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  • 2019  (21)
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  • 2019  (21)
  • 1
    In: Nucleic Acids Research, 2019, Vol.47(4), p.2075-2088
    Description: Abstract Global RNA profiling studies in bacteria have predicted the existence of many of small noncoding RNAs (sRNAs) that are processed off mRNA 3′ ends to regulate other mRNAs via the RNA chaperones Hfq and ProQ. Here, we present targets of SdhX (RybD), an Hfq-dependent sRNA that is generated by RNase E mediated 3′ processing of the ∼10 000-nt mRNA of the TCA cycle operon sdhCDAB-sucABCD in enteric bacteria. An in silico search predicted ackA mRNA, which encodes acetate kinase, as a conserved primary target of SdhX. Through base pairing, SdhX represses AckA synthesis during growth of Salmonella on acetate. Repression can be achieved by a naturally occurring 38-nucleotide SdhX variant, revealing the shortest functional Hfq-associated sRNA yet. Salmonella SdhX also targets the mRNAs of fumB (anaerobic fumarase) and yfbV , a gene of unknown function adjacent to ackA . Instead, through a slightly different seed sequence, SdhX can repress other targets in Escherichia coli , namely katG (catalase) and fdoG (aerobic formate dehydrogenase). This study illustrates how a key operon from central metabolism is functionally connected to other metabolic pathways through a 3′ appended sRNA, and supports the notion that mRNA 3′UTRs are a playground for the evolution of regulatory RNA networks in bacteria.
    Keywords: Rna And Rna-Protein Complexes
    ISSN: 0305-1048
    E-ISSN: 1362-4962
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  • 2
    Language: English
    In: RNA biology, April 2019, Vol.16(4), pp.390-396
    Description: Neisseria meningitidis, a commensal β-proteobacterium of the human nasopharynx, constitutes a worldwide leading cause of sepsis and epidemic meningitis. A recent genome-wide association study suggested an association of its type II-C CRISPR/Cas system with carriage and thus less invasive lineages. Here, we show that knock-out strains lacking the Cas9 protein are impaired in the adhesion to human nasopharyngeal cells which constitutes a central step in the pathogenesis of invasive meningococcal disease. Transcriptome sequencing data further suggest that meningococcal Cas9 does not affect the expression of surface adhesins but rather exerts its effect on cell adhesion in an indirect manner. Consequently, we speculate that the meningococcal CRISPR/Cas system exerts novel functions beyond its established role in defence against foreign DNA.
    Keywords: Crispr/Cas ; Cas9 ; RIP-Seq ; RNA-Seq ; Nasopharynx ; Virulence
    ISSN: 15476286
    E-ISSN: 1555-8584
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  • 3
    Language: English
    In: mBio, 04/30/2019, Vol.10(2)
    Description: Proteins comprised of 50 or fewer amino acids have been shown to interact with and modulate the functions of larger proteins in a range of organisms. Despite the possible importance of small proteins, the true prevalence and capabilities of these regulators remain unknown as the small size of the proteins places serious limitations on their identification, purification, and characterization. Here, we present a ribosome profiling approach with stalled initiation complexes that led to the identification of 38 new small proteins.Small proteins consisting of 50 or fewer amino acids have been identified as regulators of larger proteins in bacteria and eukaryotes. Despite the importance of these molecules, the total number of small proteins remains unknown because conventional annotation pipelines usually exclude small open reading frames (smORFs). We previously identified several dozen small proteins in the model organism Escherichia coli using theoretical bioinformatic approaches based on sequence conservation and matches to canonical ribosome binding sites. Here, we present an empirical approach for discovering new proteins, taking advantage of recent advances in ribosome profiling in which antibiotics are used to trap newly initiated 70S ribosomes at start codons. This approach led to the identification of many novel initiation sites in intergenic regions in E. coli. We tagged 41 smORFs on the chromosome and detected protein synthesis for all but three. Not only are the corresponding genes intergenic but they are also found antisense to other genes, in operons, and overlapping other open reading frames (ORFs), some impacting the translation of larger downstream genes. These results demonstrate the utility of this method for identifying new genes, regardless of their genomic context.
    Keywords: Ribo-Seq ; Small Protein ; Alternate Orfs ; Antisense ; Genome Annotation ; Leader Peptide ; Biology;
    ISSN: mBio
    ISSN: 21612129
    E-ISSN: 2150-7511
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  • 4
    Language: English
    In: Journal of Neural Engineering, 2019, Vol.16(2), p.026039 (10pp)
    Description: Objective . Currently, there are some 95 000 people in Europe suffering from upper-limb impairment. Rehabilitation should be undertaken right after the impairment occurs and should be regularly performed thereafter. Moreover, the rehabilitation process should be tailored specifically to both patient and impairment. Approach . To address this, we have developed a low-cost solution that integrates an off-the-shelf virtual reality (VR) setup with our in-house developed arm/hand intent detection system. The resulting system, called VITA, enables an upper-limb disabled person to interact in a virtual world as if her impaired limb were still functional. VITA provides two specific features that we deem essential: proportionality of force control and interactivity between the user and the intent detection core. The usage of relatively cheap commercial components enables VITA to be used in rehabilitation centers, hospitals, or even at home. The applications of VITA range from rehabilitation of patients with musculodegenerative conditions (e.g. ALS), to treating phantom-limb pain of people with limb-loss and prosthetic training. Main results . We present a multifunctional system for upper-limb rehabilitation in VR. We tested the system using a VR implementation of a standard hand assessment tool, the Box and Block test and performed a user study on this standard test with both intact subjects and a prosthetic user. Furthermore, we present additional applications, showing the versatility of the system. Significance . The VITA system shows the applicability of a combination of our experience in intent detection with state-of-the art VR system for rehabilitation purposes. With VITA, we have an easily adaptable experimental tool available, which allows us to quickly and realistically simulate all kind of real-world problems and rehabilitation exercises for upper-limb impaired patients. Additionally, other scenarios such as prostheses simulations and control modes can be quickly implemented and tested.
    ISSN: 1741-2560
    E-ISSN: 1741-2552
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  • 5
    In: mBio, 2019, Vol.10(1)
    Description: The protein ProQ has recently been discovered as the centerpiece of a previously overlooked “third domain” of small RNA-mediated control of gene expression in bacteria. As in vitro work continues to reveal molecular mechanisms, it is also important to understand how ProQ affects the life cycle of bacterial pathogens as these pathogens infect eukaryotic cells. Here, we have determined how ProQ shapes Salmonella virulence and how the activities of this RNA-binding protein compare with those of Hfq, another central protein in RNA-based gene regulation in this and other bacteria. To this end, we apply global transcriptomics of pathogen and host cells during infection. In doing so, we reveal ProQ-dependent transcript changes in key virulence and host immune pathways. Moreover, we differentiate the roles of ProQ from those of Hfq during infection, for both coding and noncoding transcripts, and provide an important resource for those interested in ProQ-dependent small RNAs in enteric bacteria. ABSTRACT FinO domain proteins such as ProQ of the model pathogen Salmonella enterica have emerged as a new class of major RNA-binding proteins in bacteria. ProQ has been shown to target hundreds of transcripts, including mRNAs from many virulence regions, but its role, if any, in bacterial pathogenesis has not been studied. Here, using a Dual RNA-seq approach to profile ProQ-dependent gene expression changes as Salmonella infects human cells, we reveal dysregulation of bacterial motility, chemotaxis, and virulence genes which is accompanied by altered MAPK (mitogen-activated protein kinase) signaling in the host. Comparison with the other major RNA chaperone in Salmonella , Hfq, reinforces the notion that these two global RNA-binding proteins work in parallel to ensure full virulence. Of newly discovered infection-associated ProQ-bound small noncoding RNAs (sRNAs), we show that the 3′UTR-derived sRNA STnc540 is capable of repressing an infection-induced magnesium transporter mRNA in a ProQ-dependent manner. Together, this comprehensive study uncovers the relevance of ProQ for Salmonella pathogenesis and highlights the importance of RNA-binding proteins in regulating bacterial virulence programs.
    Keywords: Research Article ; Molecular Biology And Physiology ; Editor'S Pick ; Hfq ; Noncoding Rna ; Proq ; Rna-Seq ; Bacterial Pathogen ; Posttranscriptional Control
    ISSN: 21612129
    E-ISSN: 2150-7511
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  • 6
    Language: English
    In: mBio, 04/30/2019, Vol.10(2)
    Description: The ability of bacteria to sense and respond to environmental signals is critical for survival. Bacteria use cyclic dinucleotides as second messengers to regulate a number of physiological processes, such as the fundamental life style transition between motility and sessility (biofilm formation). cGAMP, which is synthesized by a dinucleotide cyclase called DncV, is a newly discovered second messenger involved in virulence and chemotaxis in the Vibrio cholerae biovar El Tor causing the current 7th cholera pandemic. However, to what extent cGAMP exists and participates in physiological processes in other bacteria is still unknown. In this study, we found an elevated cGAMP level to possibly regulate biofilm formation and motility in the animal commensal E. coli strain ECOR31. Thus, we detected a novel role for cGAMP signaling in regulation of physiological processes other than those previously reported in proteobacterial species.Cyclic dinucleotides (cDNs) act as intracellular second messengers, modulating bacterial physiology to regulate the fundamental life style transition between motility and sessility commonly known as biofilm formation. Cyclic GMP-AMP (cGAMP), synthesized by the dinucleotide cyclase DncV, is a newly discovered cDN second messenger involved in virulence and chemotaxis in Vibrio cholerae O1 biovar El Tor. Here we report a novel role for horizontally transferred DncV in cGAMP production and regulation of biofilm formation and motility in the animal commensal strain Escherichia coli ECOR31. ECOR31 expresses a semiconstitutive temperature-independent rdar (red, dry, and rough) morphotype on Congo red agar plates characterized by the extracellular matrix components cellulose and curli fimbriae which requires activation by the major biofilm regulator CsgD and cyclic di-GMP signaling. In contrast, C-terminal His-tagged DncV negatively regulates the rdar biofilm morphotype and cell aggregation via downregulation of csgD mRNA steady-state level. Furthermore, DncV sequentially promotes and inhibits adhesion to the abiotic surface after 24 h and 48 h of growth, respectively. DncV also suppresses swimming and swarming motility posttranscriptional of the class 1 flagellum regulon gene flhD. Purified DncV produced different cDNs, cyclic di-GMP, cyclic di-AMP, an unknown product(s), and the dominant species 3′3′-cGAMP. In vivo, only the 3′3′-cGAMP concentration was elevated upon short-term overexpression of dncV, making this work a first report on cGAMP production in E. coli. Regulation of rdar biofilm formation and motility upon overexpression of untagged DncV in combination with three adjacent cotransferred gene products suggests a novel temperature-dependent cGAMP signaling module in E. coli ECOR31.
    Keywords: Csgd ; Dncv ; Biofilm Formation ; Cgamp ; Cyclic Di-Gmp ; Motility ; Rdar ; Biology;
    ISSN: mBio
    ISSN: 21612129
    E-ISSN: 2150-7511
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  • 7
    In: mBio, 2019, Vol.10(3)
    Description: Dual RNA sequencing (RNA-seq) offers the promise of determining an interactome at a transcriptional level between a bacterium and the host but has yet to be done on any bacterial infection in human tissue. We performed dual RNA-seq and metabolomics analyses on wounded and infected sites following experimental infection of the arm with H. ducreyi . Our results suggest that H. ducreyi survives in an abscess by utilizing l -ascorbate as an alternative carbon source, possibly taking advantage of host ascorbic acid recycling, and that H. ducreyi also adapts by upregulating genes involved in anaerobic metabolism and inorganic ion and nutrient transport. To our knowledge, this is the first description of an interaction network between a bacterium and the human host at a site of infection. ABSTRACT A major gap in understanding infectious diseases is the lack of information about molecular interaction networks between pathogens and the human host. Haemophilus ducreyi causes the genital ulcer disease chancroid in adults and is a leading cause of cutaneous ulcers in children in the tropics. We developed a model in which human volunteers are infected on the upper arm with H. ducreyi until they develop pustules. To define the H. ducreyi and human interactome, we determined bacterial and host transcriptomic and host metabolomic changes in pustules. We found that in vivo H. ducreyi transcripts were distinct from those in the inocula, as were host transcripts in pustule and wounded control sites. Many of the upregulated H. ducreyi genes were found to be involved in ascorbic acid and anaerobic metabolism and inorganic ion/nutrient transport. The top 20 significantly expressed human pathways showed that all were involved in immune responses. We generated a bipartite network for interactions between host and bacterial gene transcription; multiple positively correlated networks contained H. ducreyi genes involved in anaerobic metabolism and host genes involved with the immune response. Metabolomic studies showed that pustule and wounded samples had different metabolite compositions; the top ion pathway involved ascorbate and aldarate metabolism, which correlated with the H. ducreyi transcriptional response and upregulation of host genes involved in ascorbic acid recycling. These data show that an interactome exists between H. ducreyi and the human host and suggest that H. ducreyi exploits the metabolic niche created by the host immune response.
    Keywords: Research Article ; Host-Microbe Biology ; Dual Rna-Seq ; Haemophilus Ducreyi ; Human Infection Model ; Interactome ; Metabolome
    E-ISSN: 2150-7511
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  • 8
    Language: English
    In: Geoderma, 1 July 2019, Vol.345, pp.63-71
    Description: Soil structure is not static but undergoes continuous changes due to a wide range of biotic and abiotic drivers such as bioturbation and the mechanical disturbance by tillage. This continuous alteration of soil structure beyond the pure swelling and shrinking of some stable structure is what we refer to as soil structure dynamics. It has important consequences for carbon turnover in soil as it controls how quickly soil organic matter gets occluded from or exposed to mineralization. So far there are hardly any direct observations of the rate at which soil pores are formed and destroyed.Here we employ are recently introduced labeling approach for soil structure that measures how quickly the locations of small garnet particles get randomized in soil as a measure for soil structure dynamics. We investigate the effect of desiccation crack dynamics on pore space attributes in general and soils structure turnover in particular using X-ray microtomography for repeated wetting-drying cycles. This is explored for three different soils with a range of soil organic matter content, clay content and different clay mineralogy that were sieved to a certain aggregate size fraction (0.63–2 mm) and repacked at two different bulk density levels.The total magnitude of desiccation crack formation mainly depended on the clay content and clay mineralogy. Higher soil organic matter content led to a denser crack pattern with smaller aperture. Wetting-drying cycles did not only effect visible macroporosity (〉8 μm), but also unresolved mesoporosity. The changes in macroporosity were higher at lower bulk density. Most importantly, repeated wetting-drying cycles did not lead to a randomization of distances between garnet particles and pores. This demonstrates that former failure zones are reactivated during subsequent drying cycles. Hence, wetting-drying resulted in reversible particle displacement and therefore would not have triggered the exposure of occluded carbon that was not already exposed during the previous drying event. •3D crack dynamics in structured soil during WD cycles observed with X-ray CT•Soil structure dynamics measured via structure labeling with garnet particles•Soil structure dynamics dependent on, bulk density, SOM and clay content•Higher SOM content led to a higher density of cracks with smaller aperture•Soil structure dynamics is negligible due to reactivation of old cracks
    Keywords: Soil Structure ; Desiccation Cracks ; X-Ray Tomography ; Macropores ; Clay Mineralogy ; Carbon Turnover
    ISSN: 0016-7061
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  • 9
    Language: English
    In: Biogeosciences Discussions, 03/08/2019, pp.1-31
    ISSN: Biogeosciences Discussions
    E-ISSN: 1810-6285
    Source: CrossRef
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  • 10
    Language: English
    In: Geoderma, 15 September 2019, Vol.350, pp.61-72
    Description: During soil formation, the interaction of different biota (plants, soil fauna, microbes) with weathered mineral material shapes unique structures depending on the parental material and the site specific climatic conditions. While many of these interactions are known, the relative importance of the different biota is difficult to unravel and therefore difficult to quantify. Biological soil structure formation is often superimposed by soil management and swell-shrink dynamics, making it even more difficult to derive mechanistic understanding.We here explore soil structure formation within a “space-for-time” chronosequence in the Rhenish lignite mining area. Loess material from a depth of 4–10 m has been used for reclamation in a standardized procedure for 24 years.Changes in soil pore system are characterized by properties such as connectivity (Euler number) and pore size distribution using undisturbed soil columns with a diameter of 10 cm. They were taken from two different depths (0–20 cm and 40–60 cm) at different sites ranging in age from 0 to 24 years. X-ray CT is used for scanning the original columns as well as undisturbed subsamples of 3 and 0.7 cm diameter. This hierarchical sampling scheme was developed to overcome the trade-off between sample size and resolution.For the first time also information on the development of biopores could be measured by separating them from other structural pores based on their unique shape. The data were complemented by destructive sampling and determination of root length with WinRHIZO to give an estimate of how many biopores are filled with roots. Furthermore HYPROP measurements of water retention curves were conducted and showed a general agreement with the image-derived pore size distribution merged across three scales.An increase in biopore density throughout year zero to year 12, in particular in 40–60 cm soil depth, was observed. The biopore length densities of approximately 17 cm/cm3 obtained in year 12 was similar to the one measured in year 24, suggesting that equilibrium was reached. Only about 10% of these biopores were filled with roots. In the topsoil (0–20 cm) the equilibrium value in biopore density is already reached after six years due to a higher root length density. Ploughing lead to higher mean pore size and to lower connectivity compared to the well-connected, very stable pore network in 40–60 cm depth.This study shows how fast plant roots create a stable and connected biopore system and how this is disrupted by soil tillage, which produces completely contrasting pore characteristics. Unlabelled Image •X-ray μCT disentangles effect of tillage and plant roots on soil structure over time.•A new segmentation method allowed for quantification of biopore-network•A maximum biopore density (18 cm cm−3) was already reached after 6 years 18 cm cm−3.•Tillage led to total different macropore characteristics.
    Keywords: Biopores ; Structure Formation ; X-Ray CT ; Tilled Soil
    ISSN: 0016-7061
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