enzyme predicted that it exhibits a typical α/ß hydrolase fold that seems to include a "subdomain insertion", which is similar to the one present in its closest homolog of known function and structure, the cinnamoyl esterase Lj0536 from Lactobacillus johnsonii. As it was found in the case of Lj0536, this structural feature is expected to be an important determinant of the catalytic properties of EstDZ3. The high levels of esterolytic activity of EstDZ3, combined with its remarkable thermostability and good stability against a range of organic solvents and other denaturing agents, render this new enzyme a candidate biocatalyst for high-temperature biotechnological applications.