doi.org/10.26434/chemrxiv.12317480.v1 A Cyclic Di-GMP Network Is Present in Gram-Positive Streptococcus and Gram-Negative Proteus Species Ying Liu, Changhan Lee, Fengyang Li, Janja Trček, Heike Bähre, Rey-Ting Guo, Chun-Chi Chen, Alexey Chernobrovkin, Roman Zubarev, Ute Romling Submitted date: 16/05/2020 • Posted date: 18/05/2020 Licence: CC BY-NC-ND 4.0 Citation information: Liu, Ying; Lee, Changhan; Li, Fengyang; Trček, Janja; Bähre, Heike; Guo, Rey-Ting; et al. (2020): A Cyclic Di-GMP Network Is Present in Gram-Positive Streptococcus and Gram-Negative Proteus Species. ChemRxiv. Preprint. https://doi.org/10.26434/chemrxiv.12317480.v1 Cyclic di-GMP is a ubiquitous second messenger in bacteria. This work describes the occurrence of a cyclic di-GMP signaling network in Gram-positive Streptococcus species and Gram-negative Proteus. After identification of candidate diguanylate cyclases by homology search in the respective species, the open reading frames were cloned and proteins expressed. Production of cyclic di-GMP was demonstrated by riboswitch assays, detection of cyclic di-GMP in cell lysates by MALDI-FTMS and in cell extracts by standard LC-MS/MS. Expression of the diguanylate cyclases in the heterologous host Salmonella typhimurium showed the expected physiological activity, namely up regulation of biofilm formation and down regulation of motility. The co-localisation of both sole diguanylate cyclases with cellulose or cellulose-like synthases indicates exopolysaccharide biosynthesis to be a conserved trait of cyclic di-GMP signaling. File list (1) CdiGMPManuscript_PS.pdf (17.47 MiB) view on ChemRxiv download file 1 2 3 4 5 A cyclic di-GMP network is present in Gram-positive Streptococcus and Gram- 6 negative Proteus species 7 8 9 10 Ying Liu1,#, Changhan Lee1##, Fengyang Li1, Janja Trček2, Heike Bähre3, Rey-Ting Guo4, 11 Chun-Chi Chen4, Alexey Chernobrovkin5, Roman Zubarev5,7 and Ute Römling1 12 13 14 15 1Department of Microbiology, Tumor and Cell Biology, and 5Department of Medical 16 Biochemistry and Biophysics, Biomedicum, Karolinska Institutet, SE-171 65 Stockholm, 17 Sweden 2 18 Faculty of Natural Sciences and Mathematics, Department of Biology, University of 19 Maribor, 2000 Maribor, Slovenia 20 3 Research Core Unit Metabolomics, Hannover Medical School, Hannover, Germany 21 4State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Collaborative 22 Innovation Center for Green Transformation of Bio-Resources, Hubei Key Laboratory of 23 Industrial Biotechnology, School of Life Sciences, Hubei University, Wuhan, 430062, P.R. 24 China 25 5Department of Pharmacological & Technological Chemistry, I.M. Sechenov First Moscow 26 State Medical University, Moscow, 119146, Russia 27 28 #Current address: Department of Microbiology, Philips University Marburg, Marburg, 29 Germany 30 31 ##Current address: Department of Molecular, Cellular and Developmental Biology, 32 University of Michigan, Ann Arbor, MI 48109, USA 1 33 ABSTRACT 34 The ubiquitous cyclic di-GMP (c-di-GMP) network is highly redundant with numerous 35 GGDEF domain containing proteins as diguanylate cyclases and EAL domain containing 36 proteins as c-di-GMP specific phosphodiesterases comprising those domains as two of the 37 most abundant bacterial protein superfamilies. One hallmark of the c-di-GMP network is its 38 outmost plasticity as c-di-GMP turnover proteins can rapidly vanish from species within a 39 genus and possess an above average transmissibility. With the long-term goal to address the 40 evolutionary forces of c-di-GMP turnover protein diversity, maintenance and conservation, 41 we investigated as an example a Gram-positive and a Gram-negative species, which 42 preserved only one single clearly identifiable GGDEF domain protein. Species 43 representatives of most sequenced genera of the family Morganellaceae of the order 44 Enterobacterales exceptionally show disappearance of the c-di-GMP signaling network, but 45 some species such as Proteus spp. still retain one candidate diguanylate cyclase. As another 46 example, in species of one of the two major branches in the genus Streptococcus, one 47 candidate diguanylate cyclase was frequently identified. We demonstrate that both proteins 48 encompass PAS (Per-ARNT-Sim)-GGDEF domains, possess diguanylate cyclase catalytic 49 activity and are suggested to signal via a PilZ receptor domain at the C-terminus of a type 2 50 glycosyltransferase. Specifically, one of them being the cellulose synthase BcsA. 51 Preservation of the ancient link between production of cellulose-like exopolysaccharides and 52 c-di-GMP signaling indicates that this functionality is even of high ecological importance 53 upon maintenance of the last remnants of the c-di-GMP network in some of today’s free- 54 living bacteria. 55 56 Keywords: Streptococcus gallolyticus subsp. gallolyticus, Proteus mirabilis, GGDEF 57 domain, EAL domain, cyclic di-GMP signaling, cellulose biosynthesis 2 58 INTRODUCTION 59 Signaling systems couple sensing and information transmission and amplification in 60 order to adapt physiology and metabolism to changing external and internal stimuli. Thus, 61 those modules are highly prone to mutational adaptation and/or horizontal gene transfer. The 62 cyclic dinucleotide (CDN) molecule bis-(3’, 5’)-cyclic diguanosine monophosphate (c-di- 63 GMP), identified in 1987 as an allosteric activator of the cellulose synthase in the bacterium 64 Komagataeibacter xylinus (previously Gluconacetobacter (Acetobacter) xylinus (G. xylinus)) 65 is the most abundant CDN based second messenger signaling system in bacteria1-2. Cyclic- 66 di-GMP regulates a multitude of fundamental physiological and metabolic processes, such 67 as single cell motility-to-sessility transition leading to biofilm formation, chronic versus 68 acute virulence, antimicrobial and detergent tolerance, cell cycle progression and cell 69 morphology2. Essential signaling modules of this pathway comprise the GG(D/E)EF domain 70 with diguanylate cyclase (DGC) activity and the EAL and HD-GYP domain with 71 phosphodiesterases (PDE) activity. The GG(D/E)EF domain synthesizes c-di-GMP in a two- 72 step reaction with 5’-pppGpG as intermediate and two molecules of pyrophosphate as 73 byproducts3. The EAL- and HD-GYP domain hydrolyzes c-di-GMP into linear 5’-pGpG and 74 GMP, respectively4-5. Numerous proteins are bifunctional through a combination of GGDEF 75 with EAL/HD-GYP domains. 76 In these three domain superfamilies, catalytic domains have evolved into receptors or act 77 though protein-protein interactions6. Although an intact GG(D/E)EF motif is usually an 78 indicator for catalytic activity, however due to the requirement of extended consensus motifs, 79 the presence of such a motif and even the presence of extended consensus signature motif(s), 80 including divalent ion binding ligands required for catalytic activity, is not a guarantee for 81 the preservation of catalytic activity7-8 or substrate specificity9-10 and vice versa11-12. 82 The activity of DGCs and PDEs is controlled by a diversity of N-terminal sensory 83 domains that can receive various ligands, such as oxygen, nucleotide based small molecules, 84 and light13-17. Thereby, the most frequently used N-terminal signaling domain in this context 85 is the versatile PAS domain18-20. The compact PAS domains with diverse primary sequences 86 of less than 150 amino acids in size, possess an interior pocket flanked characteristically by 87 five antiparallel beta-strands and a few alpha helices to host a variety of prosthetic groups 88 and ligands, with few functional amino acids to determine the binding specificity21-22. 89 Furthermore, signaling of c-di-GMP is translated through protein and RNA-based 90 receptors such as the PilZ domain, MshEN domain, the inhibitory I-site of GGDEF domains, 91 inactive EAL/HD-GYP domains, various classes of transcription regulators and distinct 3 92 RNA aptamers6, 23-25. PilZ domains, the first c-di-GMP receptors discovered, are widespread 93 among bacteria26-27. For example, the catalytic activity of the cellulose synthase BcsA and 94 other exopolysaccharide synthases is regulated by PilZ domains28-29. Riboswitches, 95 consisting of a high affinity CDN binding RNA aptamer and an expression platform located 96 within the 5’-untranslated region (5’-UTR) respond to c-di-GMP binding with 97 conformational changes that alter downstream transcriptional termination and translation23-24. 98 Two classes of c-di-GMP responsive riboswitches, type I and type II, with Genes for the 99 Environment, for Membranes and for Motility (GEMM) motifs have been identified in 100 Gram-negative and Gram-positive species, such as Vibrio cholerae, Geobacter 101 metallireducens and Clostridium difficile. Compared with protein receptors, which have 102 dissociation constants (Kd) in the low µM range for c-di-GMP, RNA aptamers have 103 dissociation constants in the nanomolar range. 104 In many instances, cyclic di-GMP activates biofilm formation, a ubiquitous multicellular 105 sessile life style of bacteria and represses motility30. This physiological regulation by c-di- 106 GMP is evolutionary conserved from ancient thermophiles to human pathogens where 107 biofilm formation contributes to chronic infections31-32. 108 In this study, we identified novel DGCs in the Gram-positive pathogen Streptococcus 109 gallolyticus subsp. gallolyticus UCN34 and Gram-negative Proteus mirabilis UEB50. 110 Previously, c-di-GMP signaling network has not been reported in those two species. 111 Moreover,