The global RNA-RNA interactome of Klebsiella pneumoniae unveils a small RNA regulator of cell division.

The ubiquitous RNA chaperone Hfq is involved in the regulation of key biological processes in many species across the bacterial kingdom. In the opportunistic human pathogen Klebsiella pneumoniae, deletion of the hfq gene affects the global transcriptome, virulence, and stress resistance; however, the ligands of the major RNA-binding protein in this species have remained elusive. In this study, we have combined transcriptomic, co-immunoprecipitation, and global RNA interactome analyses to compile an inventory of conserved and species-specific RNAs bound by Hfq and to monitor Hfq-mediated RNA-RNA interactions. In addition to dozens of RNA-RNA pairs, our study revealed an Hfq-dependent small regulatory RNA (sRNA), DinR, which is processed from the 3' terminal portion of dinI mRNA. Transcription of dinI is controlled by the master regulator of the SOS response, LexA. As DinR accumulates in K. pneumoniae in response to DNA damage, the sRNA represses translation of the ftsZ transcript by occupation of the ribosome binding site. Ectopic overexpression of DinR causes depletion of ftsZ mRNA and inhibition of cell division, while deletion of dinR antagonizes cell elongation in the presence of DNA damage. Collectively, our work highlights the important role of RNA-based gene regulation in K. pneumoniae and uncovers the central role of DinR in LexA-controlled division inhibition during the SOS response.

Evaluation of reproducible cryogel preparation based on automated image analysis using deep learning.

Cryogels represent a class of porous sponge-like materials possessing unique properties including high-fidelity reproduction of tissue structure and maximized permeability. Their architecture is mainly based on an interconnected network of macropores that provides sufficient stability while allowing the movement of substances through the material. In most cryogel applications, the pore size is very important, especially when the material is used as a 3D scaffold for tissue culture, applied as a filter, or utilized as a membrane. In this study, poly(dimethylacrylamide-co-2-hydroxyethyl methacrylate) cryogels have been prepared by two preparation methods to investigate the reproducibility of homogeneous pore structures and pore sizes. Automated image analysis algorithms were developed to rapidly evaluate cryogel pore sizes based on scanning electron microscopy (SEM) images. The quantification approach contained a unique combination of classical and deep learning-based algorithms. To validate the accuracy of the two models, we compared the results obtained from automated SEM image analysis with those from manual pore size determinations and mercury intrusion porosimetry (MIP) measurements. Effect sizes were calculated to compare the results from manual and automated pore size measurements for the cryogel reproducibility series. 81% of the values obtained revealed only trivial differences, which strongly suggests that automated image analysis can reliably substitute the manual evaluation of cryogel pore sizes. The use of an adapted reactor setup yielded cryogels with heterogeneous morphologies in the absence of recognizable pore structures. With the conventional cryogel preparation using plastic syringes, the obtained cryogels represented highly reproducible morphologies and pore sizes in the range between 17 and 22 µm. Calculated effect sizes within the cryogel replicate series revealed only trivial differences between the obtained pore sizes in 83.5% or 99.4% of the data (classical approach and deep learning-based approach, respectively).

Toxin-Producing Endosymbionts Shield Pathogenic Fungus against Micropredators.

The fungus Rhizopus microsporus harbors a bacterial endosymbiont (Mycetohabitans rhizoxinica) for the production of the antimitotic toxin rhizoxin. Although rhizoxin is the causative agent of rice seedling blight, the toxinogenic bacterial-fungal alliance is, not restricted to the plant disease. It has been detected in numerous environmental isolates from geographically distinct sites covering all five continents, thus raising questions regarding the ecological role of rhizoxin beyond rice seedling blight. Here, we show that rhizoxin serves the fungal host in fending off protozoan and metazoan predators. Fluorescence microscopy and coculture experiments with the fungivorous amoeba Protostelium aurantium revealed that ingestion of R. microsporus spores is toxic to P. aurantium. This amoebicidal effect is caused by the dominant bacterial rhizoxin congener rhizoxin S2, which is also lethal toward the model nematode Caenorhabditis elegans. By combining stereomicroscopy, automated image analysis, and quantification of nematode movement, we show that the fungivorous nematode Aphelenchus avenae actively feeds on R. microsporus that is lacking endosymbionts, whereas worms coincubated with symbiotic R. microsporus are significantly less lively. This study uncovers an unexpected ecological role of rhizoxin as shield against micropredators. This finding suggests that predators may function as an evolutionary driving force to maintain toxin-producing endosymbionts in nonpathogenic fungi. IMPORTANCE The soil community is a complex system characterized by predator-prey interactions. Fungi have developed effective strategies to defend themselves against predators. Understanding these strategies is of critical importance for ecology, medicine, and biotechnology. In this study, we shed light on the defense mechanisms of the phytopathogenic Rhizopus-Mycetohabitans symbiosis that has spread worldwide. We report an unexpected role of rhizoxin, a secondary metabolite produced by the bacterium M. rhizoxinica residing within the hyphae of R. microsporus. We show that this bacterial secondary metabolite is utilized by the fungal host to successfully fend off fungivorous protozoan and metazoan predators and thus identified a fundamentally new function of this infamous cytotoxic compound. This endosymbiont-dependent predator defense illustrates an unusual strategy employed by fungi that has broader implications, since it may serve as a model for understanding how animal predation acts as an evolutionary driving force to maintain endosymbionts in nonpathogenic fungi.

Spatial quantification of clinical biomarker pharmacokinetics through deep learning-based segmentation and signal-oriented analysis of MSOT data.

Although multispectral optoacoustic tomography (MSOT) significantly evolved over the last several years, there is a lack of quantitative methods for analysing this type of image data. Current analytical methods characterise the MSOT signal in manually defined regions of interest outlining selected tissue areas. These methods demand expert knowledge of the sample anatomy, are time consuming, highly subjective and prone to user bias. Here we present our fully automated open-source MSOT cluster analysis toolkit Mcat that was designed to overcome these shortcomings. It employs a deep learning-based approach for initial image segmentation followed by unsupervised machine learning to identify regions of similar signal kinetics. It provides an objective and automated approach to quantify the pharmacokinetics and extract the biodistribution of biomarkers from MSOT data. We exemplify our generally applicable analysis method by quantifying liver function in a preclinical sepsis model whilst highlighting the advantages of our new approach compared to the severe limitations of existing analysis procedures.

Bacterial endosymbionts protect beneficial soil fungus from nematode attack.

Fungi of the genus Mortierella occur ubiquitously in soils where they play pivotal roles in carbon cycling, xenobiont degradation, and promoting plant growth. These important fungi are, however, threatened by micropredators such as fungivorous nematodes, and yet little is known about their protective tactics. We report that Mortierella verticillata NRRL 6337 harbors a bacterial endosymbiont that efficiently shields its host from nematode attacks with anthelmintic metabolites. Microscopic investigation and 16S ribosomal DNA analysis revealed that a previously overlooked bacterial symbiont belonging to the genus Mycoavidus dwells in M. verticillata hyphae. Metabolic profiling of the wild-type fungus and a symbiont-free strain obtained by antibiotic treatment as well as genome analyses revealed that highly cytotoxic macrolactones (CJ-12,950 and CJ-13,357, syn necroxime C and D), initially thought to be metabolites of the soil-inhabiting fungus, are actually biosynthesized by the endosymbiont. According to comparative genomics, the symbiont belongs to a new species (Candidatus Mycoavidus necroximicus) with 12% of its 2.2 Mb genome dedicated to natural product biosynthesis, including the modular polyketide-nonribosomal peptide synthetase for necroxime assembly. Using Caenorhabditis elegans and the fungivorous nematode Aphelenchus avenae as test strains, we show that necroximes exert highly potent anthelmintic activities. Effective host protection was demonstrated in cocultures of nematodes with symbiotic and chemically complemented aposymbiotic fungal strains. Image analysis and mathematical quantification of nematode movement enabled evaluation of the potency. Our work describes a relevant role for endofungal bacteria in protecting fungi against mycophagous nematodes.

Global importance of RNA secondary structures in protein-coding sequences.

MOTIVATION: The protein-coding sequences of messenger RNAs are the linear template for translation of the gene sequence into protein. Nevertheless, the RNA can also form secondary structures by intramolecular base-pairing. RESULTS: We show that the nucleotide distribution within codons is biased in all taxa of life on a global scale. Thereby, RNA secondary structures that require base-pairing between the position 1 of a codon with the position 1 of an opposing codon (here named RNA secondary structure class c1) are under-represented. We conclude that this bias may result from the co-evolution of codon sequence and mRNA secondary structure, suggesting that RNA secondary structures are generally important in protein-coding regions of mRNAs. The above result also implies that codon position 2 has a smaller influence on the amino acid choice than codon position 1. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.