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

<p>Abstract</p> <p>Background</p> <p>Swarming motility allows microorganisms to move rapidly over surfaces. The Gram-positive bacterium <it>Paenibacillus vortex </it>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.</p> <p>Results</p> <p>Swarming by <it>P. vortex </it>was studied by real-time light microscopy, by <it>in situ </it>scanning electron microscopy and by tracking the spread of antibiotic-resistant cells within antibiotic-sensitive colonies. When swarming, <it>P. vortex </it>was found to be peritrichously flagellated. Swarming by the curved cells of <it>P. vortex </it>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. <it>P. vortex </it>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 <it>via </it>flagella. Inhibitors of swarming (<it>p</it>-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.</p> <p>Conclusion</p> <p><it>P. vortex </it>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.</p

Bacterial Swarms Recruit Cargo Bacteria To Pave the Way in Toxic Environments

Swarming bacteria are challenged by the need to invade hostile environments. Swarms of the flagellated bacterium Paenibacillus vortex can collectively transport other microorganisms. Here we show that P.ﾠvortex can invade toxic environments by carrying antibiotic-degrading bacteria; this transport is mediated by a specialized, phenotypic subpopulation utilizing a process not dependent on cargo motility. Swarms of beta-lactam antibiotic (BLA)-sensitive P.ﾠvortex used beta-lactamase-producing, resistant, cargo bacteria to detoxify BLAs in their path. In the presence of BLAs, both transporter and cargo bacteria gained from this temporary cooperation; there was a positive correlation between BLA resistance and dispersal. P.ﾠvortex transported only the most beneficial antibiotic-resistant cargo (including environmental and clinical isolates) in a sustained way. P.ﾠvortex displayed a bet-hedging strategy that promoted the colonization of nontoxic niches by P.ﾠvortex alone; when detoxifying cargo bacteria were not needed, they were lost. This work has relevance for the dispersal of antibiotic-resistant microorganisms and for strategies for asymmetric cooperation with agricultural and medical implications

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

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

Microcolony Imaging of Aspergillus fumigatus Treated with Echinocandins Reveals Both Fungistatic and Fungicidal Activities

Background: The echinocandins are lipopeptides that can be employed as antifungal drugs that inhibit the synthesis of 1,3b-glucans within the fungal cell wall. Anidulafungin and caspofungin are echinocandins used in the treatment of Candida infections and have activity against other fungi including Aspergillus fumigatus. The echinocandins are generally considered fungistatic against Aspergillus species. Methods: Culture of A. fumigatus from conidia to microcolonies on a support of porous aluminium oxide (PAO), combined with fluorescence microscopy and scanning electron microscopy, was used to investigate the effects of anidulafungin and caspofungin. The PAO was an effective matrix for conidial germination and microcolony growth. Additionally, PAO supports could be moved between agar plates containing different concentrations of echinocandins to change dosage and to investigate the recovery of fungal microcolonies from these drugs. Culture on PAO combined with microscopy and image analysis permits quantitative studies on microcolony growth with the flexibility of adding or removing antifungal agents, dyes, fixatives or osmotic stresses during growth with minimal disturbance of fungal microcolonies. Significance: Anidulafungin and caspofungin reduced but did not halt growth at the microcony level; additionally both drugs killed individual cells, particularly at concentrations around the MIC. Intact but not lysed cells showed rapid recovery when the drugs were removed. The classification of these drugs as either fungistatic or fungicidal is simplistic. Microcolon

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

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 at 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 a functional genomic screening on a bacterium of the genus Dictyoglomus, which was isolated from this hot spring after 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 retained high levels of catalytic activity after exposure to temperatures as high as 95 oC for several hours. Interestingly, EstDZ3 was found to have very little similarity to previously characterized esterolytic enzymes. Computational modelling of the three-dimensional structure of this new enzyme predicted that it exhibits a typical α/β hydrolase fold, which seems to include a subdomain insertion. This insertion is similar to the one present in its closest homologue 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 wide range of metal ions, organic solvents, and other denaturing agents, render this new enzyme a candidate biocatalyst for high-temperature biotechnological applications

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 at 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 a functional genomic screening on a bacterium of the genus Dictyoglomus, which was isolated from this hot spring after 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 retained high levels of catalytic activity after exposure to temperatures as high as 95 oC for several hours. Interestingly, EstDZ3 was found to have very little similarity to previously characterized esterolytic enzymes. Computational modelling of the three-dimensional structure of this new enzyme predicted that it exhibits a typical α/β hydrolase fold, which seems to include a subdomain insertion. This insertion is similar to the one present in its closest homologue 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 wide range of metal ions, organic solvents, and other denaturing agents, render this new enzyme a candidate biocatalyst for high-temperature biotechnological applications

Rapid Susceptibility Testing and Microcolony Analysis of Candida spp. Cultured and Imaged on Porous Aluminum Oxide

Contains fulltext : 124300.pdf (publisher's version ) (Open Access)BACKGROUND: Acquired resistance to antifungal agents now supports the introduction of susceptibility testing for species-drug combinations for which this was previously thought unnecessary. For pathogenic yeasts, conventional phenotypic testing needs at least 24 h. Culture on a porous aluminum oxide (PAO) support combined with microscopy offers a route to more rapid results. METHODS: Microcolonies of Candida species grown on PAO were stained with the fluorogenic dyes Fun-1 and Calcofluor White and then imaged by fluorescence microscopy. Images were captured by a charge-coupled device camera and processed by publicly available software. By this method, the growth of yeasts could be detected and quantified within 2 h. Microcolony imaging was then used to assess the susceptibility of the yeasts to amphotericin B, anidulafungin and caspofungin (3.5 h culture), and voriconazole and itraconazole (7 h culture). SIGNIFICANCE: Overall, the results showed good agreement with EUCAST (86.5% agreement; n = 170) and E-test (85.9% agreement; n = 170). The closest agreement to standard tests was found when testing susceptibility to amphotericin B and echinocandins (88.2 to 91.2%) and the least good for the triazoles (79.4 to 82.4%). Furthermore, large datasets on population variation could be rapidly obtained. An analysis of microcolonies revealed subtle effects of antimycotics on resistant strains and below the MIC of sensitive strains, particularly an increase in population heterogeneity and cell density-dependent effects of triazoles. Additionally, the method could be adapted to strain identification via germ tube extension. We suggest PAO culture is a rapid and versatile method that may be usefully adapted to clinical mycology and has research applications

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