scholarly journals Genome-Scale Transcription-Translation Mapping Reveals Features of Zymomonas mobilis Transcription Units and Promoters

mSystems ◽  
2020 ◽  
Vol 5 (4) ◽  
Author(s):  
Jessica M. Vera ◽  
Indro Neil Ghosh ◽  
Yaoping Zhang ◽  
Alex S. Hebert ◽  
Joshua J. Coon ◽  
...  
Keyword(s):  
Synthetic Biology ◽  
Zymomonas Mobilis ◽  
Plant Biomass ◽  
Caulobacter Crescentus ◽  
Functional Characterization ◽  
Ribosome Profiling ◽  
Start Codon ◽  
Label Free ◽  
Content Type ◽  
Functional Sites

ABSTRACT Zymomonas mobilis is an ethanologenic alphaproteobacterium with promise for the industrial conversion of renewable plant biomass into fuels and chemical bioproducts. Limited functional annotation of the Z. mobilis genome is a current barrier to both fundamental studies of Z. mobilis and its development as a synthetic biology chassis. To gain insight, we collected sample-matched multiomics data, including RNA sequencing (RNA-seq), transcription start site (TSS) sequencing (TSS-seq), termination sequencing (term-seq), ribosome profiling, and label-free shotgun proteomic mass spectrometry, across different growth conditions and used these data to improve annotation and assign functional sites in the Z. mobilis genome. Proteomics and ribosome profiling informed revisions of protein-coding genes, which included 44 start codon changes and 42 added proteins. We developed statistical methods for annotating transcript 5′ and 3′ ends, enabling the identification of 3,940 TSSs and their corresponding promoters and 2,091 transcription termination sites, which were distinguished from RNA processing sites by the lack of an adjacent RNA 5′ end. Our results revealed that Z. mobilis σA −35 and −10 promoter elements closely resemble canonical Escherichia coli −35 and −10 elements, with one notable exception: the Z. mobilis −10 element lacks the highly conserved −7 thymine observed in E. coli and other previously characterized σA promoters. The σA promoters of another alphaproteobacterium, Caulobacter crescentus, similarly lack the conservation of −7 thymine in their −10 elements. Our results anchor the development of Z. mobilis as a platform for synthetic biology and establish strategies for empirical genome annotation that can complement purely computational methods. IMPORTANCE Efforts to rationally engineer synthetic pathways in Zymomonas mobilis are impeded by a lack of knowledge and tools for predictable and quantitative programming of gene regulation at the transcriptional, posttranscriptional, and posttranslational levels. With the detailed functional characterization of the Z. mobilis genome presented in this work, we provide crucial knowledge for the development of synthetic genetic parts tailored to Z. mobilis. This information is vital as researchers continue to develop Z. mobilis for synthetic biology applications. Our methods and statistical analyses also provide ways to rapidly advance the understanding of poorly characterized bacteria via empirical data that enable the experimental validation of sequence-based prediction for genome characterization and annotation.

scholarly journals Biochemical and Functional Characterization of the NurA-HerA Complex from Deinococcus radiodurans

2015 ◽  
Vol 197 (12) ◽  
pp. 2048-2061 ◽  
Author(s):  
Kaiying Cheng ◽  
Xuanyi Chen ◽  
Guangzhi Xu ◽  
Liangyan Wang ◽  
Hong Xu ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Homologous Recombination ◽  
Deinococcus Radiodurans ◽  
Functional Characterization ◽  
Complex Function ◽  
Optimum Growth ◽  
Content Type ◽  
Functional Sites ◽  
Optimum Activity ◽  
Recombination Efficiency

ABSTRACTIn archaea, the NurA nuclease and HerA ATPase/helicase, together with the Mre11-Rad50 complex, function in 3′ single-stranded DNA (ssDNA) end processing during homologous recombination (HR). However, bacterial homologs of NurA and HerA have not been characterized. FromDeinococcus radiodurans, we identified the manganese-dependent 5′-to-3′ ssDNA/double-stranded DNA (dsDNA) exonuclease/endonuclease NurA (DrNurA) and the ATPase HerA (DrHerA). These two proteins stimulated each other's activity through direct protein-protein interactions. The N-terminal HAS domain of DrHerA was the key domain for this interaction. Several critical residues of DrNurA and DrHerA were verified by site-directed mutational analysis. Temperature-dependent activity assays confirmed that the two proteins had mesophilic features, with optimum activity temperatures 10°C to 15°C higher than their optimum growth temperatures. Knocking out eithernurAorherAaffected cell proliferation by shortening the growth phase, especially for growth at a high temperature (37°C). In addition, both mutant strains displayed almost 10-fold-reduced intermolecular recombination efficiency, indicating that DrNurA and DrHerA might be involved in homologous recombinationin vivo. However, single- and double-gene deletions did not show significantly decreased radioresistance. Our results confirmed that the biochemical activities of bacterial NurA and HerA proteins were conserved with archaea. Our phenotypical results suggested that these proteins might have different functions in bacteria.IMPORTANCEDeinococcus radioduransNurA (DrNurA) was identified as a manganese-dependent 5′-to-3′ ssDNA/dsDNA exonuclease/endonuclease, andDeinococcus radioduransHerA (DrHerA) was identified as an ATPase. Physical interactions between DrNurA and DrHerA explained mutual stimulation of their activities. The N-terminal HAS domain on DrHerA was identified as the interaction domain. Several essential functional sites on DrNurA and DrHerA were characterized. Both DrHerA and DrNurA showed mesophilic biochemical features, with their optimum activity temperatures 10°C to 15°C higher than their optimum growth temperaturesin vitro. Knockout ofnurAorherAled to abnormal cell proliferation and reduced intermolecular recombination efficiency but no obvious effect on radioresistence.

scholarly journals Heterologous glycosyl hydrolase expression and cellular reprogramming resembling sucrose-induction enable Zymomonas mobilis growth on cellobiose

2019 ◽  
Author(s):  
Nagendra P. Kurumbang ◽  
Jessica M. Vera ◽  
Alexander S. Hebert ◽  
Joshua J. Coon ◽  
Robert Landick
Keyword(s):  
Zymomonas Mobilis ◽  
Plant Biomass ◽  
Caulobacter Crescentus ◽  
Glycosyl Hydrolase ◽  
Surface Display ◽  
Genetic Mutation ◽  
Cellular Reprogramming ◽  
Lag Phase ◽  
Renewable Biomass ◽  
Sucrose Induction

ABSTRACTPlant derived fuels and chemicals from renewable biomass have significant potential to replace reliance on petroleum and improve global carbon balance. However, plant biomass contains significant fractions of oligosaccharides that are not usable natively by many industrial microorganisms, including Escherichia coli, Saccharomyces cerevisiae, and Zymomonas mobilis. Even after chemical or enzymatic hydrolysis, some carbohydrate remains as non-metabolizable oligosaccharides (e.g., cellobiose or longer cellulose-derived oligomers), thus reducing the efficiency of conversion to useful products. To begin to address this problem for Z. mobilis, we engineered a strain (Z. mobilis GH3) that expresses a glycosyl hydrolase (GH) with β-glucosidase activity from Caulobacter crescentus and subjected it to an adaptation in cellobiose medium. Growth on cellobiose was achieved after a prolonged lag phase in cellobiose medium that induced changes in gene expression and cell composition, including increased expression and secretion of GH. These changes were reversible upon growth in glucose-containing medium, meaning they did not result from genetic mutation but could be retained upon transfer of cells to fresh cellobiose medium. After adaptation to cellobiose, our GH-expressing strain was able to convert about 50% of cellobiose to glucose within 24 hours and use it for growth and ethanol production. Alternatively, pre-growth of Z. mobilis GH3 in sucrose medium enabled immediate growth on cellobiose. Proteomic analysis of cellobiose- and sucrose-adapted strains revealed upregulation of secretion-, transport-, and outer membrane-related proteins, which may aid secretion or surface display of GHs, entry of cellobiose into the periplasm, or both. Our two key findings are that Z. mobilis can be reprogrammed to grow on cellobiose as a sole carbon source and that this reprogramming is related to a natural response of Z. mobilis to sucrose that enables sucrose secretion.

scholarly journals Absolute Measurements of mRNA Translation inCaulobacter crescentusReveal Important Fitness Costs of Vitamin B12Scavenging

mSystems ◽  
2019 ◽  
Vol 4 (4) ◽  
Author(s):  
James R. Aretakis ◽  
Alisa Gega ◽  
Jared M. Schrader
Keyword(s):  
Cell Cycle ◽  
Protein Synthesis ◽  
Cell Division ◽  
Asymmetric Cell Division ◽  
Caulobacter Crescentus ◽  
Mrna Translation ◽  
Genetic Network ◽  
Ribosome Profiling ◽  
Content Type ◽  
Bacterial Cell Cycle

ABSTRACTCaulobacter crescentusis a model for the bacterial cell cycle which culminates in asymmetric cell division, yet little is known about the absolute levels of protein synthesis of the cellular parts needed to complete the cell cycle. Here we utilize ribosome profiling to provide absolute measurements of mRNA translation inC. crescentus, providing an important resource with quantitative genome-wide measurements of protein output across individual genes. Analysis of protein synthesis rates revealed ∼4.5% of cellular protein synthesis is for genes related to vitamin B12import (btuB) and B12-independent methionine biosynthesis (metE) when grown in common growth media lacking B12. While its facultative B12lifestyle provides a fitness advantage in the absence of B12, we find that it provides a fitness disadvantage of the cells in the presence of B12, potentially explaining why manyCaulobacterspecies have lost themetEgene and become obligates for B12.IMPORTANCECaulobacter crescentusis a model system of the bacterial cell cycle culminating in asymmetric cell division, with each daughter cell inheriting a distinct set of proteins. While a genetic network of master transcription factors coordinates the cell cycle timing of transcription for nearly 20% ofCaulobactergenes, we lack knowledge of how many of each protein “part” encoded in the genome are synthesized. Therefore, to determine the absolute production rates across the genome, we performed ribosome profiling, providing, for the first time, a quantitative resource with measurements of each protein “part” needed to generate daughter cells. This resource furthers the goal of a systems-level understanding of the genetic network controlling asymmetric cell division. To highlight the utility of this data set, we probe the protein synthesis cost of a B12utilization pathway and provide new insights intoCaulobacter’s adaptation to its natural environments.

scholarly journals CATH functional families predict functional sites in proteins

2020 ◽  
Author(s):  
Sayoni Das ◽  
Harry M Scholes ◽  
Neeladri Sen ◽  
Christine Orengo
Keyword(s):  
Functional Characterization ◽  
Functional Site ◽  
Training Data ◽  
Supplementary Information ◽  
Conserved Residues ◽  
Functional Sites ◽  
Protein Protein Interaction ◽  
Evolutionary Features ◽  
Functional Families

Abstract Motivation Identification of functional sites in proteins is essential for functional characterization, variant interpretation and drug design. Several methods are available for predicting either a generic functional site, or specific types of functional site. Here, we present FunSite, a machine learning predictor that identifies catalytic, ligand-binding and protein–protein interaction functional sites using features derived from protein sequence and structure, and evolutionary data from CATH functional families (FunFams). Results FunSite’s prediction performance was rigorously benchmarked using cross-validation and a holdout dataset. FunSite outperformed other publicly available functional site prediction methods. We show that conserved residues in FunFams are enriched in functional sites. We found FunSite’s performance depends greatly on the quality of functional site annotations and the information content of FunFams in the training data. Finally, we analyze which structural and evolutionary features are most predictive for functional sites. Availabilityand implementation https://github.com/UCL/cath-funsite-predictor. Contact [email protected] or [email protected] Supplementary information Supplementary data are available at Bioinformatics online.

scholarly journals Association of the Cold Shock DEAD-Box RNA Helicase RhlE to the RNA Degradosome in Caulobacter crescentus

2017 ◽  
Vol 199 (13) ◽  
Author(s):  
Angel A. Aguirre ◽  
Alexandre M. Vicente ◽  
Steven W. Hardwick ◽  
Daniela M. Alvelos ◽  
Ricardo R. Mazzon ◽  
...  
Keyword(s):  
Low Temperature ◽  
Caulobacter Crescentus ◽  
Immunoblot Analysis ◽  
Rna Helicase ◽  
Rnase E ◽  
Rna Helicases ◽  
Content Type ◽  
Dead Box ◽  
Rna Degradosome ◽  
The Dead

ABSTRACT In diverse bacterial lineages, multienzyme assemblies have evolved that are central elements of RNA metabolism and RNA-mediated regulation. The aquatic Gram-negative bacterium Caulobacter crescentus, which has been a model system for studying the bacterial cell cycle, has an RNA degradosome assembly that is formed by the endoribonuclease RNase E and includes the DEAD-box RNA helicase RhlB. Immunoprecipitations of extracts from cells expressing an epitope-tagged RNase E reveal that RhlE, another member of the DEAD-box helicase family, associates with the degradosome at temperatures below those optimum for growth. Phenotype analyses of rhlE, rhlB, and rhlE rhlB mutant strains show that RhlE is important for cell fitness at low temperature and its role may not be substituted by RhlB. Transcriptional and translational fusions of rhlE to the lacZ reporter gene and immunoblot analysis of an epitope-tagged RhlE indicate that its expression is induced upon temperature decrease, mainly through posttranscriptional regulation. RNase E pulldown assays show that other proteins, including the transcription termination factor Rho, a second DEAD-box RNA helicase, and ribosomal protein S1, also associate with the degradosome at low temperature. The results suggest that the RNA degradosome assembly can be remodeled with environmental change to alter its repertoire of helicases and other accessory proteins. IMPORTANCE DEAD-box RNA helicases are often present in the RNA degradosome complex, helping unwind secondary structures to facilitate degradation. Caulobacter crescentus is an interesting organism to investigate degradosome remodeling with change in temperature, because it thrives in freshwater bodies and withstands low temperature. In this study, we show that at low temperature, the cold-induced DEAD-box RNA helicase RhlE is recruited to the RNA degradosome, along with other helicases and the Rho protein. RhlE is essential for bacterial fitness at low temperature, and its function may not be complemented by RhlB, although RhlE is able to complement for rhlB loss. These results suggest that RhlE has a specific role in the degradosome at low temperature, potentially improving adaptation to this condition.

scholarly journals N-Acetylglucosamine-1-Phosphate Transferase, WecA, as a Validated Drug Target in Mycobacterium tuberculosis

2017 ◽  
Vol 61 (11) ◽  
Author(s):  
Stanislav Huszár ◽  
Vinayak Singh ◽  
Alica Polčicová ◽  
Peter Baráth ◽  
María Belén Barrio ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Target ◽  
Functional Characterization ◽  
Drug Candidate ◽  
Content Type ◽  
Chemical Libraries ◽  
Whole Cells ◽  
Encoding Gene

ABSTRACT The mycobacterial phosphoglycosyltransferase WecA, which initiates arabinogalactan biosynthesis in Mycobacterium tuberculosis, has been proposed as a target of the caprazamycin derivative CPZEN-45, a preclinical drug candidate for the treatment of tuberculosis. In this report, we describe the functional characterization of mycobacterial WecA and confirm the essentiality of its encoding gene in M. tuberculosis by demonstrating that the transcriptional silencing of wecA is bactericidal in vitro and in macrophages. Silencing wecA also conferred hypersensitivity of M. tuberculosis to the drug tunicamycin, confirming its target selectivity for WecA in whole cells. Simple radiometric assays performed with mycobacterial membranes and commercially available substrates allowed chemical validation of other putative WecA inhibitors and resolved their selectivity toward WecA versus another attractive cell wall target, translocase I, which catalyzes the first membrane step in the biosynthesis of peptidoglycan. These assays and the mutant strain described herein will be useful for identifying potential antitubercular leads by screening chemical libraries for novel WecA inhibitors.

scholarly journals Ribosome Profiling Reveals HSP90 Inhibitor Effects on Stage-Specific Protein Synthesis in Leishmania donovani

mSystems ◽  
2018 ◽  
Vol 3 (6) ◽  
Author(s):  
Eugenia Bifeld ◽  
Stephan Lorenzen ◽  
Katharina Bartsch ◽  
Juan-José Vasquez ◽  
T. Nicolai Siegel ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Synthesis ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Leishmania Donovani ◽  
Specific Protein ◽  
Ribosome Profiling ◽  
Severe Illness ◽  
Content Type ◽  
Hsp90 Activity

ABSTRACT The 90-kDa heat shock protein (HSP90) of eukaryotes is a highly abundant and essential chaperone required for the maturation of regulatory and signal proteins. In the protozoan parasite Leishmania donovani, causative agent of the fatal visceral leishmaniasis, HSP90 activity is essential for cell proliferation and survival. Even more importantly, its inhibition causes life cycle progression from the insect stage to the pathogenic, mammalian stage. To unravel the molecular impact of HSP90 activity on the parasites’ gene expression, we performed a ribosome profiling analysis of L. donovani, comparing genome-wide protein synthesis patterns in the presence and absence of the HSP90-specific inhibitor radicicol and an ectopically expressed radicicol-resistant HSP90 variant. We find that ribosome-protected RNA faithfully maps open reading frames and represents 97% of the annotated protein-coding genes of L. donovani. Protein synthesis was found to correlate poorly with RNA steady-state levels, indicating a regulated translation as primary mechanism for HSP90-dependent gene expression. The results confirm inhibitory effects of HSP90 on the synthesis of Leishmania proteins that are associated with the pathogenic, intracellular stage of the parasite. Those include heat shock proteins, redox enzymes, virulence-enhancing surface proteins, proteolytic pathways, and a complete set of histones. Conversely, HSP90 promotes fatty acid synthesis enzymes. Complementing radicicol treatment with the radicicol-resistant HSP90rr variant revealed important off-target radicicol effects that control a large number of the above-listed proteins. Leishmania lacks gene-specific transcription regulation and relies on regulated translation instead. Our ribosome footprinting analysis demonstrates a controlling function of HSP90 in stage-specific protein synthesis but also significant, HSP90-independent effects of the inhibitor radicicol. IMPORTANCE Leishmania parasites cause severe illness in humans and animals. They exist in two developmental stages, insect form and mammalian form, which differ in shape and gene expression. By mapping and quantifying RNA fragments protected by protein synthesis complexes, we determined the rates of protein synthesis for >90% of all Leishmania proteins in response to the inhibition of a key regulatory protein, the 90-kDa heat shock protein. We find that Leishmania depends on a regulation of protein synthesis for controlling its gene expression and that heat shock protein 90 inhibition can trigger the developmental program from insect form to mammalian form of the pathogen.

scholarly journals Efficient Plant Biomass Degradation by Thermophilic Fungus Myceliophthora heterothallica

2012 ◽  
Vol 79 (4) ◽  
pp. 1316-1324 ◽  
Author(s):  
Joost van den Brink ◽  
Gonny C. J. van Muiswinkel ◽  
Bart Theelen ◽  
Sandra W. A. Hinz ◽  
Ronald P. de Vries
Keyword(s):  
Wheat Straw ◽  
Plant Biomass ◽  
Hydrolytic Enzymes ◽  
Reaction Times ◽  
Giant Reed ◽  
Thermophilic Fungi ◽  
Biomass Degradation ◽  
Thermostable Enzymes ◽  
Content Type

ABSTRACTRapid and efficient enzymatic degradation of plant biomass into fermentable sugars is a major challenge for the sustainable production of biochemicals and biofuels. Enzymes that are more thermostable (up to 70°C) use shorter reaction times for the complete saccharification of plant polysaccharides compared to hydrolytic enzymes of mesophilic fungi such asTrichodermaandAspergillusspecies. The genusMyceliophthoracontains four thermophilic fungi producing industrially relevant thermostable enzymes. Within this genus, isolates belonging toM. heterothallicawere recently separated from the well-described speciesM. thermophila. We evaluate here the potential ofM. heterothallicaisolates to produce efficient enzyme mixtures for biomass degradation. Compared to the other thermophilicMyceliophthoraspecies, isolates belonging toM. heterothallicaandM. thermophilagrew faster on pretreated spruce, wheat straw, and giant reed. According to their protein profiles andin vitroassays after growth on wheat straw, (hemi-)cellulolytic activities differed strongly betweenM. thermophilaandM. heterothallicaisolates. Compared toM. thermophila,M. heterothallicaisolates were better in releasing sugars from mildly pretreated wheat straw (with 5% HCl) with a high content of xylan. The high levels of residual xylobiose revealed that enzyme mixtures ofMyceliophthoraspecies lack sufficient β-xylosidase activity. Sexual crossing of twoM. heterothallicashowed that progenies had a large genetic and physiological diversity. In the future, this will allow further improvement of the plant biomass-degrading enzyme mixtures ofM. heterothallica.

scholarly journals Development of oriC-Based Plasmids for Mesoplasma florum

2017 ◽  
Vol 83 (7) ◽  
Author(s):  
Dominick Matteau ◽  
Marie-Eve Pepin ◽  
Vincent Baby ◽  
Samuel Gauthier ◽  
Mélissa Arango Giraldo ◽  
...  
Keyword(s):  
Systems Biology ◽  
Synthetic Biology ◽  
Genetic Engineering ◽  
Genome Engineering ◽  
Antibiotic Resistance Genes ◽  
Viable Cell ◽  
Pathogenic Potential ◽  
Strong Basis ◽  
Content Type ◽  
Basic Biology

ABSTRACT The near-minimal bacterium Mesoplasma florum constitutes an attractive model for systems biology and for the development of a simplified cell chassis in synthetic biology. However, the lack of genetic engineering tools for this microorganism has limited our capacity to understand its basic biology and modify its genome. To address this issue, we have evaluated the susceptibility of M. florum to common antibiotics and developed the first generation of artificial plasmids able to replicate in this bacterium. Selected regions of the predicted M. florum chromosomal origin of replication (oriC) were used to create different plasmid versions that were tested for their transformation frequency and stability. Using polyethylene glycol-mediated transformation, we observed that plasmids harboring both rpmH-dnaA and dnaA-dnaN intergenic regions, interspaced or not with a copy of the dnaA gene, resulted in a frequency of ∼4.1 × 10−6 transformants per viable cell and were stably maintained throughout multiple generations. In contrast, plasmids containing only one M. florum oriC intergenic region or the heterologous oriC region of Mycoplasma capricolum, Mycoplasma mycoides, or Spiroplasma citri failed to produce any detectable transformants. We also developed alternative transformation procedures based on electroporation and conjugation from Escherichia coli, reaching frequencies up to 7.87 × 10−6 and 8.44 × 10−7 transformants per viable cell, respectively. Finally, we demonstrated the functionality of antibiotic resistance genes active against tetracycline, puromycin, and spectinomycin/streptomycin in M. florum. Taken together, these valuable genetic tools will facilitate efforts toward building an M. florum-based near-minimal cellular chassis for synthetic biology. IMPORTANCE Mesoplasma florum constitutes an attractive model for systems biology and for the development of a simplified cell chassis in synthetic biology. M. florum is closely related to the mycoides cluster of mycoplasmas, which has become a model for whole-genome cloning, genome transplantation, and genome minimization. However, M. florum shows higher growth rates than other Mollicutes, has no known pathogenic potential, and possesses a significantly smaller genome that positions this species among some of the simplest free-living organisms. So far, the lack of genetic engineering tools has limited our capacity to understand the basic biology of M. florum in order to modify its genome. To address this issue, we have evaluated the susceptibility of M. florum to common antibiotics and developed the first artificial plasmids and transformation methods for this bacterium. This represents a strong basis for ongoing genome engineering efforts using this near-minimal microorganism.

scholarly journals Complete Genome Sequence of Caulobacter crescentus Bacteriophage φCbK

2012 ◽  
Vol 86 (18) ◽  
pp. 10234-10235 ◽  
Author(s):  
Gaël Panis ◽  
Christophe Lambert ◽  
Patrick H. Viollier
Keyword(s):  
Genome Sequence ◽  
Complete Genome Sequence ◽  
Complete Genome ◽  
Caulobacter Crescentus ◽  
Content Type ◽  
Functional Studies ◽  
Bacterial Cell Cycle ◽  
Potential Interactions ◽  
Complete Genome Analysis ◽  
Insight Into

φCbK is a B3 morphotype bacteriophage of theSiphoviridaefamily that infectsCaulobacter crescentus, the preeminent model system for bacterial cell cycle studies. The last 4 decades of research with φCbK as a genetic and cytological tool to study the biology of the host warrant an investigation of the phage genome composition. Herein, we report the complete genome sequence of φCbK and highlight unusual features that emerged from its annotation. The complete genome analysis of the φCbK phage provides new insight into its characteristics and potential interactions with itsCaulobacter crescentushost, setting the stage for future functional studies with φCbK.

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