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    Local c-di-GMP Signaling in the Control of Synthesis of the E. coli Biofilm Exopolysaccharide pEtN-Cellulose. Richter Anja M,Possling Alexandra,Malysheva Nadezhda,Yousef Kaveh P,Herbst Susanne,von Kleist Max,Hengge Regine Journal of molecular biology In many bacteria, the biofilm-promoting second messenger c-di-GMP is produced and degraded by multiple diguanylate cyclases (DGC) and phosphodiesterases (PDE), respectively. High target specificity of some of these enzymes has led to theoretical concepts of "local" c-di-GMP signaling. In Escherichia coli K-12, which has 12 DGCs and 13 PDEs, a single DGC, DgcC, is specifically required for the biosynthesis of the biofilm exopolysaccharide pEtN-cellulose without affecting the cellular c-di-GMP pool, but the mechanistic basis of this target specificity has remained obscure. DGC activity of membrane-associated DgcC, which is demonstrated in vitro in nanodiscs, is shown to be necessary and sufficient to specifically activate cellulose biosynthesis in vivo. DgcC and a particular PDE, PdeK (encoded right next to the cellulose operon), directly interact with cellulose synthase subunit BcsB and with each other, thus establishing physical proximity between cellulose synthase and a local source and sink of c-di-GMP. This arrangement provides a localized, yet open source of c-di-GMP right next to cellulose synthase subunit BcsA, which needs allosteric activation by c-di-GMP. Through mathematical modeling and simulation, we demonstrate that BcsA binding from the low cytosolic c-di-GMP pool in E. coli is negligible, whereas a single c-di-GMP molecule that is produced and released in direct proximity to cellulose synthase increases the probability of c-di-GMP binding to BcsA several hundred-fold. This local c-di-GMP signaling could provide a blueprint for target-specific second messenger signaling also in other bacteria where multiple second messenger producing and degrading enzymes exist. 10.1016/j.jmb.2020.06.006
    Under Elevated c-di-GMP in Escherichia coli, YcgR Alters Flagellar Motor Bias and Speed Sequentially, with Additional Negative Control of the Flagellar Regulon via the Adaptor Protein RssB. Nieto Vincent,Partridge Jonathan D,Severin Geoffrey B,Lai Run-Zhi,Waters Christopher M,Parkinson John S,Harshey Rasika M Journal of bacteriology In and , the c-di-GMP effector YcgR inhibits flagellar motility by interacting directly with the motor to alter both its bias and speed. Here, we demonstrate that in both of these bacteria, YcgR acts sequentially, altering motor bias first and then decreasing motor speed. We show that when c-di-GMP levels are high, deletion of restores wild-type motor behavior in , indicating that YcgR is the only motor effector in this bacterium. Yet, motility and chemotaxis in soft agar do not return to normal, suggesting that there is a second mechanism that inhibits motility under these conditions. In , c-di-GMP-induced synthesis of extracellular cellulose has been reported to entrap flagella and to be responsible for the YcgR-independent motility defect. We found that this is not the case in Instead, we found through reversion analysis that deletion of , which codes for a response regulator/adaptor protein that normally directs ClpXP protease to target σ for degradation, restored wild-type motility in the mutant. Our data suggest that high c-di-GMP levels may promote altered interactions between these proteins to downregulate flagellar gene expression. Flagellum-driven motility has been studied in and for nearly half a century. Over 60 genes control flagellar assembly and function. The expression of these genes is regulated at multiple levels in response to a variety of environmental signals. Cues that elevate c-di-GMP levels, however, inhibit motility by direct binding of the effector YcgR to the flagellar motor. In this study conducted mainly in , we show that YcgR is the only effector of motor control and tease out the order of YcgR-mediated events. In addition, we find that the σ regulator protein RssB contributes to negative regulation of flagellar gene expression when c-di-GMP levels are elevated. 10.1128/JB.00578-19
    High c-di-GMP promotes expression of fpr-1 and katE involved in oxidative stress resistance in Pseudomonas putida KT2440. Xiao Yujie,Zhu Wenjing,He Meina,Nie Hailing,Chen Wenli,Huang Qiaoyun Applied microbiology and biotechnology Oxidative stress is an unavoidable consequence of interactions with various reactive oxygen species (ROS)-inducing agents that would damage cells or even cause cell death. Bacteria have developed defensive systems, including induction of stress-sensing proteins and detoxification enzymes, to handle oxidative stress. Cyclic diguanylate (c-di-GMP) is a ubiquitous intracellular bacterial second messenger that coordinates diverse aspects of bacterial growth and behavior. In this study, we revealed a mechanism by which c-di-GMP regulated bacterial oxidative stress resistance in Pseudomonas putida KT2440. High c-di-GMP level was found to enhance bacterial resistance towards hydrogen peroxide. Transcription assay showed that expression of two oxidative stress resistance genes, fpr-1 and katE, was promoted under high c-di-GMP level. Deletion of fpr-1 and katE both decreased bacterial tolerance to hydrogen peroxide and weakened the effect of c-di-GMP on oxidative stress resistance. The promoted expression of fpr-1 under high c-di-GMP level was caused by increased cellular ROS via a transcriptional regulator FinR. We further demonstrated that the influence of high c-di-GMP on cellular ROS depend on the existence of FleQ, a transcriptional regulatory c-di-GMP effector. Besides, the regulation of katE by c-di-GMP was also FleQ dependent in an indirect way. Our results proved a connection between c-di-GMP and oxidative stress resistance and revealed a mechanism by which c-di-GMP regulated expression of fpr-1 and katE in P. putida KT2440. 10.1007/s00253-019-10178-6
    c-di-GMP Arms an Anti-σ to Control Progression of Multicellular Differentiation in Streptomyces. Gallagher Kelley A,Schumacher Maria A,Bush Matthew J,Bibb Maureen J,Chandra Govind,Holmes Neil A,Zeng Wenjie,Henderson Max,Zhang Hengshan,Findlay Kim C,Brennan Richard G,Buttner Mark J Molecular cell Streptomyces are our primary source of antibiotics, produced concomitantly with the transition from vegetative growth to sporulation in a complex developmental life cycle. We previously showed that the signaling molecule c-di-GMP binds BldD, a master repressor, to control initiation of development. Here we demonstrate that c-di-GMP also intervenes later in development to control differentiation of the reproductive hyphae into spores by arming a novel anti-σ (RsiG) to bind and sequester a sporulation-specific σ factor (σ). We present the structure of the RsiG-(c-di-GMP)-σ complex, revealing an unusual, partially intercalated c-di-GMP dimer bound at the RsiG-σ interface. RsiG binds c-di-GMP in the absence of σ, employing a novel E(X)S(X)R(X)Q(X)D motif repeated on each helix of a coiled coil. Further studies demonstrate that c-di-GMP is essential for RsiG to inhibit σ. These findings reveal a newly described control mechanism for σ-anti-σ complex formation and establish c-di-GMP as the central integrator of Streptomyces development. 10.1016/j.molcel.2019.11.006
    Homologous c-di-GMP-Binding Scr Transcription Factors Orchestrate Biofilm Development in Vibrio parahaemolyticus. Kimbrough John H,Cribbs J Thomas,McCarter Linda L Journal of bacteriology The marine bacterium and human pathogen rapidly colonizes surfaces by using swarming motility and forming robust biofilms. Entering one of the two colonization programs, swarming motility or sessility, involves differential regulation of many genes, resulting in a dramatic shift in physiology and behavior. has evolved complex regulation to control these two processes that have opposing outcomes. One mechanism relies on the balance of the second messenger c-di-GMP, where high c-di-GMP favors biofilm formation. possesses four homologous regulators, the Scr transcription factors, that belong in a -specific family of W[F/L/M][T/S]R motif transcriptional regulators, some members of which have been demonstrated to bind c-di-GMP. In this work, we explore the role of these Scr regulators in biofilm development. We show that each protein binds c-di-GMP, that this binding requires a critical R in the binding motif, and that the biofilm-relevant activities of CpsQ, CpsS, and ScrO but not ScrP are dependent upon second messenger binding. ScrO and CpsQ are the primary drivers of biofilm formation, as biofilms are eliminated when both of these regulators are absent. ScrO is most important for capsule expression. CpsQ is most important for RTX-matrix protein expression, although it contributes to capsule expression when c-di-GMP levels are high. Both regulators contribute to O-antigen ligase expression. ScrP works oppositely in a minor role to repress the ligase gene. CpsS plays a regulatory checkpointing role by negatively modulating expression of these biofilm-pertinent genes under fluctuating c-di-GMP conditions. Our work further elucidates the multifactorial network that contributes to biofilm development in can inhabit open ocean, chitinous shells, and the human gut. Such varied habitats and the transitions between them require adaptable regulatory networks controlling energetically expensive behaviors, including swarming motility and biofilm formation, which are promoted by low and high concentrations of the signaling molecule c-di-GMP, respectively. Here, we describe four homologous c-di-GMP-binding Scr transcription factors in Members of this family of regulators are present in many vibrios, yet their numbers and the natures of their activities differ across species. Our work highlights the distinctive roles that these transcription factors play in dynamically controlling biofilm formation and architecture in and serves as a powerful example of regulatory network evolution and diversification. 10.1128/JB.00723-19
    Analysis of two Mexican Pectobacterium brasiliense strains reveals an inverted relationship between c-di-GMP levels with exopolysaccharide production and swarming motility. Narváez-Barragán Delia A,de Sandozequi Andrés,Rodríguez Mabel,Estrada Karel,Tovar-Herrera Omar E,Martínez-Anaya Claudia Microbiological research Pectobacterium is a diverse genus of phytopathogenic species from soil and water that cause infection either to restricted or multiple plant hosts. Phylogenetic analysis and metabolic fingerprinting of large numbers of genomes have expanded classification of Pectobacterium members. Pectobacterium brasiliense sp. nov has been elevated to the species level having detached from P. carotovorum. Here we present two P. brasiliense strains BF20 and BF45 isolated in Mexico from Opuntia and tobacco, respectively, which cluster into two different groups in whole genome comparisons with other Pectobacterium. We found that BF20 and BF45 strains are phenotypically different as BF45 showed more severe and rapid symptoms in comparison to BF20 in the host models celery and broccoli. Both strains produced similar levels of the main autoinducers, but BF45 shows an additional low abundant autoinducer compared to strain BF20. The two strains had different levels of c-di-GMP, which regulates the transition from motile to sessile lifestyle. In contrast to BF45, BF20 had the highest levels of c-di-GMP, was more motile (swarming), non-flocculant and less proficient in biofilm formation and exopolysaccharide production. Genomic comparisons revealed that differences in c-di-GMP accumulation and perhaps the associated phenotypes might be due to unique c-di-GMP metabolic genes in these two strains. Our results improve our understanding of the associations between phenotype and genotype and how this has shaped the physiology of Pectobacterium strains. 10.1016/j.micres.2020.126427