The filamentous fungus Magnaporthe oryzae has the potential to be developed as an alternative platform organism for the heterologous production of industrially important enzymes. M. oryzae is easy to handle, fast-growing and unlike yeast, posttranslational modifications like N-glycosylations are similar to the human organism. Here, we established M. oryzae as a host for the expression of the unspecific peroxygenase from the basidiomycete Agrocybe aegerita (AaeUPO). UPOs are attractive biocatalysts for selective oxyfunctionalization of non-activated carbon-hydrogen bonds. To improve and simplify the isolation of AaeUPO in M. oryzae, we fused a Magnaporthe signal peptide for protein secretion and set it under control of the strong EF1-promotor. The success of the heterologous production of full-length AaeUPO in M. oryzae and the secretion of the functional enzyme was confirmed by a peroxygenase-specific enzyme assay. These results offer the possibility to establish the filamentous ascomycete M. oryzae as a broad applicable alternative expression system. This is in particular valid for proteins that cannot or not in sufficient yields produced in established systems.

Arsenic is a toxic metalloid that affects human health by causing numerous diseases and by being used in the treatment of acute promyelocytic leukemia. Saccharomyces cerevisiae (budding yeast) has been extensively utilized to elucidate the molecular mechanisms underlying arsenic toxicity and resistance in eukaryotes. In this study, we applied a genomic DNA overexpression strategy to identify yeast genes that provide arsenic resistance in wild-type and arsenic-sensitive S. cerevisiae cells. In addition to known arsenic-related genes, our genetic screen revealed novel genes, including PHO86, VBA3, UGP1, and TUL1, whose overexpression conferred resistance. To gain insights into possible resistance mechanisms, we addressed the contribution of these genes to cell growth, intracellular arsenic, and protein aggregation during arsenate exposure. Overexpression of PHO86 resulted in higher cellular arsenic levels but no additional effect on protein aggregation, indicating that these cells efficiently protect their intracellular environment. VBA3 overexpression caused resistance despite higher intracellular arsenic and protein aggregation levels. Overexpression of UGP1 led to lower intracellular arsenic and protein aggregation levels whilst TUL1 overexpression had no impact on intracellular arsenic or protein aggregation levels. Thus, the identified genes appear to confer arsenic resistance through distinct mechanisms but the molecular details remain to be elucidated.

While plant pathogens are traditionally controlled using synthetic agrochemicals the availability of commercial bactericides is still limited. One potential control strategy could be the use of plant-growth-promoting bacteria (PGPBs) to suppress pathogens via resource competition or the production of antimicrobial compounds. This study aimed to conduct in vitro and in vivo screening of eight Pseudomonas strains against Ralstonia solanacearum (the causative agent of bacterial wilt) and to investigate underlying mechanisms of potential pathogen suppression. We found that inhibitory effects were Pseudomonas strain-specific, with strain CHA0 showing the highest pathogen suppression. Genomic screening identified 2, 4-diacetylphloroglucinol (DAPG), pyoluteorin, and orfamides A and B secondary metabolite clusters in the genomes of the most inhibitory strains, which were investigated further. While all these compounds suppressed R. solanacearum growth, only Orfamide A was produced in the growth media based on mass spectrometry. Moreover, orfamide variants extracted from Pseudomonas cultures showed high pathogen suppression. Using the Micro Tom tomato cultivar, it was found that CHA0 could reduce bacterial wilt disease incidence with one of the two tested pathogen strains. Together, these findings suggest that a better understanding of Pseudomonas-Ralstonia interactions in the rhizosphere is required to successfully translate in vitro findings into agricultural applications.

The relatively long turnaround time and low sensitivity of traditional blood culture may delay the effective antibiotic therapy in patients with bloodstream infection (BSI). To reduce the morbidity and mortality of BSI, a rapid and sensitive pathogen detection method is urgently required. Acinetobacter baumannii and Klebsiella pneumonia are two major microorganisms responsible for BSI. Here we reported a novel droplet digital PCR (ddPCR) method that can detect A. baumannii and K. pneumonia in whole blood samples within 4 h, with a specificity of 100% for each strain and limit of detection at 0.93 copies/microliter for A. baumannii and 0.27 copies/microliter for K. pneumonia. Clinical validation in 170 patients with suspected BSIs showed that, compared with blood culture that reported 4 (2.4%) A. baumannii cases and 7 (4.1%) K. pneumonia cases, ddPCR detected 23 (13.5%) A. baumannii cases, 26 (15.3%) K. pneumonia cases, and 4 (2.4%) dual infection cases, including the 11 positive patients reported by blood culture. In addition, the positive patients reported by ddPCR alone (n = 42) had significantly lower serum concentrations of procalcitonin and lactate, SOFA and APACHE II scores, and 28-day mortality than those reported by both blood culture and ddPCR (n = 11), suggesting that patients with less severe manifestations can potentially benefit from the guidance of ddPCR results. In conclusion, our study suggests that ddPCR represents a sensitive and rapid method to identify causal pathogens in blood samples and to guide the treatment decisions in the early stage of BSI.

Biofilms are intricate communities of microorganisms encapsulated within a self-produced matrix of extra-polymeric substances (EPS), creating complex three-dimensional structures allowing for liquid and nutrient transport through them. These aggregations offer constituent microorganisms enhanced protection from environmental stimuli - like fluid flow - and are also associated with higher resistance to antimicrobial compounds, providing a persistent cause of concern in numerous sectors like the marine (biofouling, aquaculture), medical (infections, antimicrobial resistance), dentistry (plaque on teeth), food safety, as well as causing energy loss and corrosion. Recent studies have demonstrated that biofilms interact with microplastics, often influencing their pathway to higher trophic levels. Previous research has shown that initial bacterial attachment is affected by surface properties. Using a microfluidic flow cell, we have investigated the relationship between both wall shear stress (τw) and surface properties (surface wettability) upon biofilm formation of two species (Cobetia marina and Pseudomonas aeruginosa). We investigated biofilm development on low-density polyethylene (LDPE) membranes, Permanox® slides, and glass slides, using nucleic acid staining and end-point confocal laser scanning microscopy (CLSM). The results show that flow conditions affect biomass, maximum thickness, and surface area of biofilms, with higher τw (5.6 Pa) resulting in thinner biofilms than lower τw (0.2 Pa). In addition, we observed differences in biofilm development across the surfaces tested, with LDPE typically demonstrating more overall biofilm in comparison to Permanox® and glass. Moreover, we demonstrate the formation of biofilm streamers under laminar flow conditions within straight micro-channels.

To understand antibiotic resistance in pathogenic bacteria, we need to monitor environmental microbes as reservoirs of antimicrobial resistance genes (ARGs). These bacteria are present in the air and can be investigated with the whole metagenome shotgun sequencing approach. This study aimed to investigate the feasibility of a method for metagenomic analysis of microbial composition and ARGs in the outdoor air. Air samples were collected with a Harvard impactor in the PM10 range at 50 m from a hospital in Budapest. From the DNA yielded from samples of PM10 fraction single-end reads were generated with an Ion Torrent sequencer. During the metagenomic analysis, reads were classified taxonomically. The core bacteriome was defined. Reads were assembled to contigs and the ARG content was analyzed. The dominant genera in the core bacteriome were Bacillus, Acinetobacter, Leclercia and Paenibacillus. Among the identified ARGs best hits were vanRA, Bla1, mphL, Escherichia coli EF-Tu mutants conferring resistance to Pulvomycin, BcI, FosB, and mphM. Despite the low DNA content of the samples of PM10 fraction, the number of detected airborne ARGs was surprisingly high.

Geobacter species have great application potential in remediation processes and electrobiotechnology. In all applications, understanding the metabolism will enable target-oriented optimization of the processes. The typical electron donor and carbon source of the Geobacter species is acetate, while fumarate is the usual electron acceptor. Here, we could show that depending on the donor/acceptor ratio in batch cultivation of G. sulfurreducens different product patterns occur. With a donor/acceptor ratio of 1:2.5 malate accumulated as an intermediate product but was metabolized to succinate subsequently. At the end of the cultivation, the ratio of fumarate consumed and succinate produced was approximately 1:1. When fumarate was added in excess, malate accumulated in the fermentation broth without further metabolization. After the addition of acetate to stationary cells, malate concentration decreased immediately and additional succinate was synthesized. Finally, it was shown that also resting cells of G. sulfurreducens could efficiently convert fumarate to malate without an additional electron donor. Overall, it was demonstrated that by altering the donor/acceptor ratio, targeted optimization of the metabolite conversion by G. sulfurreducens can be realized.

Subsurface chlorophyll maxima layers (SCML) are ubiquitous features of stratified aquatic systems. Availability of the micronutrient iron is known to influence marine SCML, but iron has not been explored in detail as a factor in the development of freshwater SCML. This study investigates the relationship between dissolved iron and the SCML within the dimictic, ferruginous lake Grosses Heiliges Meer in northern Germany. The occurrence of the SCML under non-ferruginous conditions in the spring and ferruginous conditions in the fall are context to explore temporal changes in the phytoplankton community and indicators of primary productivity. Results indicate that despite more abundant chlorophyll in the spring, the SCML sits below a likely primary productivity maximum within the epilimnion, inferred based on co-located dissolved oxygen, δ13CDIC, and pH maxima. The peak amount of chlorophyll in the SCML is lower in the fall than in the spring, but in the fall the SCML is co-located with elevated dissolved iron concentrations and a local δ13CDIC maximum. Cyanobacteria and Chlorophyta have elevated abundances within the SCML in the fall. Further investigation of the relationship of iron to primary productivity within ferruginous SCML may help to understand the environmental controls on primary productivity in past ferruginous oceans.

Denitrification plays a central role in the global nitrogen cycle, reducing and removing nitrogen from marine and terrestrial ecosystems. The flux of nitrogen species through this pathway has a widespread impact, affecting ecological carrying capacity, agriculture, and climate. Nitrite reductase (Nir) and nitric oxide reductase (NOR) are the two central enzymes in this pathway. Here we present a previously unreported Nir domain architecture in members of Phylum Chloroflexi. Phylogenetic analyses of protein domains within Nir indicate that an ancestral horizontal transfer and fusion event produced this chimeric domain architecture. We also identify an expanded genomic diversity of a rarely reported nitric oxide reductase subtype, eNOR. Together, these results suggest a greater diversity of denitrification enzyme arrangements exist than have been previously reported.

The CRISPR-Cas system of Prokaryotes is an adaptive immune defense mechanism to protect themselves from invading genetic elements (e.g. phages and plasmids). Studies that describe the genetic organization of these prokaryotic systems have mainly reported on the Enterobacteriaceae family (now reorganized within the order Enterobacteriales). For some genera, data on CRISPR-Cas systems remain poor, as in the case of Serratia (now part of the Yersiniaceae family) where data are limited to a few genomes of the species marcescens. This study describes the detection, in silico, of CRISPR loci in 146 Serratia complete genomes and 336 high-quality assemblies available for the species ficaria, fonticola, grimesii, inhibens, liquefaciens, marcescens, nematodiphila, odorifera, oryzae, plymuthica, proteomaculans, quinivorans, rubidaea, symbiotic, and ureilytica. Apart from subtypes I-E and I-F1, which had previously been identified in marcescens, we report that of I-C and the variants I-ES1, I-ES2 and I-F1S1. Analysis of the genomic contexts for CRISPR loci revealed mdtN-phnP as the region mostly shared (grimesii, inhibens, marcescens, nematodiphila, plymuthica, rubidaea, and Serratia sp.). Three new contexts detected in genomes of rubidaea and fonticola (puu genes-mnmA) and rubidaea (osmE-soxG and ampC-yebZ) were also found. Plasmid and/or phage origin of spacers was also established.

Identification, source tracking, and surveillance of food pathogens is a crucial factor for the food-producing industry. Over the last decade, the techniques used for this have moved from conventional enrichment methods, through species-specific detection by PCR to sequencing-based methods, whole-genome sequencing (WGS) being the ultimate method. However, using WGS requires the right infrastructure, high computational power, and bioinformatics expertise. Therefore, there is a need for faster, more cost-effective, and more user-friendly methods. A newly developed method, ON-rep-seq, combines the classical rep-PCR method with nanopore sequencing, resulting in a highly discriminating set of sequences that can be used for species identification and also strain discrimination. This study is essentially a real industry case from a salmon processing plant. Twenty Listeria monocytogenes isolates were analyzed both by ON-rep-seq and WGS to identify and differentiate putative L. monocytogenes from a routine sampling of processing equipment and products, and finally, compare the strain-level discriminatory power of ON-rep-seq to different analyzing levels delivered from the WGS data. The analyses revealed that among the isolates tested there were three different strains. The isolates of the most frequently detected strain (n=15) were all detected in the problematic area in the processing plant. The strain level discrimination done by ON-rep-seq was in full accordance with the interpretation of WGS data. Our findings also demonstrate that ON-rep-seq may serve as a primary screening method alternative to WGS for identification and strain-level differentiation for surveillance of potential pathogens in a food-producing environment.

The transcription machinery of archaea can be roughly classified as a simplified version of eukaryotic organisms. The basal transcription factor machinery binds to the TATA-box found around 28 nucleotides upstream of the transcription start site; however, some transcription units lack a clear TATA-box and still have TBP/TFB binding over them. This apparent absence of conserved sequences could be a consequence of sequence divergence associated with the upstream region, operonic and gene organization. Furthermore, earlier studies have found that a structural analysis gains more information compared to a simple sequence inspection. In this work, we evaluated and coded 3630 archaeal promoter sequences of three organisms, Haloferax volcanii, Thermococcus kodakarensis, and Sulfolobus solfataricus into DNA duplex stability, enthalpy, curvature, and bendability parameters. We also split our dataset into conserved TATA and degenerated TATA promoters in order to identify differences among these two classes of promoters. The structural analysis reveals variations in archaeal promoters’ architecture, i.e., a distinctive signal is observed in the TFB, TBP, and TFE binding sites independently of these being TATA-conserved or TATA-degenerated. In addition, the promoter encountering method was validated with upstream regions of 13 other archaea, suggesting that there might be promoter sequences among them. Therefore, we suggest a novel method for locating promoters within the genome of archaea based on energetic/structural features.

Machine learning methods can be used as robust techniques to provide invaluable information for analyzing biological samples in pharmaceutical industries, such as predicting the concentration of viral particles of interest in biological samples. Here, we utilized both convolutional neural networks and random forests to predict the concentration of the samples containing measles, mumps, rubella, and varicella-zoster viruses (ProQuad®) based on Raman and absorption spectroscopy. We prepared Raman and absorption spectra datasets with known concentration values, then used the Raman and absorption signals individually and together to train RFs and CNNs. We demonstrated that both RFs and CNNs can make predictions with R2 values as high as 95%. We proposed two different networks to jointly use the Raman and absorption spectra, where our results demonstrated that concatenating the Raman and absorption data increases the prediction accuracy compared to using either Raman or absorption spectrum alone. Additionally, we further verified the advantage of using joint Raman-absorption with principal component analysis (PCA). Furthermore, our method can be extended to characterize properties other than concentration, such as the type of viral particles.

Interspecific interactions within biofilms determine relative species abundance, growth dynamics, community resilience, and success or failure of invasion by an extraneous organism. However, deciphering interspecific interactions and assessing their contribution to biofilm properties and function remain a challenge. Here, we describe the constitution of a model biofilm composed of four bacterial species belonging to four different genera (Rhodocyclus sp., Pseudomonas fluorescens, Kocuria varians, and Bacillus cereus), derived from a biofilm isolated from an industrial milk pasteurization unit. We demonstrate that the growth dynamics and equilibrium composition of this biofilm are highly reproducible. Based on its equilibrium composition, we show that the establishment of this 4-species biofilm is highly robust against initial, transient perturbations but less so towards continuous perturbations. By comparing biofilms formed from different numbers and combinations of the constituent species and by fitting a growth model to the experimental data, we reveal a network of dynamic, positive, and negative interactions that determine the final composition of the biofilm. Furthermore, we reveal that the molecular determinant of one negative interaction is the thiocillin I synthesized by the B. cereus strain, and demonstrate its importance for species distribution and its impact on robustness by mutational analysis of the biofilm ecosystem.

The enormous complexity of the eukaryotic ribosome has been a real challenge in unlocking the mechanistic aspects of its amazing molecular function during mRNA translation and many non-canonical activities of ribosomal proteins in eukaryotic cells. While exploring the uncanny nature of ribosomal P proteins in malaria parasites Plasmodium falciparum, the 60S stalk ribosomal P2 protein has been shown to get exported to the infected erythrocyte (IE) surface as an SDS resistant oligomer during the early to mid trophozoite stage. Inhibiting IE surface P2 either by monoclonal antibody or through genetic knockdown resulted in nuclear division arrest of the parasite. This very strange and serendipitous finding has led us to explore more about un-canonical cell biology and structural involvement of P2 protein in Plasmodium in the search for a novel biochemical role during parasite propagation in the human host.

Om45 is a major protein of the yeast outer mitochondrial membrane under respiratory conditions. However, the cellular role of the protein has remained obscure. Previously, deletion mutant phenotypes have not been found, and clear amino acid sequence similarities that would allow inferring its functional role are not available. In this work, we describe synthetic petite mutants of UGO1 and GEM1 that depend on the presence of OM45 for respiratory growth, as well as the identification of several multicopy suppressors of the synthetic petite phenotypes. In the analysis of our mutants, we demonstrate that Om45 and Gem1 have a collaborative role in the maintenance of mitochondrial morphology, cristae structure, and mitochondrial DNA maintenance. A group of multicopy suppressors rescuing the synthetic lethal phenotypes of the mutants on non-fermentable carbon sources additionally supports this result. Our results imply that the synthetic petite phenotypes we observed are due to the disturbance of the inner mitochondrial membrane and point to this mitochondrial sub-compartment as the main target of action of Om45, Ugo1, and the yeast Miro GTPase Gem1.

Actinobacteria are important cave inhabitants, but knowledge of how anthropization and anthropization-related visual marks affect this community on cave walls is lacking. We compared Actinobacteria communities among four French limestone caves (Mouflon, Reille, Rouffignac, Lascaux) ranging from pristine to anthropized, and within Lascaux Cave between marked (wall visual marks) and unmarked areas in different rooms (Sas-1, Passage, Apse, Diaclase). In addition to the 16S rRNA gene marker, 441 bp fragments of the hsp65 gene were used and an hsp65-related taxonomic database was constructed for identification of Actinobacteria to the species level by Illumina-MiSeq analysis. The hsp65 marker revealed higher resolution for species and higher richness (99% OTU cutoff) versus 16S rRNA gene; however, more taxa were identified at higher taxonomic ranks. Actinobacteria communities varied between Mouflon and Reille caves (both pristine), and Rouffignac and Lascaux (both anthropized). Rouffignac displayed high diversity of Nocardia, suggesting human inputs, and Lascaux exhibited high Mycobacterium relative abundance, whereas Gaiellales were typical in pristine caves and the Diaclase (least affected area of Lascaux Cave). Within Lascaux, Pseudonocardiaceae dominated on unmarked walls and Streptomycetaceae (especially Streptomyces mirabilis) on marked walls, indicating a possible role in mark formation. A new taxonomic database (https://zenodo.org/record/5576074) was developed. Although not all Actinobacteria species were represented, the use of the hsp65 marker enabled species-level variations of the Actinobacteria community to be documented based on the extent of anthropogenic pressure. This approach proved effective when comparing different limestone caves or specific conditions within one cave.

Background. Symbioses between Geosmithia fungi and wood-boring and bark beetles seldom result in disease induction within the plant host. Yet exceptions exist such as Geosmithia morbida, the causal agent of Thousand Cankers Disease (TCD) of walnuts and wingnuts, and Geosmithia sp. 41, the causal agent of Foamy Bark Canker disease of oaks. Isolates of G. obscura were recovered from black walnut trees in eastern Tennessee and at least one isolate induced cankers following artificial inoculation. Due to the putative pathogenicity and lack of recovery of G. obscura from natural lesions, a molecular diagnostic screening tool was developed using microsatellite markers mined from the G. obscura genome. Results. A total of 3,256 candidate microsatellite markers were identified (2236, 789, 137 di-, tri-, and tetra- motifs were identified, respectively), with 2011, 703, 101 di-, tri-, and tetra- motifs containing markers with primers. From these, 75 microsatellite markers were randomly selected, screened, and optimized, resulting in 28 polymorphic markers that yielded single, consistently recovered bands which were used in downstream analyses. Five of these microsatellite markers were found to be specific to G. obscura and did not cross-amplify into other, closely related species. Although the remaining tested markers could be useful, they cross-amplified within different Geosmithia species, making them not reliable for G. obscura detection. Conclusion. Five novel microsatellite markers (GOBS9, GOBS10, GOBS41, GOBS43, GOBS50) were developed based on the G. obscura genome. These species-specific microsatellite markers are available as a tool for use in molecular diagnostics and can assist future surveillance studies.