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ABSTRACT Escherichia coli is a commensal inhabitant of the mammalian gut microbiota, frequently associated with various gastrointestinal diseases. There is increasing interest in comprehending the variety of bacteriophages (phages) that target this bacterium, as such insights could pave the way for their potential use in therapeutic applications. Here, we report the isolation and characterization of four newly identified E. coli infecting tailed phages (W70, A7-1, A5-4, and A73) that were found to constitute a novel genus, Septuagintavirus, within the subfamily Vequintavirinae. Genomes of these phages ranged from 137 kbp to 145 kbp, with a GC content of 41 mol%. They possess a maximum nucleotide similarity of 30% with phages of the closest phylogenetic genus, Certrevirus, while displaying limited homology to other genera of the Vequintavirinae family. Host range analysis showed that these phages have limited activity against a panel of E. coli strains, infecting 6 out of 16 tested isolates, regardless of their phylotype. Electrospray ionization-tandem mass spectrometry (ESI-MS/MS) was performed on the virion of phage W70, allowing the identification of 28 structural proteins, 19 of which were shared with phages of other genera of Vequintavirinae family. The greatest diversity was identified with proteins forming tail fiber structures, likely indicating the adaptation of virions of each phage genus of this subfamily for the recognition of their target receptor on host cells. The findings of this study provide greater insights into the phages of the subfamily Vequintavirinae, contributing to the pool of knowledge currently known about these phages.IMPORTANCEEscherichia coli is a well-known bacterium that inhabits diverse ecological niches, including the mammalian gut microbiota. Certain strains are associated with gastrointestinal diseases, and there is a growing interest in using bacteriophages, viruses that infect bacteria, to combat bacterial infections. Here, we describe the isolation and characterization of four novel E. coli bacteriophages that constitute a new genus, Septuagintavirus, within the subfamily Vequintavirinae. We conducted mass spectrometry on virions of a representative phage of this novel clade and compared it to other phages within the subfamily. Our analysis shows that virion structure is highly conserved among all phages, except for proteins related to tail fiber structures implicated in the host range. These findings provide greater insights into the phages of the subfamily Vequintavirinae, contributing to the existing pool of knowledge about these phages.

Abstract NAD is a coenzyme central to metabolism that also serves as a 5′-terminal cap for bacterial and eukaryotic transcripts. Thermal degradation of NAD can generate nicotinamide and ADP-ribose (ADPR). Here, we use LC-MS/MS and NAD captureSeq to detect and identify NAD-RNAs in the thermophilic model archaeon Sulfolobus acidocaldarius and in the halophilic mesophile Haloferax volcanii. None of the four Nudix proteins of S. acidocaldarius catalyze NAD-RNA decapping in vitro, but one of the proteins (Saci_NudT5) promotes ADPR-RNA decapping. NAD-RNAs are converted into ADPR-RNAs, which we detect in S. acidocaldarius total RNA. Deletion of the gene encoding the 5′−3′ exonuclease Saci-aCPSF2 leads to a 4.5-fold increase in NAD-RNA levels. We propose that the incorporation of NAD into RNA acts as a degradation marker for Saci-aCPSF2. In contrast, ADPR-RNA is processed by Saci_NudT5 into 5′-p-RNAs, providing another layer of regulation for RNA turnover in archaeal cells.

Abstract The current threat of multidrug resistant strains necessitates development of alternatives to antibiotics such as bacteriophages. This study describes the isolation and characterization of a novel Salmonella Typhimurium phage ‘Arash’ from hospital wastewater in Leuven, Belgium. Arash has a myovirus morphology with a 95 nm capsid and a 140 nm tail. The host range of Arash is restricted to its isolation host. Approximately 86% of the phage particles are adsorbed to a host cell within 10 min. Arash has latent period of 65 min and burst size of 425 PFU/cell. Arash has a dsDNA genome of 180,819 bp with GC content of 53.02% with no similarities to any characterized phages, suggesting Arash as a novel species in the novel ‘Arashvirus’ genus. Arash carries no apparent lysogeny-, antibiotic resistance- nor virulence-related genes. Proteome analysis revealed 116 proteins as part of the mature phage particles of which 27 could be assigned a function. Therefore, the present findings shed light on the morphological, microbiological and genomic characteristics of Arash and suggest its potential application as therapeutic and/or biocontrol agent.

ABSTRACT Protein Ser/Thr kinases are posttranslational regulators of key molecular processes in bacteria, such as cell division and antibiotic tolerance. Here, we characterize the E. coli toxin YjjJ (HipH), a putative protein kinase annotated as a member of the family of HipA-like Ser/Thr kinases, which are involved in antibiotic tolerance. Using SILAC-based phosphoproteomics we provide experimental evidence that YjjJ is a Ser/Thr protein kinase and its primary protein substrates are the ribosomal protein RpmE (L31) and the carbon storage regulator CsrA. YjjJ activity impacts ribosome assembly, cell division, and central carbon metabolism but it does not increase antibiotic tolerance as does its homologue HipA. Intriguingly, overproduction of YjjJ and its kinase-deficient variant can activate HipA and other kinases, pointing to a cross talk between Ser/Thr kinases in E. coli. IMPORTANCE Adaptation to growth condition is the key for bacterial survival, and protein phosphorylation is one of the strategies adopted to transduce extracellular signal in physiological response. In a previous work, we identified YjjJ, a putative kinase, as target of the persistence-related HipA kinase. Here, we performed the characterization of this putative kinase, complementing phenotypical analysis with SILAC-based phosphoproteomics and proteomics. We provide the first experimental evidence that YjjJ is a Ser/Thr protein kinase, having as primary protein substrates the ribosomal protein RpmE (L31) and the carbon storage regulator CsrA. We show that overproduction of YjjJ has a major influence on bacterial physiology, impacting DNA segregation, cell division, glycogen production, and ribosome assembly.

Bacteriophages are highly abundant viruses of bacteria. The major role of phages in shaping bacterial communities and their emerging medical potential as antibacterial agents has triggered a rebirth of phage research. To understand the molecular mechanisms by which phages hijack their host, omics technologies can provide novel insights into the organization of transcriptional and translational events occurring during the infection process. In this study, we apply transcriptomics and proteomics to characterize the temporal patterns of transcription and protein synthesis during the T4 phage infection of E. coli. We investigated the stability of E. coli-originated transcripts and proteins in the course of infection, identifying the degradation of E. coli transcripts and the preservation of the host proteome. Moreover, the correlation between the phage transcriptome and proteome reveals specific T4 phage mRNAs and proteins that are temporally decoupled, suggesting post-transcriptional and translational regulation mechanisms. This study provides the first comprehensive insights into the molecular takeover of E. coli by bacteriophage T4. This data set represents a valuable resource for future studies seeking to study molecular and regulatory events during infection. We created a user-friendly online tool, POTATO4, which is available to the scientific community and allows access to gene expression patterns for E. coli and T4 genes.

Bacteria are dependent on rapid alterations in gene expression. A prerequisite for rapid adaptations is efficient RNA turnover, with endonuclease RNase Y playing a crucial role in mRNA stability as well as in maturation. In Bacillus subtilis, RNase Y in turn interacts with the so-called “Y-complex” consisting of three proteins, which play important functions in sporulation, natural transformation and biofilm formation. It is thought that the Y-complex acts as an accessory factor in RNase Y regulation but might also have independent functions. Using single-molecule tracking, we show that all three Y-complex proteins exhibit three distinct mobilities, including movement through the cytosol and confined motion, predominantly at membrane-proximal sites but also within the cell center. A transcriptional arrest leads to a strong change in localization and dynamics of YmcA, YlbF and YaaT, supporting their involvement in global RNA degradation. However, Y-complex proteins show distinguishable protein dynamics, and the deletion of yaaT or ylbF shows a minor effect on the dynamics of YmcA. Cell fractionation reveals that YaaT displays a mixture of membrane association and presence in the cytosol, while YlbF and YmcA do not show direct membrane attachment. Taken together, our experiments reveal membrane-associated and membrane-independent activities of Y-complex proteins and a dynamic interplay between them with indirect membrane association of YmcA and YlbF via YaaT.

RNA 5′-modifications are known to extend the functional spectrum of ribonucleotides. In recent years, numerous non-canonical 5′-modifications, including adenosine-containing cofactors from the group of B vitamins, have been confirmed in all kingdoms of life. The structural component of thiamine adenosine triphosphate (thiamine-ATP), a vitamin B1 derivative found to accumulate in Escherichia coli and other organisms in response to metabolic stress conditions, suggests an analogous function as a 5′-modification of RNA. Here, we report the synthesis of thiamine adenosine dinucleotides and the preparation of pure 5′-thiamine-capped RNAs based on phosphorimidazolide chemistry. Furthermore, we present the incorporation of thiamine-ATP and thiamine adenosine diphosphate (thiamine-ADP) as 5′-caps of RNA by T7 RNA polymerase. Transcripts containing the thiamine modification were modified specifically with biotin via a combination of thiazole ring opening, nucleophilic substitution and copper-catalyzed azide-alkyne cycloaddition. The highlighted methods provide easy access to 5′-thiamine RNA, which may be applied in the development of thiamine-specific RNA capture protocols as well as the discovery and confirmation of 5′-thiamine-capped RNAs in various organisms.

The complexity of the transcriptome is governed by the intricate interplay of transcription, RNA processing, translocation, and decay. In eukaryotes, the removal of the 5’-RNA cap is essential for the initiation of RNA degradation. In addition to the canonical 5’-N7-methyl guanosine cap in eukaryotes, the ubiquitous redox cofactor nicotinamide adenine dinucleotide (NAD) was identified as a new 5’-RNA cap structure in prokaryotic and eukaryotic organisms. So far, two classes of NAD-RNA decapping enzymes have been identified, namely Nudix enzymes that liberate nicotinamide mononucleotide (NMN) and DXO-enzymes that remove the entire NAD cap. Herein, we introduce 8-(furan-2-yl)-substituted NAD-capped-RNA (FurNAD-RNA) as a new research tool for the identification and characterization of novel NAD-RNA decapping enzymes. These compounds are found to be suitable for various enzymatic reactions that result in the release of a fluorescence quencher, either nicotinamide (NAM) or nicotinamide mononucleotide (NMN), from the RNA which causes a fluorescence turn-on. FurNAD-RNAs allow for real-time quantification of decapping activity, parallelization, high-throughput screening and identification of novel decapping enzymes in vitro. Using FurNAD-RNAs, we discovered that the eukaryotic glycohydrolase CD38 processes NAD-capped RNA in vitro into ADP-ribose-modified-RNA and nicotinamide and therefore might act as a decapping enzyme in vivo. The existence of multiple pathways suggests that the decapping of NAD-RNA is an important and regulated process in eukaryotes.

Recent evidence indicates that regulatory small non-coding RNAs are not only components of eukaryotic cells and vesicles, but also reside within a number of different viruses including retroviral particles. Using ultra-deep sequencing we have comprehensively analyzed the content of simian immunodeficiency virions (SIV), which were compared to mock-control preparations. Our analysis revealed that more than 428,000 sequence reads matched the SIV mac239 genome sequence. Among these we could identify 12 virus-derived small RNAs (vsRNAs) that were highly abundant. Beside known retrovirus-enriched small RNAs, like 7SL-RNA, tRNA(Lys3) and tRNA(Lys) isoacceptors, we also identified defined fragments derived from small ILF3/NF90-associated RNA snaR-A14, that were enriched more than 50 fold in SIV. We also found evidence that small nucleolar RNAs U2 and U12 were underrepresented in the SIV preparation, indicating that the relative number or the content of co-isolated exosomes was changed upon infection. Our comprehensive atlas of SIV-incorporated small RNAs provides a refined picture of the composition of retrovirions, which gives novel insights into viral packaging.

Abstract Background Fatty acid-derived products such as fatty alcohols (FAL) find growing application in cosmetic products, lubricants, or biofuels. So far, FAL are primarily produced petrochemically or through chemical conversion of bio-based feedstock. Besides the well-known negative environmental impact of using fossil resources, utilization of bio-based first-generation feedstock such as palm oil is known to contribute to the loss of habitat and biodiversity. Thus, the microbial production of industrially relevant chemicals such as FAL from second-generation feedstock is desirable. Results To engineer Corynebacterium glutamicum for FAL production, we deregulated fatty acid biosynthesis by deleting the transcriptional regulator gene fasR, overexpressing a fatty acyl-CoA reductase (FAR) gene of Marinobacter hydrocarbonoclasticus VT8 and attenuating the native thioesterase expression by exchange of the ATG to a weaker TTG start codon. C. glutamicum ∆fasR cg2692TTG (pEKEx2-maqu2220) produced in shaking flasks 0.54 ± 0.02 gFAL L−1 from 20 g glucose L−1 with a product yield of 0.054 ± 0.001 Cmol Cmol−1. To enable xylose utilization, we integrated xylA encoding the xylose isomerase from Xanthomonas campestris and xylB encoding the native xylulose kinase into the locus of actA. This approach enabled growth on xylose. However, adaptive laboratory evolution (ALE) was required to improve the growth rate threefold to 0.11 ± 0.00 h−1. The genome of the evolved strain C. glutamicum gX was re-sequenced, and the evolved genetic module was introduced into C. glutamicum ∆fasR cg2692TTG (pEKEx2-maqu2220) which allowed efficient growth and FAL production on wheat straw hydrolysate. FAL biosynthesis was further optimized by overexpression of the pntAB genes encoding the membrane-bound transhydrogenase of E. coli. The best-performing strain C. glutamicum ∆fasR cg2692TTG CgLP12::(P tac -pntAB-T rrnB ) gX (pEKEx2-maqu2220) produced 2.45 ± 0.09 gFAL L−1 with a product yield of 0.054 ± 0.005 Cmol Cmol−1 and a volumetric productivity of 0.109 ± 0.005 gFAL L−1 h−1 in a pulsed fed-batch cultivation using wheat straw hydrolysate. Conclusion The combination of targeted metabolic engineering and ALE enabled efficient FAL production in C. glutamicum from wheat straw hydrolysate for the first time. Therefore, this study provides useful metabolic engineering principles to tailor this bacterium for other products from this second-generation feedstock.

In the face of emerging technological achievements, cyber security remains a significant issue. Despite the new possibilities that arise with such development, these do not come without a drawback. Attackers make use of the new possibilities to take advantage of possible security defects in new systems. Advanced-persistent-threat (APT) attacks represent sophisticated attacks that are executed in multiple steps. In particular, network systems represent a common target for APT attacks where known or yet undiscovered vulnerabilities are exploited. For this reason, intrusion detection systems (IDS) are applied to identify malicious behavioural patterns in existing network datasets. In recent times, machine-learning (ML) algorithms are used to distinguish between benign and anomalous activity in such datasets. The application of such methods, especially autoencoders, has received attention for achieving good detection results for APT attacks. This paper builds on this fact and applies several autoencoder-based methods for the detection of such attack patterns in two datasets created by combining two publicly available benchmark datasets. In addition to that, statistical analysis is used to determine features to supplement the anomaly detection process. An anomaly detector is implemented and evaluated on a combination of both datasets, including two experiment instances–APT-attack detection in an independent test dataset and in a zero-day-attack test dataset. The conducted experiments provide promising results on the plausibility of features and the performance of applied algorithms. Finally, a discussion is provided with suggestions of improvements in the anomaly detector.

Epidemiology of Fusarium Head Blight (FHB) of spring barley is relatively little understood. In a five-year study, we assessed quantitative resistance to FHB in an assortment of 17 spring barley genotypes in the field in southern Germany. To this end, we used soil and spray inoculation of plants with F. culmorum and F. avenaceum. This increased disease pressure and provoked genotypic differentiation. To normalize effects of variable weather conditions across consecutive seasons, we used a disease ranking of the genotypes based on quantification of fungal DNA contents and multiple Fusarium toxins in harvested grain. Together, this allowed for assessment of stable quantitative FHB resistance of barley in several genotypes. Fungal DNA contents were positively associated with species-specific Fusarium toxins in single years and over several years in plots with soil inoculation. In those plots, plant height limited FHB; however, this was not observed after spray inoculation. A multiple linear regression model of recorded weather parameter and fungal DNA contents over five years identified time periods during the reproductive phase of barley, in which weather strongly influenced fungal colonization measured in mature barley grain. Environmental conditions before heading and late after anthesis showed strongest associations with F. culmorum DNA in all genotypes, whereas for F. avenaceum, this was less consistent where we observed weather-dependent associations, depending on the genotype. Based on this study, we discuss aspects of practical resistance breeding in barley relevant to improve quantitative resistance to FHB and associated mycotoxin contaminations.

Smart Grids (SGs) represent electrical power systems that incorporate increased information processing and efficient technological solutions. The integration of local prosumers, demand response systems and storage allows novel possibilities with regard to energy balancing and optimization of grid operations. Unfortunately, the dependence on IT leaves the SG exposed to security violations. In this paper, we contribute to this challenge and provide a methodology for systematic risk assessment of cyber attacks in SG systems. We propose a threat model and identify possible vulnerabilities in low-voltage distribution grids. Then, we calculate exploitation probabilities from realistic attack scenarios. Lastly, we apply formal verification to check the stochastic model against attack properties. The obtained results provide insight into potential threats and the likeliness of successful attacks. We elaborate on the effects of a security violation with regard to security and privacy of energy clients. In the aftermath, we discuss future considerations for improving security in the critical energy sector.

Financial technology, or Fintech, represents an emerging industry on the global market. With online transactions on the rise, the use of IT for automation of financial services is of increasing importance. Fintech enables institutions to deliver services to customers worldwide on a 24/7 basis. Its services are often easy to access and enable customers to perform transactions in real-time. In fact, advantages such as these make Fintech increasingly popular among clients. However, since Fintech transactions are made up of information, ensuring security becomes a critical issue. Vulnerabilities in such systems leave them exposed to fraudulent acts, which cause severe damage to clients and providers alike. For this reason, techniques from the area of Machine Learning (ML) are applied to identify anomalies in Fintech applications. They target suspicious activity in financial datasets and generate models in order to anticipate future frauds. We contribute to this important issue and provide an evaluation on anomaly detection methods for this matter. Experiments were conducted on several fraudulent datasets from real-world and synthetic databases, respectively. The obtained results confirm that ML methods contribute to fraud detection with varying success. Therefore, we discuss the effectiveness of the individual methods with regard to the detection rate. In addition, we provide an analysis on the influence of selected features on their performance. Finally, we discuss the impact of the observed results for the security of Fintech applications in the future.