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Purpose: The current study aimed to identify metabolites associated with age-related macular degeneration (AMD) by performing the largest metabolome association analysis in AMD to date, as well as aiming to determine the effect of AMD-associated genetic variants on metabolite levels and investigate associations between the identified metabolites and activity of the complement system, one of the main AMD-associated disease pathways. Design: Case-control association analysis of metabolomics data. Participants: Five European cohorts consisting of 2267 AMD patients and 4266 control participants. Methods: Metabolomics was performed using a high-throughput proton nuclear magnetic resonance metabolomics platform, which allows quantification of 146 metabolite measurements and 79 derivative values. Metabolome–AMD associations were studied using univariate logistic regression analyses. The effect of 52 AMD-associated genetic variants on the identified metabolites was investigated using linear regression. In addition, associations between the identified metabolites and activity of the complement pathway (defined by the C3d-to-C3 ratio) were investigated using linear regression. Main Outcome Measures: Metabolites associated with AMD. Results: We identified 60 metabolites that were associated significantly with AMD, including increased levels of large and extra-large high-density lipoprotein (HDL) subclasses and decreased levels of very low-density lipoprotein (VLDL), amino acids, and citrate. Of 52 AMD-associated genetic variants, 7 variants were associated significantly with 34 of the identified metabolites. The strongest associations were identified for genetic variants located in or near genes involved in lipid metabolism (ABCA1, CETP, APOE, and LIPC) with metabolites belonging to the large and extra-large HDL subclasses. Also, 57 of 60 metabolites were associated significantly with complement activation levels, independent of AMD status. Increased large and extra-large HDL levels and decreased VLDL and amino acid levels were associated with increased complement activation. Conclusions: Lipoprotein levels were associated with AMD-associated genetic variants, whereas decreased essential amino acids may point to nutritional deficiencies in AMD. We observed strong associations between the vast majority of the AMD-associated metabolites and systemic complement activation levels, independent of AMD status. This may indicate biological interactions between the main AMD disease pathways and suggests that multiple pathways may need to be targeted simultaneously for successful treatment of AMD.

Protein-protein-interaction networks (PPINs) organize fundamental biological processes, but how oncogenic mutations impact these interactions and their functions at a network-level scale is poorly understood. Here, we analyze how a common oncogenic KRAS mutation (KRASG13D) affects PPIN structure and function of the Epidermal Growth Factor Receptor (EGFR) network in colorectal cancer (CRC) cells. Mapping >6000 PPIs shows that this network is extensively rewired in cells expressing transforming levels of KRASG13D (mtKRAS). The factors driving PPIN rewiring are multifactorial including changes in protein expression and phosphorylation. Mathematical modelling also suggests that the binding dynamics of low and high affinity KRAS interactors contribute to rewiring. PPIN rewiring substantially alters the composition of protein complexes, signal flow, transcriptional regulation, and cellular phenotype. These changes are validated by targeted and global experimental analysis. Importantly, genetic alterations in the most extensively rewired PPIN nodes occur frequently in CRC and are prognostic of poor patient outcomes. This work was supported by European Union FP7 Grant No. 278568 “PRIMES” and Science Foundation Ireland Investigator Program Grant 14/IA/2395 to W.K. B.K. is supported by SmartNanoTox (Grant no. 686098), NanoCommons (Grant no. 731032), O.R. by MSCA-IF-2016 SAMNets (Grant no. 750688). D.M. is supported by Science Foundation Ireland Career Development award 15-CDA-3495. I.J. is supported by the Canada Research Chair Program (CRC #225404), Krembil Foundation, Ontario Research Fund (GL2-01-030 and #34876), Natural Sciences Research Council (NSERC #203475), Canada Foundation for Innovation (CFI #225404, #30865), and IBM. O.S. is supported by ERC investigator Award ColonCan 311301 and CRUK. I.S. is supported by the Canadian Cancer Society Research Institute (#703889), Genome Canada via Ontario Genomics (#9427 & #9428), Ontario Research fund (ORF/ DIG-501411 & RE08-009), Consortium Québécois sur la Découverte du Médicament (CQDM Quantum Leap) & Brain Canada (Quantum Leap), and CQDM Explore and OCE (#23929). T.C. was supported by a Teagasc Walsh Fellowship

The Protein Data Bank in Europe – Knowledge Base (PDBe-KB, https://pdbe-kb.org) is an open collaboration between world-leading specialist data resources contributing functional and biophysical annotations derived from or relevant to the Protein Data Bank (PDB). The goal of PDBe-KB is to place macromolecular structure data in their biological context by developing standardised data exchange formats and integrating functional annotations from the contributing partner resources into a knowledge graph that can provide valuable biological insights. Since we described PDBe-KB in 2019, there have been significant improvements in the variety of available annotation data sets and user functionality. Here, we provide an overview of the consortium, highlighting the addition of annotations such as predicted covalent binders, phosphorylation sites, effects of mutations on the protein structure and energetic local frustration. In addition, we describe a library of reusable web-based visualisation components and introduce new features such as a bulk download data service and a novel superposition service that generates clusters of superposed protein chains weekly for the whole PDB archive., ELIXIR [IDP implementation study]; Biotechnology and Biological Sciences Research Council via the 3D-Gateway [BB/T01959X/1]; FunPDBe [BB/P024351/1]; European Molecular Biology Laboratory-European Bioinformatics Institute who supported this work; J.D. acknowledges support from the Ministry of Education, Youth and Sport of the Czech Republic [INBIO CZ.02.1.01/0.0/0.0/16_026/0008451]; R.S., K.B. and J.D. also acknowledge support from the Ministry of Education, Youth and Sport of the Czech Republic [ELIXIR-CZ LM2018131]; L.M. acknowledges support from the European Union's Horizon 2020 Programme (H2020-INFRAIA-2018-1) [823839]; Research Foundation Flanders (FWO) [G032816N, G042518N, G028821N]; W.V. acknowledges support from the Research Foundation Flanders (FWO) [G032816N, G028821N]; A.R. acknowledges support from the Fondazione Cassa Di Risparmio di Firenze [24316]; European Commission [101017567]; M.H.C. acknowledges the AIRC project to MHC [IG 23539]; J.F.-R. acknowledges support from the Spanish Ministry of Science and Innovation [PID2019-110167RB-I00]; N.R. acknowledges support from the Norwegian Research Council (Norges Forskningsråd) [288008]; E.D.L. acknowledges support from the European Union's Horizon 2020 research and innovation programme [819318]; M.J.E.S. acknowledges support from the Wellcome Trust [104955/Z/14/Z, 218242/Z/19/Z]. Funding for open access charge: Biotechnology and Biological Sciences Research Council grant [BB/T01959X/1]; Wellcome Trust [104955/Z/14/Z and 218242/Z/19/Z].

RAC1 activity is critical for intestinal homeostasis, and is required for hyperproliferation driven by loss of the tumour suppressor gene Apc in the murine intestine. To avoid the impact of direct targeting upon homeostasis, we reasoned that indirect targeting of RAC1 via RAC-GEFs might be effective. Transcriptional profiling of Apc deficient intestinal tissue identified Vav3 and Tiam1 as key targets. Deletion of these indicated that while TIAM1 deficiency could suppress Apc-driven hyperproliferation, it had no impact upon tumourigenesis, while VAV3 deficiency had no effect. Intriguingly, deletion of either gene resulted in upregulation of Vav2, with subsequent targeting of all three (Vav2−/− Vav3−/− Tiam1−/−), profoundly suppressing hyperproliferation, tumourigenesis and RAC1 activity, without impacting normal homeostasis. Critically, the observed RAC-GEF dependency was negated by oncogenic KRAS mutation. Together, these data demonstrate that while targeting RAC-GEF molecules may have therapeutic impact at early stages, this benefit may be lost in late stage disease., Loss of small GTPase RAC1 suppresses intestinal tumorigenesis caused by APC loss, but impacts normal intestinal homeostasis. Here, the authors provide an alternative method of reducing RAC1 activity by the combined targeting of three RAC-GEFs and show that this approach delays intestinal tumorigenesis without the detrimental effects on normal intestinal architecture.

The removal of unwanted genetic material is a key aspect in many synthetic biology efforts and often requires preliminary knowledge of which genomic regions are dispensable. Typically, these efforts are guided by transposon mutagenesis studies, coupled to deepsequencing (TnSeq) to identify insertion points and gene essentiality. However, epistatic interactions can cause unforeseen changes in essentiality after the deletion of a gene, leading to the redundancy of these essentiality maps. Here, we present LoxTnSeq, a new methodology to generate and catalogue libraries of genome reduction mutants. LoxTnSeq combines random integration of lox sites by transposon mutagenesis, and the generation of mutants via Cre recombinase, catalogued via deep sequencing. When LoxTnSeq was applied to the naturally genome reduced bacterium Mycoplasma pneumoniae, we obtained a mutant pool containing 285 unique deletions. These deletions spanned from > 50 bp to 28 Kb, which represents 21% of the total genome. LoxTnSeq also highlighted large regions of non-essential genes that could be removed simultaneously, and other non-essential regions that could not, providing a guide for future genome reductions., Microbial Biotechnology, 14 (6), ISSN:1751-7915, ISSN:1751-7907

The Protein Data Bank in Europe-Knowledge Base (PDBe-KB, https://pdbe-kb.org) is a community-driven, collaborative resource for literature-derived, manually curated and computationally predicted structural and functional annotations of macromolecular structure data, contained in the Protein Data Bank (PDB). The goal of PDBe-KB is two-fold: (i) to increase the visibility and reduce the fragmentation of annotations contributed by specialist data resources, and to make these data more findable, accessible, interoperable and reusable (FAIR) and (ii) to place macromolecular structure data in their biological context, thus facilitating their use by the broader scientific community in fundamental and applied research. Here, we describe the guidelines of this collaborative effort, the current status of contributed data, and the PDBe-KB infrastructure, which includes the data exchange format, the deposition system for added value annotations, the distributable database containing the assembled data, and programmatic access endpoints. We also describe a series of novel web-pages—the PDBe-KB aggregated views of structure data—which combine information on macromolecular structures from many PDB entries. We have recently released the first set of pages in this series, which provide an overview of available structural and functional information for a protein of interest, referenced by a UniProtKB accession.

RNA splicing is widely dysregulated in cancer, frequently due to altered expression or activity of splicing factors (SFs). Microexons are extremely small exons (3–27 nucleotides long) that are highly evolutionarily conserved and play critical roles in promoting neuronal differentiation and development. Inclusion of microexons in mRNA transcripts is mediated by the SF Serine/Arginine Repetitive Matrix 4 (SRRM4), whose expression is largely restricted to neural tissues. However, microexons have been largely overlooked in prior analyses of splicing in cancer, as their small size necessitates specialized computational approaches for their detection. Here, we demonstrate that despite having low expression in normal nonneural tissues, SRRM4 is further silenced in tumors, resulting in the suppression of normal microexon inclusion. Remarkably, SRRM4 is the most consistently silenced SF across all tumor types analyzed, implying a general advantage of microexon down-regulation in cancer independent of its tissue of origin. We show that this silencing is favorable for tumor growth, as decreased SRRM4 expression in tumors is correlated with an increase in mitotic gene expression, and up-regulation of SRRM4 in cancer cell lines dose-dependently inhibits proliferation in vitro and in a mouse xenograft model. Further, this proliferation inhibition is accompanied by induction of neural-like expression and splicing patterns in cancer cells, suggesting that SRRM4 expression shifts the cell state away from proliferation and toward differentiation. We therefore conclude that SRRM4 acts as a proliferation brake, and tumors gain a selective advantage by cutting off this brake., This project was funded in part by a grant from the Plan Estatal de Investigación Científica y Técnica y de Innovación to L.S. (PGC2018-101271-B-I00, http://www.ciencia.gob.es). S.A.H. is supported by a Marie Skłodowska-Curie Individual Fellowship from the European Union’s Horizon 2020 research and innovation programme (MSCA-IF-2017-794629, http://ec.europa.eu/). X.H. is supported by a PhD fellowship from the Fundación Ramón Areces (http://www.fundacionareces.es). We acknowledge the support of the Spanish Ministry of Economy and Competitiveness, ‘Centro de Excelencia Severo Ochoa’, the CERCA Programme / Generalitat de Catalunya, and the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) to the EMBL partnership.

Bacteria present a promising delivery system for treating human diseases. Here, we engineered the genome-reduced human lung pathogen Mycoplasma pneumoniae as a live biotherapeutic to treat biofilm-associated bacterial infections. This strain has a unique genetic code, which hinders gene transfer to most other bacterial genera, and it lacks a cell wall, which allows it to express proteins that target peptidoglycans of pathogenic bacteria. We first determined that removal of the pathogenic factors fully attenuated the chassis strain in vivo. We then designed synthetic promoters and identified an endogenous peptide signal sequence that, when fused to heterologous proteins, promotes efficient secretion. Based on this, we equipped the chassis strain with a genetic platform designed to secrete antibiofilm and bactericidal enzymes, resulting in a strain capable of dissolving Staphylococcus aureus biofilms preformed on catheters in vitro, ex vivo, and in vivo. To our knowledge, this is the first engineered genome-reduced bacterium that can fight against clinically relevant biofilm-associated bacterial infections., This work has been supported by the European Research Council (ERC) underthe European Union’s Horizon2020research and innovation program, undergrant agreement 670216(MYCOCHASSIS). We also acknowledge the support ofthe Spanish Ministry of Economy, Industry and Competitiveness (MEIC) to theEMBL partnership, the Centro de Excelencia Severo Ochoa, the CERCA Programfrom the Generalitat de Catalunya, the European Union’s Horizon2020research and innovation program under grant agreement 634942 (Myco-SynVac), and the LaCaixa Fundation grant (Livetherapeutics HR18-00058).M.L.-S. acknowledges the support from FEDER project from Instituto Carlos III(ISCIII, Accion Estrategica en Salud2016) (reference CP16/00094). We alsoacknowledge the staff of CRG/UPF Proteomics Unit, which is part of the Span-ish Infrastructure for Omics Technologies (ICTS OmicsTech) unit is a memberof the ProteoRed PRB3consortium, which is supported by grant PT17/0019ofthe PE I+D+i2013-2016from the Instituto de Salud Carlos III (ISCIII) and ERDF. We also acknowledge Margarita Ecay for her assistance in the PETimages analysis and Samuel Miravet-Verde for the bioinformatic support.

14 pags. 5 figs., 2 tabs., Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac disease. Variants in MYBPC3, the gene encoding cardiac myosin-binding protein C (cMyBP-C), are the leading cause of HCM. However, the pathogenicity status of hundreds of MYBPC3 variants found in patients remains unknown, as a consequence of our incomplete understanding of the pathomechanisms triggered by HCM-causing variants. Here, we examined 44 nontruncating MYBPC3 variants that we classified as HCM-linked or nonpathogenic according to cosegregation and population genetics criteria. We found that around half of the HCM-linked variants showed alterations in RNA splicing or protein stability, both of which can lead to cMyBP-C haploinsufficiency. These protein haploinsufficiency drivers associated with HCM pathogenicity with 100% and 94% specificity, respectively. Furthermore, we uncovered that 11% of nontruncating MYBPC3 variants currently classified as of uncertain significance in ClinVar induced one of these molecular phenotypes. Our strategy, which can be applied to other conditions induced by protein loss of function, supports the idea that cMyBP-C haploinsufficiency is a fundamental pathomechanism in HCM., A.-C. acknowledges funding from the Ministerio de Ciencia e Innovación (MCIN) throughgrants BIO2014-54768-P; BIO2017-83640-P/MINECO/AEI/FEDER, UE; EIN2019-102966 and RYC-2014-16604, the European Research Area Network on Cardiovascular Diseases (ERA-CVD/ISCIII, Spain MINOTAUR, AC16/00045), and the Comunidad de Madrid (consortium Tec4Bio-CM, S2018/NMT-4443, FEDER). TheCNIC is supported by the ISCIII, MCIN, and the Pro CNIC Foundation and was a Severo Ochoa Center of Excellence (SEV-2015-0505). We acknowledge funding from ISCIII to the Centro de Investigación Biomédica en Red (CB16/11/00425). L. S. acknowl-edges funding from MCIN (BFU2015-63571-P). J. S. acknowledgesfunding from AEI (PID2019-107293GB-I00), Gobierno de Aragón(E45_17R), and ERDF-InterregV-A POCTEFA, Spain (PIREPRED-EFA086/15). C. S.-C. is the recipient of an FPI-SO predoctoral fellowship BES-2016-076638. M. R. P. was the recipient of a Ph D fellowship from the Italian Ministry of Education, Universities and Research, Italy. H. G.-C. is the recipient of an FPU16/04232 doctoralcontract from MCIN.

It is well-established that FtsZ drives peptidoglycan synthesis at the division site in walled bacteria. However, the function and conservation of FtsZ in wall-less prokaryotes such as mycoplasmas are less clear. In the genome-reduced bacterium Mycoplasma genitalium, the cell division gene cluster is limited to four genes: mraZ, mraW, MG_223, and ftsZ. In a previous study, we demonstrated that ftsZ was dispensable for growth of M. genitalium under laboratory culture conditions. Herein, we show that the entire cell division gene cluster of M. genitalium is non-essential for growth in vitro. Our analyses indicate that loss of the mraZ gene alone is more detrimental for growth of M. genitalium than deletion of ftsZ or the entire cell division gene cluster. Transcriptional analysis revealed a marked upregulation of ftsZ in the mraZ mutant. Stable isotope labeling by amino acids in cell culture (SILAC)-based proteomics confirmed the overexpression of FtsZ in MraZ-deprived cells. Of note, we found that ftsZ expression was upregulated in non-adherent cells of M. genitalium, which arise spontaneously at relatively high rates. Single cell analysis using fluorescent markers showed that FtsZ localization varied throughout the cell cycle of M. genitalium in a coordinated manner with the chromosome and the terminal organelle (TMO). In addition, our results indicate a possible role for the RNA methyltransferase MraW in the regulation of FtsZ expression at the post-transcriptional level. Altogether, this study provides an extensive characterization of the cell division gene cluster of M. genitalium and demonstrates the existence of regulatory elements controlling FtsZ expression at the temporal and spatial level in mycoplasmas.

The Cre-Lox system is a highly versatile and powerful DNA recombinase mechanism, mainly used in genetic engineering to insert or remove desired DNA sequences. It is widely utilized across multiple fields of biology, with applications ranging from plants, to mammals, to microbes. A key feature of this system is its ability to allow recombination between mutant lox sites. Two of the most commonly used mutant sites are named lox66 and lox71, which recombine to create a functionally inactive double mutant lox72 site. However, a large portion of the published literature has incorrectly annotated these mutant lox sites, which in turn can lead to difficulties in replication of methods, design of proper vectors and confusion over the proper nomenclature. Here, we demonstrate common errors in annotations, the impacts they can have on experimental viability, and a standardized naming convention. We also show an example of how this incorrect annotation can induce toxic effects in bacteria that lack optimal DNA repair systems, exemplified by Mycoplasma pneumoniae. This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement 634942 (MycoSynVac) and was also financed by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme, under grant agreement 670216 (MYCOCHASSIS). We also acknowledge support of the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) to the EMBL partnership, the Centro de Excelencia Severo Ochoa and the CERCA Programme/Generalitat de Catalunya

Protein degradation is a crucial cellular process in all‐living systems. Here, using Mycoplasma pneumoniae as a model organism, we defined the minimal protein degradation machinery required to maintain proteome homeostasis. Then, we conditionally depleted the two essential ATP‐dependent proteases. Whereas depletion of Lon results in increased protein aggregation and decreased heat tolerance, FtsH depletion induces cell membrane damage, suggesting a role in quality control of membrane proteins. An integrative comparative study combining shotgun proteomics and RNA‐seq revealed 62 and 34 candidate substrates, respectively. Cellular localization of substrates and epistasis studies supports separate functions for Lon and FtsH. Protein half‐life measurements also suggest a role for Lon‐modulated protein decay. Lon plays a key role in protein quality control, degrading misfolded proteins and those not assembled into functional complexes. We propose that regulating complex assembly and degradation of isolated proteins is a mechanism that coordinates important cellular processes like cell division. Finally, by considering the entire set of proteases and chaperones, we provide a fully integrated view of how a minimal cell regulates protein folding and degradation., A minimal protein degradation machinery required for maintaining proteome homeostasis is defined in the genome‐reduced bacterium M. pneumoniae. Genetic and high‐throughput analyses identify substrates and pathways regulated by degradation.

[Background] Cancer is a rapidly evolving, multifactorial disease that accumulates numerous genetic and epigenetic alterations. This results in molecular and phenotypic heterogeneity within the tumor, the complexity of which is further amplified through specific interactions between cancer cells. We aimed to dissect the molecular mechanisms underlying the cooperation between different clones., [Methods] We produced clonal cell lines derived from the MDA-MB-231 breast cancer cell line, using the UbC-StarTrack system, which allowed tracking of multiple clones by color: GFP C3, mKO E10 and Sapphire D7. Characterization of these clones was performed by growth rate, cell metabolic activity, wound healing, invasion assays and genetic and epigenetic arrays. Tumorigenicity was tested by orthotopic and intravenous injections. Clonal cooperation was evaluated by medium complementation, co-culture and co-injection assays., [Results] Characterization of these clones in vitro revealed clear genetic and epigenetic differences that affected growth rate, cell metabolic activity, morphology and cytokine expression among cell lines. In vivo, all clonal cell lines were able to form tumors; however, injection of an equal mix of the different clones led to tumors with very few mKO E10 cells. Additionally, the mKO E10 clonal cell line showed a significant inability to form lung metastases. These results confirm that even in stable cell lines heterogeneity is present. In vitro, the complementation of growth medium with medium or exosomes from parental or clonal cell lines increased the growth rate of the other clones. Complementation assays, co-growth and co-injection of mKO E10 and GFP C3 clonal cell lines increased the efficiency of invasion and migration., [Conclusions] These findings support a model where interplay between clones confers aggressiveness, and which may allow identification of the factors involved in cellular communication that could play a role in clonal cooperation and thus represent new targets for preventing tumor progression., SRYC acknowledges support from Fondo de Investigaciones Sanitarias (FIS; PI17/02247 and PI14/01320), Centro de Investigación Biomédica en Red de Cáncer (CIBERONC; CB16/12/00363) and Generalitat de Catalunya (AGAUR; 2017 SGR 1799 and 2014 SGR 1131). AMP is funded by a FP7 Marie Sklodowska-Curie COFUND program under Grant Agreement n° 267128 (INCOMED program). ADL is funded by a “Juan Rodés” contract (JR17/00016) from ISCIII.

It is well known that in cancer gene families some members are more frequently mutated in tumor samples than their family counterparts. A paradigmatic case of this phenomenon is KRAS from the RAS family. Different explanations have been proposed ranging from differential interaction with other proteins to preferential expression or localization. Interestingly, it has been described that despite the high amino acid identity between RAS family members, KRAS employs an intriguing differential codon usage. Here, we found that this phenomenon is not exclusive to the RAS family. Indeed, in the RAS family and other oncogene families with two or three members, the most prevalently mutated gene in tumor samples employs a differential codon usage that is characteristic of genes involved in proliferation. Prompted by these observations, we chose the RAS family to experimentally demonstrate that the translation efficiency of oncogenes that are preferentially mutated in tumor samples is increased in proliferative cells compared to quiescent cells. These results were further validated by assessing the translation efficiency of KRAS in cell lines that differ in their tRNA expression profile. These differences are related to the cell division rate of the studied cells and thus suggest an important role in context-specific oncogene expression regulation. Altogether, our study demonstrates that dynamic translation programs contribute to shaping the expression profiles of oncogenes. Therefore, we propose this codon bias as a regulatory layer to control cell context-specific expression and explain the differential prevalence of mutations in certain members of oncogene families. The work of X.H.-A. has been supported by a PhD fellowship from the Fundación Ramón Areces. We acknowledge the support of the Spanish Ministry of Science and Innovation to the European Molecular Biology Laboratory partnership, the Centro de Excelencia Severo Ochoa and the Centres de Recerca de Catalunya Programme/Generalitat de Catalunya

The Cre-Lox system is a highly versatile and powerful DNA recombinase mechanism, mainly used in genetic engineering to insert or remove desired DNA sequences. It is widely utilised across multiple fields of biology, with applications ranging from plants, to mammals, to microbes. A key feature of this system is its ability to allow recombination between mutant lox sites, traditionally named lox66 and lox71, to create a functionally inactive double mutant lox72 site. However, a large portion of the published literature has incorrectly annotated these mutant lox sites, which in turn can lead to difficulties in replication of methods, design of proper vectors, and confusion over the proper nomenclature. Here, we demonstrate common errors in annotations, the impacts they can have on experimental viability, and a standardised naming convention. We also show an example of how this incorrect annotation can induce toxic effects in bacteria that lack optimal DNA repair systems, exemplified byMycoplasma pneumoniae.Data SummaryThe authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

Aggregates of Pseudomonas aeruginosa form a protective barrier against antibiotics and the immune system. These barriers, known as biofilms, are associated with several infectious diseases. One of the main components of these biofilms is alginate, a homo- and hetero-polysaccharide that consists of β-D-mannuronate (M) and α-L-guluronate (G) units. Alginate lyases degrade this sugar and have been proposed as biotherapeutic agents to dissolve P. aeruginosa biofilms. However, there are contradictory reports in the literature regarding the efficacy of alginate lyases against biofilms and their synergistic effect with antibiotics. We found that most positive reports used a commercial crude extract from Flavobacterium multivorum as the alginate lyase source. By using anion exchange chromatography coupled to nano LC MS/MS, we identified two distinct enzymes in this extract, one has both polyM and polyG (polyM/G) degradation activities and it is similar in sequence to a broad-spectrum alginate lyase from Flavobacterium sp. S20 (Alg2A). The other enzyme has only polyG activity and it is similar in sequence to AlyA1 from Zobellia galactanivorans. By characterizing both of these enzymes together with three recombinant alginate lyases (a polyM, a polyG and a polyM/G), we showed that only enzymes with polyM/G activity such as Alg2A and A1-II' (alginate lyase from Sphingomonas sp.) are effective in dissolving biofilms. Furthermore, both activities are required to have a synergistic effect with antibiotics. We acknowledge support of the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program under agreement No 670216 (MYCOCHASSIS), the Spanish Ministry of Economy and Competitiveness and Fondo Europeo de Desarrollo Regional (MINECO-FEDER) (BIO2015-63557-R), ‘Centro de Excelencia Severo Ochoa 2013-2017’, FEDER project from Instituto Carlos III (ISCIII, Acción Estratégica en Salud 2016) (reference CP16/00094) and “Secretaria d’Universitats i Recerca del Departament d’Economia i Coneixement de la Generalitat de Catalunya / CERCA programme” (2014SGR678 and 2017SGR1079). The CRG/UPF Proteomics Unit is part of the “Plataforma de Recursos Biomoleculares y Bioinformáticos (ProteoRed)” supported by grant PT13/0001 of Instituto de Salud Carlos III from the Spanish Government.

Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac disease. Mutations inMYBPC3, the gene encoding cardiac myosin-binding protein C (cMyBP-C), are a leading cause of HCM. However, it remains challenging to define whether specific gene variants found in patients are pathogenic or not, limiting the reach of cardiovascular genetics in the management of HCM. Here, we have examined cMyBP-C haploinsufficiency drivers in 68 clinically annotated non-truncating variants ofMYBPC3. We find that 45% of the pathogenic variants show alterations in RNA splicing or protein stability, which can be linked to pathogenicity with 100% and 94% specificity, respectively. Relevant for variant annotation, we uncover that 9% of non-truncating variants ofMYBPC3currently classified as of uncertain significance induce one of these molecular phenotypes. We propose that alteration of RNA splicing or protein stability caused byMYBPC3variants provide strong evidence of their pathogenicity, leading to improved clinical management of HCM patients and their families.

Comparative genomics among bacteria has been used to gain insight into the minimal number of conserved genes in biological pathways. Essentiality studies have provided information regarding which genes are non-dispensable (essential, E) for cell growth. Here, we integrated studies of gene conservation, essentiality and function, performed in 47 diverse bacterial species. We showed there is a modest positive correlation between genome size and number of essential genes. Interestingly, we observed a clear shift in the functions assigned to these essential genes as genome size increases. For instance, essential genes related to transcription and translation dominate small genomes. In contrast, in large genomes functions of essential genes are related with cellular processing and metabolism. Finally, and most intriguing, we found a group of genes that while being highly conserved are also typically non-essential. This suggests that some housekeeping genes confer a significant survival benefit in nature while being non-essential in vitro.

Mycoplasma pneumoniaeis a slow-growing, human pathogen that causes atypical pneumonia. Because it lacks a cell wall, many antibiotics are ineffective, and vaccination is required. Due to its reduced genome and dearth of many biosynthetic pathways, this fastidious bacterium depends on rich, undefined medium for growth, which makes large-scale cultivation for vaccine production challenging and expensive.To understand factors limiting growth, we developed a genome-scale, constraint-based model ofM. pneumoniaecalled iEG158_mpn to describe the metabolic potential of this bacterium. We have put special emphasis on cell membrane formation to identify key lipid components to maximize bacterial growth. We have used this knowledge to predict and validatein vitrotwo serum-free media able to sustain growth.Our findings also show that glycolysis and lipid metabolism are much less efficient under hypoxia; these findings suggest that factors other than metabolism and membrane formation alone affect the growth ofM. pneumoniae.Altogether, our modelling approach enabled us to optimize medium composition, capacitated growth in defined media and streamlined operational requirements, thereby providing the basis for stable, reproducible and less expensive vaccine production.

Mycoplasma species share a set of features, such as lack of a cell wall, streamlined genomes, simplified metabolism, and the use of a deviant genetic code, that make them attractive approximations of what a chassis strain should ideally be. Among them, Mycoplasma pneumoniae arises as a candidate for synthetic biology projects, as it is one of the most deeply characterized bacteria. However, the historical paucity of tools for editing Mycoplasma genomes has precluded the establishment of M. pneumoniae as a suitable chassis strain. Here, we developed an oligonucleotide recombineering method for this strain based on GP35, a ssDNA recombinase originally encoded by a Bacillus subtilis-associated phage. GP35-mediated oligo recombineering is able to carry out point mutations in the M. pneumoniae genome with an efficiency as high as 2.7 × 10-2, outperforming oligo recombineering protocols developed for other bacteria. Gene deletions of different sizes showed a decreasing power trend between efficiency and the scale of the attempted edition. However, the editing rates for all modifications increased when CRISPR/Cas9 was used to counterselect nonedited cells. This allowed edited clones carrying chromosomal deletions of up to 1.8 kb to be recovered with little to no screening of survivor cells. We envision this technology as a major step toward the use of M. pneumoniae, and possibly other Mycoplasmas, as synthetic biology chassis strains. This project has received funding from the European Union’s Horizon 2020 research and innovation program under Grant 634942 (MycoSynVac) and was also financed by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program under Grant 670216 (MYCOCHASSIS) and the FEDER project from Instituto Carlos III (ISCIII, Acción Estratégica en Salud 2016) (reference CP16/00094). We also acknowledge support of the Spanish Ministry of Science and Innovation, to the EMBL partnership, the Centro de Excelencia Severo Ochoa, and the CERCA Programme/Generalitat de Catalunya. Finally, we would like to thank Dr. Sarah A. Head for critical manuscript revision.

The coronavirus disease COVID-19 constitutes the most severe pandemic of the last decades having caused more than 1 million deaths worldwide. The SARS-CoV-2 virus recognizes the angiotensin converting enzyme 2 (ACE2) on the surface of human cells through its spike protein. It has been reported that the coronavirus can mildly infect cats, and ferrets, and perhaps dogs while not pigs, mice, chicken and ducks. Differences in viral infectivity among different species or individuals could be due to amino acid differences at key positions of the host proteins that interact with the virus, the immune response, expression levels of host proteins and translation efficiency of the viral proteins among other factors. Here, first we have addressed the importance that sequence variants of different animal species, human individuals and virus isolates have on the interaction between the RBD domain of the SARS-CoV-2 spike S protein and human angiotensin converting enzyme 2 (ACE2). Second, we have looked at viral translation efficiency by using the tRNA adaptation index. We find that integration of both interaction energy with ACE2 and translational efficiency explains animal infectivity. Humans are the top species in which SARS-CoV-2 is both efficiently translated as well as optimally interacting with ACE2. We have found some viral mutations that increase affinity for hACE and some hACE2 variants affecting ACE2 stability and virus binding. These variants suggest that different sensitivities to coronavirus infection in humans could arise in some cases from allelic variability affecting ACE2 stability and virus binding., Author summary In these early stages of the COVID-19 pandemic it is urgent to understand all features determining the new virus expansion. Two significant factors conditioning infection are ACE2-mediated SARS-CoV-2 cellular entry and viral proteome translation efficiency. Genomic variability across species, including humans, results in ACE2 variants that destabilize its fold, modify ACE2/SARS-CoV-2 recognition, or both. We also point out the importance of considering waters at the interface of protein-protein interactions when performing in silico mutagenesis.

Different tissues express genes with particular codon usage and anticodon tRNA repertoires. However, the codon–anticodon co‐adaptation in humans is not completely understood, nor is its effect on tissue‐specific protein levels. Here, we first validated the accuracy of small RNA‐seq for tRNA quantification across five human cell lines. We then analyzed the tRNA abundance of more than 8,000 tumor samples from TCGA, together with their paired mRNA‐seq and proteomics data, to determine the Supply‐to‐Demand Adaptation. We thereby elucidate that the dynamic adaptation of the tRNA pool is largely related to the proliferative state across tissues. The distribution of such tRNA pools over the whole cellular translatome affects the subsequent translational efficiency, which functionally determines a condition‐specific expression program both in healthy and tumor states. Furthermore, the aberrant translational efficiency of some codons in cancer, exemplified by ProCCA and GlyGGT, is associated with poor patient survival. The regulation of these tRNA profiles is partly explained by the tRNA gene copy numbers and their promoter DNA methylation., Quantification of tRNA expression over thousands of small RNA‐seq samples from The Cancer Genome Atlas unveils the existence of tissue‐specific translational efficiencies related to proliferation.

Age-related macular degeneration (AMD) is one of the commonest causes of sight loss in the elderly population and to date there is no intervention that slows or prevents early AMD disease progressing to blinding neovascularization or geographic atrophy. AMD is a complex disease and factors proposed to contribute to the development and progression of disease include aging, genetics, epigenetics, oxidative stress, pro-inflammatory state, and life-style factors such as smoking, alcohol, and high fat diet. Here, we generate a knowledge repository of pathways and protein-protein interaction (PPI) networks likely to be implicated in AMD pathogenesis, such as complement activation, lipid trafficking and metabolism, vitamin A cycle, oxidative stress, proteostasis, bioenergetics, autophagy/mitophagy, extracellular matrix (ECM) turnover, and choroidal vascular dropout. Two disctinct clusters ermerged from the networks for parainflamation and ECM homeostasis, which may represent two different disease modules underlying AMD pathology. Our analyses also suggest that the disease manifests primarily in RPE/choroid and less in neural retina. The use of standardized syntax when generating maps of these biological processes (SBGN standard) and networks (PSI standard) enables visualization of complex information in graphical programs such as CellDesigner and Cytoscape and enhances reusability and extension of data. The ability to focus onto subnetworks, multiple visualizations and simulation options will enable the AMD research community to computationally model subnetworks or to test experimentally new hypotheses arising from connectivities in the AMD pathway map. This work has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No. 634479 (EYE-RISK—to P.J.L., L.S., and C.K.). This work was supported by the Spanish Ministry of Economy and Competitiveness, Plan Nacional BIO2012-39754 and the European Fund for Regional Development (to L.S.). L.S. acknowledges the support of the Spanish Ministry of Economy and Competitiveness, “Centro de Excelencia Severo Ochoa 2013–2017” (SEV-2012-0208)

We examined possible early-effect biomarkers and polymorphisms of susceptibility in primary school children living near the Atoyac River in central Mexico, which receives waste from multiple industries. We observed a significant increase in micronucleated reticulocytes associated with the oxidative stress index (OSI) and the OGG1 GG (S326C) genotype, and a significant decrease of reticulocytes carrying the transferrin receptor, inversely correlated with OSI.

DNA methylation is an epigenetic mechanism known to affect gene expression and aberrant DNA methylation patterns have been described in cancer. However, only a small fraction of differential methylation events target genes with a defined role in cancer, raising the question of how aberrant DNA methylation contributes to carcinogenesis. As recently a link has been suggested between methylation patterns arising in ageing and those arising in cancer, we asked which aberrations are unique to cancer and which are the product of normal ageing processes. We therefore compared the methylation patterns between ageing and cancer in multiple tissues. We observed that hypermethylation preferentially occurs in regulatory elements, while hypomethylation is associated with structural features of the chromatin. Specifically, we observed consistent hypomethylation of late-replicating, lamina-associated domains. The extent of hypomethylation was stronger in cancer, but in both ageing and cancer it was proportional to the replication timing of the region and the cell division rate of the tissue. Moreover, cancer patients who displayed more hypomethylation in late-replicating, lamina-associated domains had higher expression of cell division genes. These findings suggest that different cell division rates contribute to tissue- and cancer type-specific DNA methylation profiles. German Research Foundation [SCHA 1933/1-1]; European Union Seventh Framework Programme [FP7/2007-2013] under grant agreements [HEALTH-F4-2011-278568 (PRIMES)]; Spanish Ministry of Economy, Industry and Competitiveness (MEIC) [BFU2015-63571-P] and to the EMBL partnership; the European Regional Development Fund (ERDF/FEDER); Centro de Excelencia Severo Ochoa and the support of the CERCA Programme/Generalitat de Catalunya. Funding for open access charge: Spanish Ministry of Economy, Industry and Competitiveness (MEIC) [BFU2015-63571-P].

The C‐terminal sequence of a protein is involved in processes such as efficiency of translation termination and protein degradation. However, the general relationship between features of this C‐terminal sequence and levels of protein expression remains unknown. Here, we identified C‐terminal amino acid biases that are ubiquitous across the bacterial taxonomy (1,582 genomes). We showed that the frequency is higher for positively charged amino acids (lysine, arginine), while hydrophobic amino acids and threonine are lower. We then studied the impact of C‐terminal composition on protein levels in a library of Mycoplasma pneumoniae mutants, covering all possible combinations of the two last codons. We found that charged and polar residues, in particular lysine, led to higher expression, while hydrophobic and aromatic residues led to lower expression, with a difference in protein levels up to fourfold. We further showed that modulation of protein degradation rate could be one of the main mechanisms driving these differences. Our results demonstrate that the identity of the last amino acids has a strong influence on protein expression levels., Large‐scale genomics analyses combined with high‐throughput experimental assays reveal that the C‐terminal amino acid composition has a strong influence on protein expression levels in bacteria.

Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac disease. Mutations in cardiac myosin-binding protein C (cMyBP-C) are a leading cause of HCM. However, as for many other genetic diseases, it remains challenging to define whether specific gene variants found in patients are pathogenic or not. Here, we have examined pathogenicity drivers in a group of clinically annotated exonic variants of MYBPC3, the gene encoding cMyBP-C. First, we did bioinformatics predictions of RNA splicing and of protein thermodynamic stability. To validate results, we studied RNA splicing of the MYBPC3 gene using peripheral blood from variant carriers, and circular dichroism measurements on purified recombinant proteins. Our results show that around half of the pathogenic exonic mutations alter RNA splicing or protein thermodynamic stability, both of which can lead to cMyBP-C haploinsufficiency. These molecular phenotypes are not found in control, non-pathogenic variants. Remarkably, the remaining pathogenic missense mutations appear to result in stable proteins, for which the cause of pathogenicity remains unknown. We propose that examination of protein haploinsufficiency drivers can define pathogenicity of genetic variants associated with HCM, decisive for the clinical management of patients and their families.

Identification of small open reading frames (smORFs) encoding small proteins (≤ 100 amino acids; SEPs) is a challenge in the fields of genome annotation and protein discovery. Here, by combining a novel bioinformatics tool (RanSEPs) with "-omics" approaches, we were able to describe 109 bacterial small ORFomes. Predictions were first validated by performing an exhaustive search of SEPs present in Mycoplasma pneumoniae proteome via mass spectrometry, which illustrated the limitations of shotgun approaches. Then, RanSEPs predictions were validated and compared with other tools using proteomic datasets from different bacterial species and SEPs from the literature. We found that up to 16 ± 9% of proteins in an organism could be classified as SEPs. Integration of RanSEPs predictions with transcriptomics data showed that some annotated non-coding RNAs could in fact encode for SEPs. A functional study of SEPs highlighted an enrichment in the membrane, translation, metabolism, and nucleotide-binding categories. Additionally, 9.7% of the SEPs included a N-terminus predicted signal peptide. We envision RanSEPs as a tool to unmask the hidden universe of small bacterial proteins. We thank Dr. Luca Cozzuto from the Bioinformatics Unit at CRG for providing valuable guidance for the conservation studies. Also, we would like to thank Dr. Carolina Gallo, Dr. Eva Yus, and Dr. Raul Burgos for providing MS data. Finally, we thank Dr. Marc Weber for his recommendation about how to analyze Ribo-Seq data. We acknowledge support of the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) to the EMBL partnership, the Spanish Ministry of Economy and Competitiveness, “Centro de Excelencia Severo Ochoa”, the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program under agreement No 670216 (MYCOCHASSIS), the CERCA Programme/Generalitat de Catalunya, the European Regional Development Fund (ERDF) project from Instituto Carlos III (ISCIII, Acción Estratégica en Salud 2016; reference CP16/00094), and “Secretaria d’Universitats i Recerca del Departament d’Economia i Coneixement de la Generalitat de Catalunya” (2014SGR678). The CRG/UPF Proteomics Unit is part of the “Plataforma de Recursos Biomoleculares y Bioinformáticos (ProteoRed)” supported by grant PT13/0001 of Instituto de Salud Carlos III from the Spanish Government.

Summary Here, we determined the relative importance of different transcriptional mechanisms in the genome-reduced bacterium Mycoplasma pneumoniae, by employing an array of experimental techniques under multiple genetic and environmental perturbations. Of the 143 genes tested (21% of the bacterium’s annotated proteins), only 55% showed an altered phenotype, highlighting the robustness of biological systems. We identified nine transcription factors (TFs) and their targets, representing 43% of the genome, and 16 regulators that indirectly affect transcription. Only 20% of transcriptional regulation is mediated by canonical TFs when responding to perturbations. Using a Random Forest, we quantified the non-redundant contribution of different mechanisms such as supercoiling, metabolic control, RNA degradation, and chromosome topology to transcriptional changes. Model-predicted gene changes correlate well with experimental data in 95% of the tested perturbations, explaining up to 70% of the total variance when also considering noise. This analysis highlights the importance of considering non-TF-mediated regulation when engineering bacteria., Graphical Abstract, Highlights • Full comprehensive reconstruction of a bacterial gene regulatory network achieved • Genome-reduced bacterium Mycoplasma pneumoniae is robust to genetic perturbations • Large part of transcription regulation in bacteria is transcription-factor independent • Transcription-factor-independent regulation has a smaller dynamic range, We have achieved a comprehensive reconstruction of a gene regulatory network in a genome-reduced bacterium, Mycoplasma pneumoniae. With this network, we observed that a large part of transcription regulation is determined by non-canonical factors such as DNA supercoiling, riboswitches, and genome organization or RNA-mediated regulation. This analysis highlights the importance of considering non-transcription-factor (TF)-mediated regulation when engineering bacteria.

Viruses need to hijack the translational machinery of the host cell for a productive infection to happen. However, given the dynamic landscape of tRNA pools among tissues, it is unclear whether different viruses infecting different tissues have adapted their codon usage toward their tropism. Here, we collect the coding sequences of 502 human-infecting viruses and determine that tropism explains changes in codon usage. Using the tRNA abundances across 23 human tissues from The Cancer Genome Atlas (TCGA), we build an in silico model of translational efficiency that validates the correspondence of the viral codon usage with the translational machinery of their tropism. For instance, we detect that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is specifically adapted to the upper respiratory tract and alveoli. Furthermore, this correspondence is specifically defined in early viral proteins. The observed tissue-specific translational efficiency could be useful for the development of antiviral therapies and vaccines., Graphical abstract, Viruses need to hijack the translational machinery of the host for the expression of their own proteins. Hernandez-Alias et al. show that viruses that infect different tissues use different synonymous codons, which can affect the efficiency in which they are translated across human tissues.

We examined 314 red foxes (Vulpes vulpes) from the province of Soria, Spain, for Rickettsia typhi, Rickettsia slovaca, and Borrelia burgdorferi infection. Immunofluorescence assays showed 1.9% had antibodies to R. typhi, 6.7% had antibodies to R. slovaca, and 8.3% had antibodies to B. burgdorferi. Serostatus was not correlated with sex or age. Because red foxes can be infected by Rickettsiae and B. burgdorferi, presence of red foxes may be and indicator for the presence of these pathogens.

Four hundred red foxes (Vulpes vulpes) were examined for ecto- (arthropods) and endoparasites (Leishmania spp., Trichinella spp., and intestinal parasites). Different species of flea (total prevalence, 40.50%), tick (16.25%), mite (7.25%), and fly (1.50%) were identified. The most prevalent flea was Pulex irritans (found on 29% of the foxes); the most prevalent tick, mite, and fly were Ixodes canisuga (on 5%), Sarcoptes scabiei (on 5.25%), and Hippobosca equina (on 1%), respectively. The endoparasites identified included Leishmania spp. (found in 12% of the foxes), Trichinella spp. (in 15.5%, with T. britovi the most prevalent species in 15.25%), Cestoda (in 72.75%, with Mesocestoides spp. the most prevalent in 69.50%), and intestinal ascarids (in 73.25%, with Ancylostoma caninum the most prevalent in 12.50%). No animal was free of parasites. The present results suggest that foxes can act as sentinels of diseases transmitted by ecto- and endoparasites.

Transposon sequencing is commonly applied for identifying the minimal set of genes required for cellular life; a major challenge in fields such as evolutionary or synthetic biology. However, the scientific community has no standards at the level of processing, treatment, curation and analysis of this kind data. In addition, we lack knowledge about artifactual signals and the requirements a dataset has to satisfy to allow accurate prediction. Here, we have developed FASTQINS, a pipeline for the detection of transposon insertions, and ANUBIS, a library of functions to evaluate and correct deviating factors known and uncharacterized until now. ANUBIS implements previously defined essentiality estimate models in addition to new approaches with advantages like not requiring a training set of genes to predict general essentiality. To highlight the applicability of these tools, and provide a set of recommendations on how to analyze transposon sequencing data, we performed a comprehensive study on artifacts corrections and essentiality estimation at a 1.5-bp resolution, in the genome-reduced bacterium Mycoplasma pneumoniae. We envision FASTQINS and ANUBIS to aid in the analysis of Tn-seq procedures and lead to the development of accurate genome essentiality estimates to guide applications such as designing live vaccines or growth optimization., Nucleic Acids Research, 48 (17), ISSN:1362-4962, ISSN:0301-5610

Three hundred and fourteen red foxes (Vulpes vulpes) in the province of Soria, Spain, were examined for hantavirus and lymphocytic choriomeningitis virus (LCMV) infection (and were likely to have been infected by feeding on infected rodents). Immunofluorescence and western blot assays confirmed 3.5% (11/314) to have antibodies to hantaviruses, and the immune fluorescence assay showed 2.2% (7/314) to have antibodies to LCMV. The serologic status of the animals showed no statistically significant association with sex or age. Although studies on the prevalence of hantaviruses and LCMV normally focus on rodents, our results showed that foxes can provide complementary information in determined areas.

Summary Here, we propose an approach to identify active metabolic pathways by integrating gene essentiality analysis and protein abundance. We use two bacterial species (Mycoplasma pneumoniae and Mycoplasma agalactiae) that share a high gene content similarity yet show significant metabolic differences. First, we build detailed metabolic maps of their carbon metabolism, the most striking difference being the absence of two key enzymes for glucose metabolism in M. agalactiae. We then determine carbon sources that allow growth in M. agalactiae, and we introduce glucose-dependent growth to show the functionality of its remaining glycolytic enzymes. By analyzing gene essentiality and performing quantitative proteomics, we can predict the active metabolic pathways connected to carbon metabolism and show significant differences in use and direction of key pathways despite sharing the large majority of genes. Gene essentiality combined with quantitative proteomics and metabolic maps can be used to determine activity and directionality of metabolic pathways., Graphical Abstract, Highlights • Active metabolic bacterial pathways are identified • Integration of gene essentiality and proteomics allow prediction of active pathways • Glucose-dependent growth is restored in Mycoplasma agalactiae • Two Mycoplasma species show different usage of metabolic pathways, Montero-Blay et al. identify active metabolic pathways in bacteria by integrating gene essentiality data and quantitative proteomics. Predictions agree with experimental information and show substantial differences in usage and directionality of metabolic pathways in bacteria with high degree of gene similarity.

Background: Proteins of the ubiquitously expressed core proteome are quantitatively correlated across multiple eukaryotic species. In addition, it was found that many protein paralogues exhibit expression anticorrelation, suggesting that the total level of protein with a given functionality must be kept constant. Methods: We performed Spearman’s rank correlation analyses of gene expression levels for the RAS GTPase subfamily and their regulatory GEF and GAP proteins across tissues and across individuals for each tissue. A large set of published data for normal tissues from a wide range of species, human cancer tissues and human cell lines was analysed. Results: We show that although the multidomain regulatory proteins of Ras GTPases exhibit considerable tissue and individual gene expression variability, their total amounts are balanced in normal tissues. In a given tissue, the sum of activating (GEFs) and deactivating (GAPs) domains of Ras GTPases can vary considerably, but each person has balanced GEF and GAP levels. This balance is impaired in cell lines and in cancer tissues for some individuals. Conclusions: Our results are relevant for critical considerations of knock out experiments, where functionally related homologs may compensate for the down regulation of a protein. This work was funded by the EU (PRIMES under grant agreement number FP7-HEALTH-F4- 2011-278568). This work was supported by the Spanish Ministerio de Economía y Competitividad, Plan Nacional BIO2012-39754 and Plan Estatal BFU2015-63571 and the European Fund for Regional Development. We acknowledge support of the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) to the EMBL partnership, to the Spanish Ministry of Economy and Competitiveness (MEC) ‘Centro de Excelencia Severo Ochoa’ and to the CERCA Programme / Generalitat de Catalunya.

Determining the gene regulatory network of an organism is fundamental for achieving a global understanding of cellular behaviour. Here, we determine the relative importance of different transcription regulators in the genome-reduced bacterium Mycoplasma pneumoniae, by integrating DNA affinity chromatography, chromatin isolation, ChIP-seq, transcriptomics, proteomics and growth curves experiments for 143 genes (20.8% of the bacteria´s NCBI annotated proteins). In 55% of the cases, gene overexpression or mutation does not result in transcriptional nor growth phenotypes, highlighting the robustness of even very simple cells. We identified nine TFs (besides the sigma factor) and their targets, representing 33% of the genome. Nevertheless, we find that the majority of transcription regulation in M. pneumoniae is not mediated by canonical TFs, but rather occurs at the level of supercoiling, metabolic control, RNA degradation, and chromosome topology. For the first time, we quantify the non-redundant contribution of each of these mechanisms in various environmental perturbations using a random forest model, showing that cells use different mechanisms to respond to distinct stresses. The gene changes predicted by our model correlate with the actual changes in 95% of the tested perturbations, and after accounting for experimental and gene expression noise, we can explain over 70% of the total variance. This comprehensive analysis highlights the importance of non-TF-mediated regulation in bacteria, an ancient type of regulation that allows adaptation to mild environmental changes.

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a death ligand cytokine known for its cytotoxic activity against malignantly transformed cells. TRAIL induces cell death through binding to death receptors DR4 and DR5. The inhibitory decoy receptors (DcR1 and DcR2) co-expressed with death receptor 4 (DR4)/DR5 on the same cell can block the transmission of the apoptotic signal. Here, we show that DcRs also regulate TRAIL sensitivity at a supracellular level and thus represent a mechanism by which the microenvironment can diminish tumour TRAIL sensitivity. Mathematical modelling and layered or spheroid stroma–extracellular matrix–tumour cultures were used to model the tumour microenvironment. By engineering TRAIL to escape binding by DcRs, we found that DcRs do not only act in a cell-autonomous or cis-regulatory manner, but also exert trans-cellular regulation originating from stromal cells and affect tumour cells, highlighting the potent inhibitory effect of DcRs in the tumour tissue and the necessity of selective targeting of the two death-inducing TRAIL receptors to maximise efficacy.

In this study, a new simple method for chemical modification of wood from fast-growing Eucalyptus saligna (blue gum) was tested by a two-step treatment with methacryloyl chloride. The wood samples were submerged in a solution of 10 % methacryloyl chloride in dichloromethane for 15 h and then exposed to three temperatures: air dried, 50 and 100 °C for 2 h. The chemical modifications of the wood were examined by ATR-IR and 13C CP/MAS solid-state NMR; quantitative chemical composition of wood samples by means of wet chemical quantification, HPLC, TGA; colorimetric evaluation by CIELAB method and UV–Vis spectroscopy. Water uptake tests were also performed. A partial substitution of the hydroxyl by methacrylate groups on the cell wall of blue gum wood was observed, and also loss of a hemicelluloses fraction after the treatments. The colour change increased with increasing temperature in the second step of the treatment. Dimensional stability and water repellence were improved by 40–50 and 60–75 %, respectively.

This study aims to evaluate the chemical composition of wood and extractives of Pinus taeda and Schizolobium parahyba (guapuruvu) as potential feedstock for new applications in the biorefinery industry. For this purpose, their content of α-cellulose, hemicellulose, insoluble lignin, hot water solubility, NaOH1% solubility, inorganic materials (ash), and monomeric sugars by high-performance liquid chromatography was quantified. Attenuated total reflectance infrared spectroscopy and thermogravimetric analysis were also used to complete the physicochemical characterization of the studied woods. The extractives were obtained by soxhlet extraction with ethanol:toluene and dichloromethane and identified with pyrolysis-gas chromatography/mass spectroscopy technique. The results showed that guapuruvu wood has the higher amount of hemicellulose (16%) when compared to pine wood (10%), which resulted in higher solubility in alkali solution. Furthermore, in relation to other biomasses, the two woods presented...

DNA-binding proteins are central regulators of chromosome organization; however, in genome-reduced bacteria their diversity is largely diminished. Whether the chromosomes of such bacteria adopt defined three-dimensional structures remains unexplored. Here we combine Hi-C and super-resolution microscopy to determine the structure of the Mycoplasma pneumoniae chromosome at a 10 kb resolution. We find a defined structure, with a global symmetry between two arms that connect opposite poles, one bearing the chromosomal Ori and the other the midpoint. Analysis of local structures at a 3 kb resolution indicates that the chromosome is organized into domains ranging from 15 to 33 kb. We provide evidence that genes within the same domain tend to be co-regulated, suggesting that chromosome organization influences transcriptional regulation, and that supercoiling regulates local organization. This study extends the current understanding of bacterial genome organization and demonstrates that a defined chromosomal structure is a universal feature of living systems., The three-dimensional architecture of genome-reduced bacteria is poorly understood. Here the authors combine Hi-C with super-resolution microscopy in Mycoplasma pneumoniae and provide evidence of how supercoiling and local organization influences gene regulation.

G protein-coupled receptors (GPCRs) are membrane receptors critically involved in sensing the environment and orchestrating physiological processes. As such, they transduce extracellular signals such as hormone, neurotransmitters, ions, and light into an integrated cell response. The intracellular trafficking, internalization, and signaling ability of ligand-activated GPCRs are controlled by arrestins, adaptor proteins that they interact with upon ligand binding. β-arrestins 1 and 2 in particular are now considered as hub proteins assembling multiprotein complexes to regulate receptor fate and transduce diversified cell responses. While more than 400 β-arrestin interaction partners have been identified so far, much remains to be learnt on how discrimination between so many binding partners is accomplished. Here, we gathered the interacting partners of β-arrestins through database mining and manual curation of the literature to map the β-arrestin interactome (β-arrestinome). We discussed several parameters that determine compatible (AND) or mutually exclusive (XOR) binding of β-arrestin interactors, such as structural constraints, intracellular abundance, or binding affinity. This work was funded by the Agence Nationale de la Recherche GPCRnet project, by the Centre National de la Recherche Scientifique, by the Institut de la Recherche Agronomique PHASE Department, by the Région Centre, and by the European Commission (EC) Framework programme (FP) 7 project SynSignal (contract nr. 613879). LS is supported by the Spanish Ministerio de Economía y Competitividad, Plan Nacional BIO2012-39754, and the European Fund for Economic and Regional Development. LS and CK are particularly grateful for the support of the Spanish Ministry of Economy and Competitiveness, “Centro de Excelencia Severo Ochoa 2013-2017” (SEV-2012-0208).

The understanding of interaction dynamics in signaling pathways can shed light on pathway architecture and provide insights into targets for intervention. Here, we explored the relevance of kinetic rate constants of a key upstream osmosensor in the yeast high-osmolarity glycerol-mitogen-activated protein kinase (HOG-MAPK) pathway to signaling output responses. We created mutant pairs of the Sln1-Ypd1 complex interface that caused major compensating changes in the association (kon) and dissociation (koff) rate constants (kinetic perturbations) but only moderate changes in the overall complex affinity (Kd). Yeast cells carrying a Sln1-Ypd1 mutant pair with moderate increases in kon and koff displayed a lower threshold of HOG pathway activation than wild-type cells. Mutants with higher kon and koff rates gave rise to higher basal signaling and gene expression but impaired osmoadaptation. Thus, the kon and koff rates of the components in the Sln1 osmosensor determine proper signaling dynamics and osmoadaptation. The work was supported by European Commission 7th Framework Programme (UNICELLSYS, grant 201142 to L.S., F.P., and E.K.), grants from the Spanish Ministry of Economy and Competitiveness (BFU2012-33503 and FEDER, BFU2014-52125-REDT, BFU14-51672-REDC, and BFU2015-64437-P to F.P.; BFU2014-52333-P to E.d.N.), the Catalan Government (2014 SGR 599), an ERC Advanced Grant 294294 from the EU 7th Framework Programme (SYNCOM, to F.P.), the Fundación Botín, Banco Santander (through its Santander Universities Global Division, to F.P.), and the German Research Council (RTG 1772Computational Systems Biology to E.K.). F.P. and E.d.N. are recipients of an ICREA Acadèmia (Generalitat de Catalunya). This work was supported by the Spanish Ministerio de Economía y Competitividad, Plan Nacional, and the European Fund for Regional Development (BIO2012-39754 to L.S.). We acknowledge support of the Spanish Ministry of Economy and Competitiveness, “Centro de Excelencia Severo Ochoa 2013-2017” (SEV-2012-0208). We acknowledge the support of the CERCA Programme/Generalitat de Catalunya.

Transcription is a core process of bacterial physiology, and as such it must be tightly controlled, so that bacterial cells maintain steady levels of each RNA molecule in homeostasis and modify them in response to perturbations. The major regulators of transcription in bacteria (and in eukaryotes) are transcription factors. However, in genome-reduced bacteria, the limited number of these proteins is insufficient to explain the variety of responses shown upon changes in their environment. Thus, alternative regulators may play a central role in orchestrating RNA levels in these microorganisms. These alternative mechanisms rely on intrinsic features within DNA and RNA molecules, suggesting they are ancestral mechanisms shared among bacteria that could have an increased relevance on transcriptional regulation in minimal cells. In this review, we summarize the alternative elements that can regulate transcript abundance in genome-reduced bacteria and how they contribute to the RNA homeostasis at different levels. We acknowledge support of the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program under agreement No 670216 (MYCOCHASSIS) and the Spanish Ministry of Economy and Competitiveness, 'Centro de Excelencia Severo Ochoa 2013-2017′.

In the search for effective vehicles to carry genetic material into cells, we present here new pseudodendrimers that consist of a hyperbranched polyester core surrounded by amino-terminated 2,2-bis(hydroxymethyl)propionic acid (bis-MPA) dendrons. The pseudodendrimers are readily synthesized from commercial hyperbranched bis-MPA polyesters of the second, third, and fourth generations and third-generation bis-MPA dendrons, bearing eight peripheral glycine moieties, coupled by the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC). This approach provides globular macromolecular structures bearing 128, 256, and 512 terminal amino groups, and these can complex pDNA. The toxicity of the three pseudodendrimers was studied on two cell lines, mesenchymal stem cells, and HeLa, and it was demonstrated that these compounds do not affect negatively cell viability up to 72 h. The complexation with DNA was investigated in terms of N-to-P ratio and dendriplex stability. The three generations were found to promote internalizing of pDNA into mesenchymal stem cells (MSCs), and their transfection capacity was compared with two nonviral commercial transfection agents, Lipofectamine and TransIT-X2. The highest generations were able to transfect these cells at levels comparable to both commercial reagents., This work was financially supported by the MINECO-FEDER funds (under the projects CTQ2015-70174-P and MAT2015-66208-C3-1-P) and the Gobierno de Aragón-FSE (E04 research groups). A.L. thanks the MECD for his FPU grants (FPU12/05210) R.G. thanks DGA for his PhD grant.

Quantitative analysis of the sequence determinants of transcription and translation regulation is relevant for systems and synthetic biology. To identify these determinants, researchers have developed different methods of screening random libraries using fluorescent reporters or antibiotic resistance genes. Here, we have implemented a generic approach called ELM-seq (expression level monitoring by DNA methylation) that overcomes the technical limitations of such classic reporters. ELM-seq uses DamID (Escherichia coli DNA adenine methylase as a reporter coupled with methylation-sensitive restriction enzyme digestion and high-throughput sequencing) to enable in vivo quantitative analyses of upstream regulatory sequences. Using the genome-reduced bacterium Mycoplasma pneumoniae, we show that ELM-seq has a large dynamic range and causes minimal toxicity. We use ELM-seq to determine key sequences (known and putatively novel) of promoter and untranslated regions that influence transcription and translation efficiency. Applying ELM-seq to other organisms will help us to further understand gene expression and guide synthetic biology., Quantitative analysis of how DNA sequence determines transcription and translation regulation is of interest to systems and synthetic biologists. Here the authors present ELM-seq, which uses Dam activity as reporter for high-throughput analysis of promoter and 5’-UTR regions.

Cellulose is one of the most abundant materials in nature. Besides its biological function, cellulose can be extracted from the cell wall and used in several industrial applications. Thus, it can be used in papers, pharmaceuticals, food, cosmetics and innovative materials such as nanocomposites, packaging, coatings and dispersion technology. With the aim of extending cellulose applications and producing so-called “smart” materials, new functionality can be introduced by physical or chemical modifications. Taking into account that capsaicin, the active component of chili peppers, is an excellent antifungal agent, a potential new material could be obtained by chemical reaction between this active compound and cellulose. In this work, capsaicin grafting onto cellulose using polycarboxylic acid as linking agent is proposed. The reaction occurrence was corroborated by Fourier transform infrared spectroscopy and UV–Vis spectrophotometry in reflectance mode. Modified cellulose with

Copy number alterations (CNAs) in cancer patients show a large variability in their number, length and position, but the sources of this variability are not known. CNA number and length are linked to patient survival, suggesting clinical relevance. We have identified genes that tend to be mutated in samples that have few or many CNAs, which we term CONIM genes (COpy Number Instability Modulators). CONIM proteins cluster into a densely connected subnetwork of physical interactions and many of them are epigenetic modifiers. Therefore, we investigated how the epigenome of the tissue-of-origin influences the position of CNA breakpoints and the properties of the resulting CNAs. We found that the presence of heterochromatin in the tissue-of-origin contributes to the recurrence and length of CNAs in the respective cancer type. DOI: http://dx.doi.org/10.7554/eLife.16519.001, eLife digest Cancer is a genetic disease that develops when a cell’s DNA becomes altered. There are several different types of DNA alterations and one that is frequently seen in cancer cells is known as a “copy number alteration” (or CNA for short). These CNAs arise when breaks in the DNA are repaired incorrectly, leading to some pieces of DNA being multiplied while others are lost. Ultimately, CNAs contribute to cancer growth either by providing extra copies of genes that drive tumour development or by deleting genes that normally protect against cancer. However, it is not known why patients with some types of cancer tend to have more CNAs than others and why some DNA regions are particularly susceptible to this type of alteration. Cramer et al. asked whether cancer patients have any other genetic mutations that might be linked with having many or few CNAs. Analysing datasets from almost 6000 patients with 20 different types of cancer showed that mutations in several genes are linked to a higher or lower number of CNAs in patients. Cramer et al. called the proteins encoded by these genes “copy number instability modulators” (or CONIMs for short). Further investigation revealed that several of these CONIM proteins can change the way DNA is packaged inside cells. Furthermore, many of the regions of DNA that are vulnerable to CNAs in cancer cells are tightly packaged within healthy cells. These data suggest that the three-dimensional arrangement of DNA in cells influences where CNAs occur. The next step following on from this work is to find out exactly how the CONIM proteins influence the formation of CNAs. DOI: http://dx.doi.org/10.7554/eLife.16519.002