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  • Working paper
    Schulz LM, Rothe P, Halbedel S, Gründling A, Rismondo Jet al., 2022,

    Imbalance of peptidoglycan biosynthesis alters the cell surface charge of <i>Listeria monocytogenes</i>

    <jats:title>ABSTRACT</jats:title> <jats:p> The bacterial cell wall is composed of a thick layer of peptidoglycan and cell wall polymers, which are either embedded in the membrane or linked to the peptidoglycan backbone and referred to as lipoteichoic acid (LTA) and wall teichoic acid (WTA), respectively. Modifications of the peptidoglycan or WTA backbone can alter the susceptibility of the bacterial cell towards cationic antimicrobials and lysozyme. The human pathogen <jats:italic>Listeria monocytogenes</jats:italic> is intrinsically resistant towards lysozyme, mainly due to deacetylation and <jats:italic>O</jats:italic> -acetylation of the peptidoglycan backbone via PgdA and OatA. Recent studies identified additional factors, which contribute to the lysozyme resistance of this pathogen. One of these is the predicted ABC transporter, EslABC. An <jats:italic>eslB</jats:italic> mutant is hyper-sensitive towards lysozyme, likely due to the production of thinner and less <jats:italic>O</jats:italic> -acetylated peptidoglycan. Using a suppressor screen, we show here that suppression of <jats:italic>eslB</jats:italic> phenotypes could be achieved by enhancing peptidoglycan biosynthesis, reducing peptidoglycan hydrolysis or alterations in WTA biosynthesis and modification. The lack of EslB also leads to a higher negative surface charge, which likely stimulates the activity of peptidoglycan hydrolases and lysozyme. Based on our results, we hypothesize that the portion of cell surface exposed WTA is increased in the <jats:italic>eslB</jats:italic> mutant due to the thinner peptidoglycan layer and that latter one could be caused by an impairment in UDP-
  • Journal article
    Wong J, David S, Sanchez Garrido J, Woo J, Low WW, Morecchiato F, Giani T, Rossolini GM, Beis K, Brett S, Clements A, Aaenensen D, Rouskin S, Frankel Get al., 2022,

    Recurrent emergence of Klebsiella pneumoniae carbapenem resistance mediated by an inhibitory ompK36 mRNA secondary structure

    , Proceedings of the National Academy of Sciences of USA, Vol: 119, Pages: 1-12, ISSN: 0027-8424

    Outer membrane porins in Gram-negative bacteria facilitate antibiotic influx. In Klebsiella pneumoniae (KP), modifications in the porin OmpK36 are implicated in increasing resistance to carbapenems. Analysis of large KP genome collections, encompassing major healthcare-associated clones, revealed the recurrent emergence of a synonymous cytosine to thymine transition at position 25 (25c>t) in ompK36. We show that the 25c>t transition increases carbapenem resistance through depletion of OmpK36 from the outer membrane. The mutation attenuates KP in a murine pneumonia model, which accounts for its limited clonal expansion observed by phylogenetic analysis. However, in the context of carbapenem treatment, the 25c>t transition tips the balance towards treatment failure, thus accounting for its recurrent emergence. Mechanistically, the 25c>t transition mediates an intramolecular mRNA interaction between a uracil encoded by 25t and the first adenine within the Shine-Dalgarno sequence. This specific interaction leads to the formation of an RNA stem structure, which obscures the ribosomal binding site thus disrupting translation. While mutations reducing OmpK36 expression via transcriptional silencing are known, we uniquely demonstrate the repeated selection of a synonymous ompK36 mutation mediating translational suppression in response to antibiotic pressure.
  • Journal article
    Fusco G, Bemporad F, Chiti F, Dobson CMM, De Simone Aet al., 2022,

    The role of structural dynamics in the thermal adaptation of hyperthermophilic enzymes

    , FRONTIERS IN MOLECULAR BIOSCIENCES, Vol: 9
  • Conference paper
    Chadha D, Williams D, Lukham P, Hale Cet al., 2022,

    Doing design differently: hybrid teaching in the age of Covid-19

    , American Society for Engineering Education Annual Meeting
  • Journal article
    Crone MA, Freemont PS, 2022,

    Simple, low-cost production of DNA MS2 virus-like particles as molecular diagnostic controls

    <jats:title>Abstract</jats:title> <jats:p> Suitable controls are integral for the validation and continued quality assurance of diagnostic workflows. Plasmids, DNA or <jats:italic>in vitro</jats:italic> transcribed RNA are often used to validate novel diagnostic workflows, however, they are poorly representative of clinical samples. RNA phage virus-like particles packaged with exogenous RNA have been used in clinical diagnostics as workflow controls, serving as surrogates for infectious viral particles. Comparable controls for DNA viruses are more challenging to produce, with analogous DNA phages being infectious and packaging of DNA within RNA phages requiring complex purification procedures and expensive chemical linkers. We present a simple and inexpensive method to produce MS2 virus-like particles, packaged with DNA, that makes use of affinity chromatography for purification and enzymatic production of exogenous DNA suitable for packaging. The produced virus-like particles were packaged with Hepatitis B Virus DNA and were then quantified using droplet digital PCR and calibrated against the WHO international standard using a commercial assay in an accredited clinical laboratory. </jats:p>
  • Journal article
    Nolan AC, Zeden MS, Campbell C, Kviatkovski I, Urwin L, Corrigan RM, Gründling A, OGara JPet al., 2022,

    Purine nucleosides interfere with c-di-AMP levels and act as adjuvants to re-sensitise MRSA to β-lactam antibiotics

    <jats:title>Abstract</jats:title> <jats:p> Elucidating the complex mechanisms controlling <jats:italic>mecA</jats:italic> /PBP2a-mediated β-lactam resistance in methicillin resistant <jats:italic>Staphylococcus aureus</jats:italic> (MRSA) has the potential to identify new drug targets with therapeutic potential. Here, we report that mutations that interfere with <jats:italic>de novo</jats:italic> purine synthesis ( <jats:italic>pur</jats:italic> operon), purine transport (NupG, PbuG and PbuX) and the nucleotide salvage pathway (DeoD2, Hpt) increased β-lactam resistance in MRSA strain JE2. Extrapolating from these findings, exogenous guanosine and xanthosine, which are fluxed through the GTP branch of purine biosynthesis were shown to significantly reduce MRSA β-lactam resistance. In contrast adenosine, which is fluxed to ATP, significantly increased oxacillin resistance, whereas inosine, which can be fluxed to ATP and GTP via hypoxanthine, only marginally reduced the oxacillin MIC. Increased oxacillin resistance of the <jats:italic>nupG</jats:italic> mutant was not significantly reversed by guanosine, indicating that NupG is required for guanosine transport, which in turn is required to reduce β-lactam resistance. Suppressor mutants resistant to oxacillin/guanosine combinations contained several purine salvage pathway mutations, including <jats:italic>nupG</jats:italic> and <jats:italic>hpt</jats:italic> . Microscopic analysis revealed that guanosine significantly increased cell size, a phenotype also associated with reduced levels of c-di-AMP. Consistent with this, guanosine signi
  • Journal article
    Viola S, Roseby W, Santabarbara S, Nürnberg D, Assunção R, Dau H, Sellés J, Boussac A, Fantuzzi A, Rutherford Aet al., 2022,

    Impact of energy limitations on function and resilience in long-wavelength photosystem II

    , eLife, Vol: 11, ISSN: 2050-084X

    Photosystem II (PSII) uses the energy from red light to split water and reduce quinone, an energy-demanding process based on chlorophyll a (Chl-a) photochemistry. Two types of cyanobacterial PSII can use chlorophyll d (Chl-d) and chlorophyll f (Chl-f) to perform the same reactions using lower energy, far-red light. PSII from Acaryochloris marina has Chl-d replacing all but one of its 35 Chl-a, while PSII from Chroococcidiopsis thermalis, a facultative far-red species, has just 4 Chl-f and 1 Chl-d and 30 Chl-a. From bioenergetic considerations, the far-red PSII were predicted to lose photochemical efficiency and/or resilience to photodamage. Here, we compare enzyme turnover efficiency, forward electron transfer, back-reactions and photodamage in Chl-f-PSII, Chl-d-PSII and Chl-a-PSII. We show that: i) all types of PSII have a comparable efficiency in enzyme turnover; ii) the modified energy gaps on the acceptor side of Chl-d-PSII favour recombination via PD1+Phe- repopulation, leading to increased singlet oxygen production and greater sensitivity to high-light damage compared to Chl-a-PSII and Chl-f-PSII; iii) the acceptor-side energy gaps in Chl-f-PSII are tuned to avoid harmful back reactions, favouring resilience to photodamage over efficiency of light usage. The results are explained by the differences in the redox tuning of the electron transfer cofactors Phe and QA and in the number and layout of the chlorophylls that share the excitation energy with the primary electron donor. PSII has adapted to lower energy in two distinct ways, each appropriate for its specific environment but with different functional penalties.
  • Journal article
    Chukhutsina VU, van Thor JJ, 2022,

    Molecular Activation Mechanism and Structural Dynamics of Orange Carotenoid Protein

    , Physchem, Vol: 2, Pages: 235-252

    <jats:p>Like most photosynthetic organisms, cyanobacteria are vulnerable to fluctuations in light intensity, which can damage their photosynthetic machinery. To protect against this, they use a photoprotective mechanism called non-photochemical quenching (NPQ), where excess absorbed photo-energy is dissipated as heat. In cyanobacteria, light activation of Orange Carotenoid Protein (OCP) is the critical first step in the NPQ response. OCP is also the only known photosensitive protein, which uses carotenoid for its activation. We summarize the current knowledge on the light induced reactions of OCP; the different mechanisms of activation that have been proposed; photocycle kinetics and characteristics; and the reported structural intermediates. We discuss the possible interpretations of reported experimental results, and we formulate important open questions and directions for future work, to reveal the molecular and structural basis of photosensing by OCP.</jats:p>
  • Conference paper
    Allgower F, Gamiz-Hernandez AP, Rutherford AW, Kaila VRIet al., 2022,

    Molecular Mechanisms of Redox-Coupled Protonation Dynamics in Photosystem II

    , 21st European Bioenergetics Conference (EBEC), Publisher: ELSEVIER, Pages: 83-83, ISSN: 0005-2728
  • Journal article
    Balasco N, Esposito L, De Simone A, Vitagliano Let al., 2022,

    Local Backbone Geometry Plays a Critical Role in Determining Conformational Preferences of Amino Acid Residues in Proteins

    , BIOMOLECULES, Vol: 12
  • Conference paper
    Kim TD, Oliver T, Trinugroho JP, Cordon-Preciado V, Bianchini G, Rutherford AW, Sanchez-Baracaldo P, Cardona Tet al., 2022,

    Evolution and evolvability of photosystem II

    , 21st European Bioenergetics Conference (EBEC), Publisher: ELSEVIER, Pages: 80-80, ISSN: 0005-2728
  • Conference paper
    Viola S, Roseby W, Santabarabara S, Nurnberg D, Assuncao R, Dau H, Selles J, Boussac A, Fantuzzi A, Rutherford AWet al., 2022,

    Abstract The energetics of the two types of far-red Photosystem II

    , 21st European Bioenergetics Conference (EBEC), Publisher: ELSEVIER, Pages: 81-81, ISSN: 0005-2728
  • Journal article
    Palmas MF, Etzi M, Pisanu A, Camoglio C, Sagheddu C, Santoni M, Manchinu MF, Pala M, Fusco G, De Simone A, Picci L, Mulas G, Spiga S, Scherma M, Fadda P, Pistis M, Simola N, Carboni E, Carta ARet al., 2022,

    The Intranigral Infusion of Human-Alpha Synuclein Oligomers Induces a Cognitive Impairment in Rats Associated with Changes in Neuronal Firing and Neuroinflammation in the Anterior Cingulate Cortex

    , CELLS, Vol: 11
  • Journal article
    Malagrino F, Fusco G, Pennacchietti V, Toto A, Nardella C, Pagano L, de Simone A, Gianni Set al., 2022,

    Cryptic binding properties of a transient folding intermediate in a PDZ tandem repeat

    , PROTEIN SCIENCE, Vol: 31, ISSN: 0961-8368
  • Journal article
    McIntyre SR, Hunter-Sellars E, Haycock PR, Williams DRet al., 2022,

    Considerations when determining Counter-diffusion constants in liquid phase catalytic reactions using the Zero Length column (ZLC) method

    , Chemical Engineering Science, Vol: 258, ISSN: 0009-2509

    A Zero Length Column (ZLC) method was developed using a liquid chromatographic system to calculate reactant counter-diffusion coefficients in porous, solvent swollen catalytic pellets and gate-opening support materials. Reactant diffusivities within these porous materials were determined at the reaction conditions of 80 °C and 0.3–0.75 mL min<sup>−1</sup>. For all materials, molecules of a similar size to the pore apertures, or molecules with strong interactions with the material surfaces, catalysts, were observable by the ZLC method, with both micro and macropore diffusion observed. Differences between Pd(II) and Pd(0) forms of the EnCat30 catalyst were examined to determine the effects of catalytic deactivation. The deactivated catalyst showed iodobenzene macroporous diffusion constants similar to the relatively inert toluene molecules. Finally, pulse field gradient NMR was utilised to compare and validate ZLC diffusion measurements for solvent swollen reaction systems. This study presents much needed novel findings on diffusivity data for liquid phase catalytic systems.
  • Journal article
    Herisson J, Duigou T, du Lac M, Bazi-Kabbaj K, Azad MS, Buldum G, Telle O, El Moubayed Y, Carbonell P, Swainston N, Zulkower V, Kushwaha M, Baldwin GS, Faulon J-Let al., 2022,

    The automated Galaxy-SynBioCAD pipeline for synthetic biology design and engineering

    , NATURE COMMUNICATIONS, Vol: 13
    • Cite
    • Citations: 25
  • Journal article
    Tortuel D, Tahrioui A, David A, Cambronel M, Nilly F, Clamens T, Maillot O, Barreau M, Feuilloley MGJ, Lesouhaitier O, Filloux A, Bouffartigues E, Cornelis P, Chevalier Set al., 2022,

    Pf4 Phage Variant Infection Reduces Virulence-Associated Traits in <i>Pseudomonas aeruginosa</i>

    , MICROBIOLOGY SPECTRUM, Vol: 10, ISSN: 2165-0497
    • Cite
    • Citations: 17
  • Journal article
    Fillol-Salom A, Rostøl JT, Ojiogu AD, Chen J, Douce G, Humphrey S, Penadés JRet al., 2022,

    Bacteriophages benefit from mobilizing pathogenicity islands encoding immune systems against competitors

    , Cell, Vol: 185, Pages: 3248-3262.e20, ISSN: 0092-8674

    Bacteria encode sophisticated anti-phage systems that are diverse and versatile and display high genetic mobility. How this variability and mobility occurs remains largely unknown. Here, we demonstrate that a widespread family of pathogenicity islands, the phage-inducible chromosomal islands (PICIs), carry an impressive arsenal of defense mechanisms, which can be disseminated intra- and inter-generically by helper phages. These defense systems provide broad immunity, blocking not only phage reproduction, but also plasmid and non-cognate PICI transfer. Our results demonstrate that phages can mobilize PICI-encoded immunity systems to use them against other mobile genetic elements, which compete with the phages for the same bacterial hosts. Therefore, despite the cost, mobilization of PICIs may be beneficial for phages, PICIs, and bacteria in nature. Our results suggest that PICIs are important players controlling horizontal gene transfer and that PICIs and phages establish mutualistic interactions that drive bacterial ecology and evolution.
  • Journal article
    Kaplan M, Oikonomou CM, Wood CR, Chreifi G, Ghosal D, Dobro MJ, Yao Q, Pal RR, Baidya AK, Liu Y, Maggi S, McDowall AW, Ben-Yehuda S, Rosenshine I, Briegel A, Beeby M, Chang Y-W, Shaffer CL, Jensen GJet al., 2022,

    Discovery of a Novel Inner Membrane-Associated Bacterial Structure Related to the Flagellar Type III Secretion System

    , JOURNAL OF BACTERIOLOGY, Vol: 204, ISSN: 0021-9193
  • Journal article
    Lopez-Olvera A, Pioquinto-Garcia S, Zarate JA, Diaz G, Martinez-Ahumada E, Obeso JL, Martis V, Williams DR, Lara-Garcia HA, Leyva C, Soares CV, Maurin G, Ibarra IA, Davila-Guzman NEet al., 2022,

    SO<sub>2</sub> capture in a chemical stable Al(III) MOF: DUT-4 as an effective adsorbent to clean CH<sub>4</sub>

    , FUEL, Vol: 322, ISSN: 0016-2361
  • Journal article
    Holmes AS, Kiziroglou ME, Yang SKE, Yuan C, Boyle DE, Lincoln DM, McCabe JDJ, Szasz P, Keeping SC, Williams DR, Yeatman EMet al., 2022,

    Minimally invasive online water monitor

    , IEEE Internet of Things Journal, Vol: 9, Pages: 14325-14335, ISSN: 2327-4662

    Sensor installation on water infrastructure is challenging due to requirements for service interruption, specialised personnel, regulations and reliability as well as the resultant high costs. Here, a minimally invasive installation method is introduced based on hot-tapping and immersion of a sensor probe. A modular architecture is developed that enables the use of interchangeable multi-sensor probes, non-specialist installation and servicing, low-power operation and configurable sensing and connectivity. A prototype implementation with a temperature, pressure, conductivity and flow multi-sensor probe is presented and tested on an evaluation rig. This paper demonstrates simple installation, reliable and accurate sensing capability as well as remote data acquisition. The demonstrated minimally invasive multi-sensor probes provide an opportunity for the deployment of water quality sensors that typically require immersion such as pH and spectroscopic composition analysis. This design allows dynamic deployment on existing water infrastructure with expandable sensing capability and minimal interruption, which can be key to addressing important sensing parameters such as optimal sensor network density and topology.
  • Journal article
    Meisl G, Xu CK, Taylor JD, Michaels TCT, Levin A, Otzen D, Klenerman D, Matthews S, Linse S, Andreasen M, Knowles TPJet al., 2022,

    Uncovering the universality of self-replication in protein aggregation and its link to disease

    , Science Advances, Vol: 8, Pages: 1-11, ISSN: 2375-2548

    Fibrillar protein aggregates are a hallmark of a range of human disorders, from prion diseases to dementias, but are also encountered in several functional contexts. Yet, the fundamental links between protein assembly mechanisms and their functional or pathological roles have remained elusive. Here, we analyze the aggregation kinetics of a large set of proteins that self-assemble by a nucleated-growth mechanism, from those associated with disease, over those whose aggregates fulfill functional roles in biology, to those that aggregate only under artificial conditions. We find that, essentially, all such systems, regardless of their biological role, are capable of self-replication. However, for aggregates that have evolved to fulfill a structural role, the rate of self-replication is too low to be significant on the biologically relevant time scale. By contrast, all disease-related proteins are able to self-replicate quickly compared to the time scale of the associated disease. Our findings establish the ubiquity of self-replication and point to its potential importance across aggregation-related disorders.
  • Journal article
    Browning ND, Bryan W, Clarke J, Ellis M, Kirkland AI, Maskell S, McKenzie J, Layla Mehdi B, Dwayne Miller RJ, Murooka Y, Noakes TCQ, Robinson I, Schroeder SLM, van Thor J, Welsch Cet al., 2022,

    The Design and Operation of a New Relativistic Ultrafast Electron Diffraction and Imaging (RUEDI) National Facility in the UK

    , Microscopy and Microanalysis, Vol: 28, Pages: 2764-2765, ISSN: 1431-9276
  • Journal article
    Nixon P, Telfer A, 2022,

    Remembering James Barber (1940-2020)

    , Photosynthesis Research, Vol: 153, Pages: 1-20, ISSN: 0166-8595

    James Barber, known to colleagues and friends as Jim, passed away in January 2020 after a long battle against cancer. During his long and distinguished career in photosynthesis research, Jim made many outstanding contributions with the pinnacle achieving his dream of determining the first detailed structure of the Mn cluster involved in photosynthetic water oxidation. Here, colleagues and friends remember Jim and reflect upon his scientific career and the impact he had on their lives and the scientific community.
  • Conference paper
    Ahmed A, Bickel JK, Andrei SA, Couves E, McAllister T, Kawamura A, Bubeck DA, Tate EWet al., 2022,

    Optimisation of Macrocyclic Peptide Hits Identified as CD59 Binders to Overcome Resistance in Cancer Immunotherapy

    , Publisher: WILEY, ISSN: 1075-2617
  • Journal article
    Fadini A, Van Thor J, 2022,

    Photoactivation in a Fluorescent Protein Proceeds via the Hula-Twist : Mechanism Revealed through TR-SFX

    , ACTA CRYSTALLOGRAPHICA A-FOUNDATION AND ADVANCES, Vol: 78, Pages: E784-E784, ISSN: 2053-2733
  • Conference paper
    Fadini A, Van Thor J, 2022,

    Photoactivation in a Fluorescent Protein Proceeds via the Hula-Twist Mechanism : Revealed through TR-SFX

    , Publisher: INT UNION CRYSTALLOGRAPHY, Pages: E64-E64, ISSN: 2053-2733
  • Journal article
    Malpartida-Cardenas K, Baum J, Cunnington A, Georgiou P, Rodriguez-Manzano Jet al., 2022,

    Electricity-free nucleic acid extraction method from dried blood spots on filter paper for point-of-care diagnostics

    <jats:title>Abstract</jats:title> <jats:sec> <jats:title>Background</jats:title> <jats:p>Nucleic acid extraction is a crucial step for molecular biology applications, being a determinant for any diagnostic test procedure. Dried blood spots (DBS) have been used for decades for serology, drug monitoring, environmental investigations, and molecular studies. Nevertheless, nucleic acid extraction from DBS remains one of the main challenges to translate them to the point-of-care (POC).</jats:p> </jats:sec> <jats:sec> <jats:title>Method</jats:title> <jats:p> We have developed a fast nucleic acid extraction (NAE) method from DBS which is electricity-free and relies on cellulose filter papers (DBSFP). The performance of NAE was assessed with loop-mediated isothermal amplification (LAMP), targeting the human reference gene beta-actin. The developed method was evaluated against FTA cards and magnetic bead-based purification, using time-to-positive (min) for comparative analysis. We optimised and validated the developed method for elution ( <jats:italic>eluted disk</jats:italic> ) and disk directly in the reaction ( <jats:italic>in-situ disk)</jats:italic> , RNA and DNA detection, and whole blood stored in anticoagulants (K <jats:sub>2</jats:sub> EDTA and lithium heparin). Furthermore, the compatibility of DBSFP with colourimetric detection was studied to show the transferability to the POC. </jats:p> </jats:sec> <jats:sec> <jats:title>Results</jats:title> <jats:p> The proposed DBSFP is based on g
  • Journal article
    Olivieri C, Li GC, Wang Y, Manu VS, Walker C, Kim J, Camilloni C, De Simone A, Vendruscolo M, Bernlohr DA, Taylor SS, Veglia Get al., 2022,

    ATP-competitive inhibitors modulate the substrate binding cooperativity of a kinase by altering its conformational entropy

    , SCIENCE ADVANCES, Vol: 8, ISSN: 2375-2548
  • Journal article
    Eilers K, Kuok Hoong Yam J, Morton R, Mei Hui Yong A, Brizuela J, Hadjicharalambous C, Liu X, Givskov M, Rice SA, Filloux Aet al., 2022,

    Phenotypic and integrated analysis of a comprehensive Pseudomonas aeruginosa PAO1 library of mutants lacking cyclic-di-GMP-related genes

    , Frontiers in Microbiology, Vol: 13, ISSN: 1664-302X

    Pseudomonas aeruginosa is a Gram-negative bacterium that is able to survive and adapt in a multitude of niches as well as thrive within many different hosts. This versatility lies within its large genome of ca. 6 Mbp and a tight control in the expression of thousands of genes. Among the regulatory mechanisms widely spread in bacteria, cyclic-di-GMP signaling is one which influences all levels of control. c-di-GMP is made by diguanylate cyclases and degraded by phosphodiesterases, while the intracellular level of this molecule drives phenotypic responses. Signaling involves the modification of enzymes’ or proteins’ function upon c-di-GMP binding, including modifying the activity of regulators which in turn will impact the transcriptome. In P. aeruginosa, there are ca. 40 genes encoding putative DGCs or PDEs. The combined activity of those enzymes should reflect the overall c-di-GMP concentration, while specific phenotypic outputs could be correlated to a given set of dgc/pde. This notion of specificity has been addressed in several studies and different strains of P. aeruginosa. Here, we engineered a mutant library for the 41 individual dgc/pde genes in P. aeruginosa PAO1. In most cases, we observed a significant to slight variation in the global c-di-GMP pool of cells grown planktonically, while several mutants display a phenotypic impact on biofilm including initial attachment and maturation. If this observation of minor changes in c-di-GMP level correlating with significant phenotypic impact appears to be true, it further supports the idea of a local vs global c-di-GMP pool. In contrast, there was little to no effect on motility, which differs from previous studies. Our RNA-seq analysis indicated that all PAO1 dgc/pde genes were expressed in both planktonic and biofilm growth conditions and our work suggests that c-di-GMP networks need to be reconstructed for each strain separately and cannot be extrapolated from one to another.

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