FRET monitoring of transcription factor activities in living bacteria

Bacteria adapt to the constantly changing environments largely by transcriptional regulation through the activities of various transcription factors (TFs). However, techniques that monitor the in situ TF-promoter interactions in living bacteria are lacking. Herein, we developed a whole-cell TF-promoter binding assay based on the intermolecular Förster resonance energy transfer (FRET) between a fluorescent unnatural amino acid CouA which is genetically encoded into defined sites in TFs and the live cell fluorescent nucleic acid stain SYTO 9. We show that this new FRET pair monitors the intricate TF-promoter interactions elicited by various types of signal transduction systems with specificity and sensitivity. Furthermore, the assay is applicable to identify novel modulators of the regulatory systems of interest and monitor TF activities in bacteria colonized in C. elegans. In conclusion, we established a tractable and sensitive TF-promoter binding assay in living bacteria which not only complements currently available approaches for DNA-protein interactions but also provides novel opportunities for functional annotation of bacterial signal transduction systems and studies of the bacteria-host interface.

Evaluating H2O2 in Living Bacteria by Ratiometric Fluorescent Probe

Hydrogen peroxide (H2O2) is a significant signal molecule in physiological and pathological processes. Levels of H2O2 in bacteria are proved to be a key factor in immune response. To sum up, detection of H2O2 levels in living bacteria is remarkable for further study of its physiological and pathological effects. Herein, we propose a novel ratiometric fluorescent probe (Nahp) to detect H2O2 in living cells and bacteria. In addition, based on boronate, Nahp has satisfactory selectivity and sensitivity toward H2O2 (LOD = 0.158 μM). Furthermore, with excellent detection performance to H2O2, Nahp is successfully used for fluorescent bioimaging of H2O2 and measuring H2O2 accumulation in bacteria. Most importantly, the probe was also used to image H2O2 in three Gram-negative bacteria, clearly revealing for the first time significant differences in H2O2 expression levels in live bacteria.

Physiological and Biochemical Characterization of Isolated Bacteria from a Coccolithophore Chrysotila dentata (Prymnesiophyceae) Culture

Coccolithophores are involved in oceanic carbon and nitrogen cycles, and they also have an impact on global climate change. Chrysotila dentata have a complex and close relationship with phycosphere bacteria. In this study, culturable phycosphere bacteria (free-living bacteria and attached bacteria) are isolated from C. dentata by a gradient dilution method and identified based on the 16S rRNA gene sequencing analysis. The phylogenetic tree (neighbor-joining tree, N-J tree) was constructed using the bacterial sequences and closest related sequences from GenBank. Colony characteristics, Gram nature, and physiological and biochemical characteristics were obtained based on a series of tests, such as the sugar utilization (glucose, arabinose, xylose, maltose, and mannitol) test, Voges–Proskauer reaction, urease tests, gelatin liquefaction, Gram test, starch hydrolysis, among others. In this study, seven strains (CF1, CF2, CF3, CF5, CF6, and CF7) of free-living bacteria (CF) and five strains (CA1, CA2, CA3, CA4, and CA5) of attached bacteria (CA) are isolated and identified. We found that the culturable phycosphere bacteria of C. dentata were mainly α-proteobacteria and γ-proteobacteria, with a small part of the CFB (Cytophaga-Flexibacter-Bacteroides) group bacteria and firmicutes. In this study, most α-proteobacteria can utilize malonate and positive in the urease test, meanwhile they can grow in a 7% NaCl medium. Differently to α-proteobacteria, γ-proteobacteria are more reactive, and can utilize maltose, glucose, arabinose, malonate, aesculin, and starch hydrolysis. Meanwhile, γ-proteobacteria can growth in a 7% NaCl and pH 5.7 medium, and some bacteria of this strain were positive in nitrate reduction. Firmicutes are similar to γ-proteobacteria: they are similar in reactivity, as they can utilize maltose, glucose, arabinose, malonate, aesculin, and starch hydrolysis, and can growth in a 7% NaCl and pH 5.7 medium. The difference is that some of firmicutes were positive in gelatin liquefaction and can utilize mannitol. The CFB group of bacteria were only positive in malonate, aesculin, and starch hydrolysis. The above results provide basic experimental data for further studies on the relationship between the coccolithophores and culturable phycosphere bacteria.

Benefic organisms in agricultural crops: Towards a safety and healthy food in response to COVID-19 and future syndemics

<p>A population with an adequate immunity is key to reduce the effects of COVID-19. Moreover, a healthy diet and an innocuous environment are factors for an adequate immunity. Healthier and more innocuous foods could be obtained with the extensive use of beneficial organisms on agricultural crops, helping reduce the use of agrochemicals and increasing the tolerance of plants to stress caused by abiotic and biotic factors. Nitrogen-fixating bacteria or free-living bacteria, mycorrhizae, endosymbiotic microorganisms, endophytes, entomopathogenic fungi and bacteria, pest predators and parasitoids, hyper parasitic viruses of pests and pathogens are some of the organisms that can induce the natural suppression of parasites, fixate nitrogen and optimize the capture of nutrients and water, among other ecosystemic benefits. This revision presents functions and properties of beneficial organisms and proposals are made for their use to benefit farmers and consumers, with the intention of contributing to the productive processes towards a sustainable agriculture.</p>

A salvaging strategy enables stable metabolite provisioning among free-living bacteria

All organisms rely on complex metabolites such as amino acids, nucleotides, and cofactors for essential metabolic processes. Some microbes synthesize these fundamental ingredients of life de novo, while others rely on uptake to fulfill their metabolic needs. Although certain metabolic processes are inherently 'leaky', the mechanisms enabling stable metabolite provisioning among microbes in the absence of a host remain largely unclear. In particular, how can metabolite provisioning among free-living bacteria be maintained under the evolutionary pressure to economize resources? Salvaging, the process of 'recycling and reusing', can be a metabolically efficient route to obtain access to required resources. Here, we show experimentally how precursor salvaging in engineered Escherichia coli populations can lead to stable, long-term metabolite provisioning. We find that salvaged cobamides (vitamin B12 and related enzyme cofactors) are readily made available to non-productive population members, yet salvagers are strongly protected from overexploitation due to partial metabolite privatization. We also describe a previously unnoted benefit of precursor salvaging, namely the removal of the non-functional, proliferation-inhibiting precursor. As long as compatible precursors are present, any microbe possessing the terminal steps of a biosynthetic process can, in principle, forgo de novo biosynthesis in favor of salvaging. Consequently, precursor salvaging likely represents a potent, yet overlooked, alternative to de novo biosynthesis for the acquisition and provisioning of metabolites in free-living bacterial populations.

Self-floating Capsule of Algicidal Bacteria Bacillus sp. HL and its Performance in the Dissolution of Microcystis Aeruginosa

Algicidal bacteria is known as efficient and environmentally friendly in treating Microcystis aeruginosa (M. aeruginosa). However, the practical application of algicidal bacteria in the natural water is limited by the interference of external factors and the low reuse capability. In this study, a biodegradation capsule for M. aeruginosa is prepared by biocompatible sodium alginate (SA) compositing with eco-friendly ethyl cellulose (EC). Bacterial strain HL was immobilized and the capsule was obtained under optimal usage concentrations of SA, Calcium chloride (CaCl2) and EC at 2%, 3% and 3%. It has been observed that capsules immobilizing bacteria HL shows considerable advantages over traditional bio-treatment systems (free-living bacteria) and good reusability performance. A better dissolution rate of 77.67% ± 1.14% on the 7th day was obtained with the embedding of algicidal bacteria at 50 mL, which enhanced algae dissolution rate by 11.05% compared with free-living bacteria, and the dissolution rate for M. aeruginosa still reaches 68.57% ± 2.88% after three times repetitive use. Algicidal bacteria capsules were examined on the fluorescence and antioxidant system of M. aeruginosa. It was indicated that photosynthetic mechanisms of M. aeruginosa were destroyed, superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) were all significantly induced as antioxidant response, and malondialdehyde (MDA) content increased. Overall, capsules prepared in this study can provide a desirable environment for algicidal bacteria HL and ensure algicidal bacteria to in-situ work well in the inhibition of algae.

Jäger und Gejagte — wie sich Pilze gegen räuberische Amöben wehren

What seems obvious for most free-living bacteria, also appeals for yeast and filamentous fungi: their natural reservoirs include a variety of micropredators, such as members of the Amoebozoa kingdom. Not only do they share a predatory lifestyle, but their amoeboid motility and way of ingesting living microbial food reveals several similarities to innate immune cells. Understanding how fungi have learned to cope with such environmental phagocytes will shed new light on the evolutionary driving forces of fungal diversity and virulence.

Presence of carbepenemase-producing Enterobacteriaceae in the River Lambro basin, Italy: might sediment represent an important resistance reservoir?

In the last years, the rapid spread in anthropized ecosystems of pathogens which are resistant to carbapenem antibiotics has raised great concern. In this study, KPC-producing Klebsiella pneumoniae was found in the River Lambro in June 2019, whereas KPC-producing Klebsiella oxytoca and Citrobacter braakii were identified in untreated wastewaters. Susceptibility profiles indicated resistance to imipenem, ertapenem and meropenem. Different carbapenamase genes (blaKPC, blaNDM, blaVIM, blaOXA-48) were also found in the River Lambro, although not associated to living bacteria. The presence of a wide set of carbapenemase genes and resistant pathogens show that river sediments could act as a reservoir of antibiotic resistance potentially threatening human health.

Dynamics of Free-Living and Attached Bacterial Assemblages in Skeletonema sp. Diatom Cultures at Elevated Temperatures

In the context of global warming, changes in phytoplankton-associated bacterial communities have the potential to change biogeochemical cycling and food webs in marine ecosystems. Skeletonema is a cosmopolitan diatom genus in coastal waters worldwide. Here, we grew a Skeletonema strain with its native bacterial assemblage at different temperatures and examined cell concentrations of Skeletonema sp. and free-living bacteria, dissolved organic carbon (DOC) concentrations of cultures, and the community structure of both free-living and attached bacteria at different culture stages. The results showed that elevated temperature increased the specific growth rates of both Skeletonema and free-living bacteria. Different growth stages had a more pronounced effect on community structure compared with temperatures and different physical states of bacteria. The effects of temperature on the structure of the free-living bacterial community were more pronounced compared with diatom-attached bacteria. Carbon metabolism genes and those for some specific amino acid pathways were found to be positively correlated with elevated temperature, which may have profound implications on the oceanic carbon cycle and the marine microbial loop. Network analysis revealed evidence of enhanced cooperation with an increase in positive interactions among different bacteria at elevated temperature. This may help the whole community to overcome the stress of elevated temperature. We speculate that different bacterial species may build more integrated networks with a modified functional profile of the whole community to cope with elevated temperature. This study contributes to an improved understanding of the response of diatom-associated bacterial communities to elevated temperature.