MrkH, a Novel c-di-GMP-Dependent Transcriptional Activator, Controls Klebsiella pneumoniae Biofilm Formation by Regulating Type 3 Fimbriae Expression

Klebsiella pneumoniae causes significant morbidity and mortality worldwide, particularly amongst hospitalized individuals. The principle mechanism for pathogenesis in hospital environments involves the formation of biofilms, primarily on implanted medical devices. In this study, we constructed a transposon mutant library in a clinical isolate, K. pneumoniae AJ218, to identify the genes and pathways implicated in biofilm formation. Three mutants severely defective in biofilm formation contained insertions within the mrkABCDF genes encoding the main structural subunit and assembly machinery for type 3 fimbriae. Two other mutants carried insertions within the yfiN and mrkJ genes, which encode GGDEF domain- and EAL domain-containing c-di-GMP turnover enzymes, respectively. The remaining two isolates contained insertions that inactivated the mrkH and mrkI genes, which encode for novel proteins with a c-di-GMP-binding PilZ domain and a LuxR-type transcriptional regulator, respectively. Biochemical and functional assays indicated that the effects of these factors on biofilm formation accompany concomitant changes in type 3 fimbriae expression. We mapped the transcriptional start site of mrkA, demonstrated that MrkH directly activates transcription of the mrkA promoter and showed that MrkH binds strongly to the mrkA regulatory region only in the presence of c-di-GMP. Furthermore, a point mutation in the putative c-di-GMP-binding domain of MrkH completely abolished its function as a transcriptional activator. In vivo analysis of the yfiN and mrkJ genes strongly indicated their c-di-GMP-specific function as diguanylate cyclase and phosphodiesterase, respectively. In addition, in vitro assays showed that purified MrkJ protein has strong c-di-GMP phosphodiesterase activity. These results demonstrate for the first time that c-di-GMP can function as an effector to stimulate the activity of a transcriptional activator, and explain how type 3 fimbriae expression is coordinated with other gene expression programs in K. pneumoniae to promote biofilm formation to implanted medical devices

Genome-scale co-expression network comparison across escherichia coli and salmonella enterica serovar typhimurium reveals significant conservation at the regulon level of local regulators despite their dissimilar lifestyles

Availability of genome-wide gene expression datasets provides the opportunity to study gene expression across different organisms under a plethora of experimental conditions. In our previous work, we developed an algorithm called COMODO (COnserved MODules across Organisms) that identifies conserved expression modules between two species. In the present study, we expanded COMODO to detect the co-expression conservation across three organisms by adapting the statistics behind it. We applied COMODO to study expression conservation/divergence between Escherichia coli, Salmonella enterica, and Bacillus subtilis. We observed that some parts of the regulatory interaction networks were conserved between E. coli and S. enterica especially in the regulon of local regulators. However, such conservation was not observed between the regulatory interaction networks of B. subtilis and the two other species. We found co-expression conservation on a number of genes involved in quorum sensing, but almost no conservation for genes involved in pathogenicity across E. coli and S. enterica which could partially explain their different lifestyles. We concluded that despite their different lifestyles, no significant rewiring have occurred at the level of local regulons involved for instance, and notable conservation can be detected in signaling pathways and stress sensing in the phylogenetically close species S. enterica and E. coli. Moreover, conservation of local regulons seems to depend on the evolutionary time of divergence across species disappearing at larger distances as shown by the comparison with B. subtilis. Global regulons follow a different trend and show major rewiring even at the limited evolutionary distance that separates E. coli and S. enterica

A moonlighting enzyme imposes second messenger bistability to drive lifestyle decisions in E. coli.

Bacteria preferentially colonize surfaces and air-liquid interfaces as matrix embedded communities called biofilms. Biofilms exhibit specific physiological properties, including general stress tolerance, increased antibiotic recalcitrance and tolerance against phagocytic clearance. Together this largely accounts for increased biofilm persistence, chronic infections and infection relapses. One of the principle regulators of biofilm formation is c-di-GMP, a bacterial second messenger controlling various cellular processes. Cellular levels of c-di-GMP are controlled by two antagonistic enzyme families, diguanylate cyclases and phosphodiesterases. But despite the identification and characterization of an increasing number of components of the c-di-GMP network in different bacterial model organisms, details of c-di- GMP mediated decision-making have remained unclear. In particular, how cells shuttle between specific c-di-GMP regimes at the population and single cell level is largely unknown and moreover how these transitions are deterministically made in time and space, given that bacterial networks of diguanylate cyclases and phosphodiesterases show a high degree of complexity. Here we describe a novel mechanism regulating c-di-GMP mediated biofilm formation in E. coli. This mechanism relies on the bistable expression of a key phosphodiesterase that acts both as catalyst for c- di-GMP degradation and as a transcription factor promoting its own production. Bistability results from two interconnected positive feedback loops operating on the catalytic and gene expression level. Based on genetic, structural and biochemical analyses we postulate a simple substrate-induced switch mechanism through which this enzyme can sense changing concentration of c-di-GMP and convert this information into a bistable c-di-GMP response. This mechanism may explain how cellular heterogeneity of small signaling molecules is generated in bacteria and used as a bet hedging strategy for important lifestyle transitions

Roles of the second messenger cyclic di-GMP in environmental adaptation of Sinorhizobium meliloti

Bacteria have evolved various systems for the integration of environmental signals to rapidly coordinate cellular pathways and adapt to changes in their environment. In the quickly advancing field of nucleotide-based second messengers, cyclic dimeric guanosine monophosphate (c-di-GMP) has emerged as a key regulatory player whose underlying signaling networks control major adaptational and lifestyle changes. Enzymes that catalyze synthesis and degradation of c-di-GMP, named diguanylate cylases (DGCs) and phosphodiesterases (PDEs), respectively, are near-ubiquitous in the bacterial kingdom. Despite the numerous studies aiming to better understand the role of c-di-GMP in bacteria, knowledge on integration of c-di-GMP networks into other regulatory networks, the molecular inventory of c-di-GMP receptors and molecular mechanisms underlying c-di-GMP-dependent regulation is limited. This study investigated roles of c-di-GMP in environmental adaptation of soil-dwelling Sinorhizobium meliloti, a rod-shaped alphaproteobacterium from the order Rhizobiales that exists either in free-living states or in symbiosis with leguminous plant hosts. The S. meliloti genome encodes 22 proteins putatively involved in synthesis, degradation and binding of c-di-GMP. Single mutations in 21 of these genes did not cause evident changes in surface attachment, swimming motility or exopolysaccharide (EPS) production. Moreover, screening the different phenotypes of S. meliloti c-di-GMP0 mutants revealed no defects in cell viability and symbiotic potency. In contrast, artificially increasing c-di-GMP levels by overproduction of several DGCs promoted production of extracellular matrix components and surface attachment, whereas swimming motility and extracellular accumulation of N-Acyl-homoserine lactones (AHLs) was reduced. The identification of genetic determinants responsible for observed phenotypic changes at elevated c-di-GMP levels proved c-di-GMP-dependent regulation at both transcriptional and post-translational levels. The SMc01790-SMc01796 locus, homologous to the Agrobacterium tumefaciens uppABCDEF cluster governing biosynthesis of a unipolar polysaccharide (UPP), was required for c-di-GMP-stimulated surface attachment, while the stand-alone PilZ domain protein SMc00507 (renamed McrA) acted as c-di-GMP receptor protein involved in regulation of swimming motility. Transcriptome profiling of S. meliloti at elevated c-di-GMP levels revealed upregulation of the uxs1-SMb20463 gene cluster governing biosynthesis of an extracellular polysaccharide (referred to as CUP). Resulting from this finding, AraC-like transcriptional activator SMb20457 (renamed CuxR) was shown to bind c-di-GMP by a mechanism similar to that of PilZ domains, which provided an example of convergent evolution in two distinct protein families. This study demonstrates that the c-di-GMP network in S. meliloti is integrated into other cellular systems, particularly the well-characterized regulatory network for opposing control of EPS biosynthesis and motility. For instance, CuxR-mediated activation of CUP production was counteracted by the global repressor MucR, while both MucR and the AHL-sensitive master regulator ExpR reduced UPP-mediated surface attachment at elevated c-di-GMP levels. Moreover, a new cellular function was assigned to the essential PDE SMc00074 (renamed GdcP), which is linked to cell envelope biogenesis in alpha-rhizobial species. Overall, c-di-GMP-dependent regulation of multiple cellular functions indicated that high c-di-GMP levels favor a sedentary lifestyle of free-living S. meliloti. The switch of single motile bacteria from a planktonic state to a structured community of cells might contribute to environmental adaptation and long-term survival of S. meliloti in its natural soil habitat

Identification And Structure-Function Analyses Of Bacterial C-Di-Gmp Receptors

In recent years a novel, nucleotide-based small molecule, c-di-GMP, has emerged in the spotlight of scientific investigation as a second messenger unique to the bacterial world. The discovery that its intracellular levels strictly regulate cell adhesion and persistence of bacterial biofilms on one hand, and motility and virulence of planktonic cells on the other, has related this RNA molecule to a variety of disease states including both chronic and acute bacterial infections. Interestingly, intracellular signaling mechanisms involving c-di-GMP appear to be spatially restricted, yet cellular targets for this nucleotide remain mostly unknown. Here we set out to identify and provide comprehensive structure-function analyses of putative or known c-di-GMP receptors. By using structural biology methods we would first determine the atomic resolution structures and conformational states of appropriate targets and then use these molecular blueprints to guide our research into their mechanistic role in the big picture of intracellular signaling networks. We identified VpsT of V.cholerae as a novel c-di-GMP receptor and solved the crystal structures of the nucleotide-free and c-di-GMP-bound states. Our studies identified two biologically relevant dimerization interfaces and the potential formation of higher order oligomeric species assembling upon nucleotide recognition. VpsT defines a novel class of response regulators that utilize a characteristic structural feature to dimerize upon a signaling input regardless of concurrent phosphorylation events. The relative orientation of the DNA-binding domains of VpsT favors a model in which gene regulation is likely accompanied by major changes in DNA architecture. We showed that VpsT is a master regulator of biofilm formation, integrating c-di-GMP signaling events to inversely control exopolysaccharide production and flagellar motility. In a separate study, we provide a complete mechanistic analysis of the structure and function of LapD, a transmembrane c-di-GMP receptor in P. fluorescens which directly translates intracellular c-di-GMP levels in a signal for biofilm dispersal or initiation. We solved three novel crystal structures capturing distinct intermediates in the inside-out signaling process. Most importantly, our structural and functional analyses helped us identify homologous systems in a number of free-living and pathogenic species, likely controlling biofilm formation or toxin expression in a largely similar manner

Regulation by cyclic di-GMP in Myxococcus xanthus

The nucleotide-based second messenger bis-(3’-5’)-cyclic dimeric GMP (c-di-GMP) is involved in regulating a plethora of processes in bacteria that are typically associated with lifestyle changes. Myxococcus xanthus undergoes major lifestyle changes in response to nutrient availability with the formation of spreading colonies in the presence of nutrients and spore-filled fruiting bodies in the absence of nutrients. Here, we investigated the function of c-di-GMP in M. xanthus. We show that this bacterium synthesizes c-di-GMP. Manipulation of the cellular c-di-GMP level by expression of either an active, heterologous diguanylate cyclase or an active, heterologous phosphodiesterase in vegetative cells caused defects in type IV pili (T4P)-dependent motility whereas gliding motility was unaffected. An increased level of c-di-GMP caused reduced transcription of the pilA gene that encodes the major pilin of T4P, reduced assembly of T4P and altered cell agglutination whereas a decreased level of c-di-GMP caused altered cell agglutination. The systematic inactivation of the 24 genes in M. xanthus encoding proteins containing GGDEF, EAL or HD-GYP domains, which are associated with c-di-GMP synthesis, degradation or binding, identified three genes encoding proteins important for T4P-dependent motility. These three proteins named DmxA, TmoK and SgmT all contain a GGDEF domain. Purified DmxA had diguanylate cyclase activity whereas the TmoK and SgmT (both hybrid histidine protein kinases) did not have diguanylate cyclase activity. During starvation, the c-di-GMP level in M. xanthus increases significantly. Manipulation of this level revealed that a low c-di-GMP level negatively affects the developmental program while an increased level does not interfere with development. Moreover, among the 24 genes encoding proteins containing GGDEF, EAL or HD-GYP domains, we identified two which are specifically involved in development: pmxA and dmxB. pmxA codes for an enzymatically active phosphodiesterase with an HD-GYP domain. dmxB codes for a developmentally induced, enzymatically active diguanylate cyclase. DmxB is essential for the increased c-di-GMP level and regulates exopolysaccharide accumulation during starvation. Our results show that c-di-GMP acts as an important signaling molecule during M. xanthus development, and suggest a model in which a minimal threshold level of c-di-GMP is essential for the successful progression and completion of the developmental program. Additionally, candidates for c-di-GMP effectors in M. xanthus were identified using a capture compound mass spectrometry approach. Some of the candidates were confirmed to bind c-di-GMP in vitro and deletion mutants for genes encoding those proteins were characterized in terms of T4P-dependent motility and development

Signals, Regulatory Networks, And Materials That Build And Break Bacterial Biofilms

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REGULATION OF BACTERIAL ADHESION FACTORS BY THE SIGNAL MOLECULE C-DI-GMP: SPECIFIC EFFECTS AT GENE EXPRESSION LEVELS AND SEARCH FOR NOVEL INHIBITORS

Bacteria are able to switch between a single cell (planktonic) lifestyle and a biofilm (community) lifestyle. In pathogenic bacteria, growth as biofilm protects bacterial cells against the host immune system and increases tolerance to antibiotic treatment, thus resulting in chronic infections. The bacterial second messenger cyclic-di-GMP (c-di-GMP) plays a pivotal role in biofilm formation, by promoting production of adhesion factors such as extracellular polysaccharides (EPS). Two classes of enzymes are involved in c-di-GMP metabolism: diguanylate cyclases (DGCs), which synthesize c-di-GMP, and phosphodiesterases (PDEs) that hydrolyze the signal molecule. Usually, a high intracellular c-di-GMP concentration correlates with EPS production and biofilm formation. The enzymes involved in c-di-GMP metabolism are widely conserved in Bacteria, but they are not present in upper eukaryotes. Thus, the proteins involved in c-di-GMP metabolism are a very interesting target for antimicrobial compounds with anti-biofilm activity. In first part of my thesis I developed a screening system for specific inhibitors of DCGs based on a set of microbiological assays that rely on detection of c-di-GMP-dependent EPS production using specific dyes such as Congo Red. Intracellular c-di-GMP levels can then be measured directly by HPLC determination. I tested over 1,000 chemical compounds in my screening system: I found that azathioprine and sulfathiazole two antimetabolites able to inhibit nucleotide biosynthesis impair c-di-GMP production. My results confirm previous literature data showing that perturbation in intracellular nucleotide pools negatively affect biofilm formation in Gram negative bacteria. In second part of this thesis I discussed the role of yddV-dos operon which encodes a DGC and a PDE acting as a protein complex. Both YddV and Dos proteins affect the production of the main adhesion factors of Escherichia coli: curli and the EPS poly-N-acetylglucosamine (PNAG). In particular, the YddV-Dos complex regulates transcription of the csgBAC operon, which encodes curli structural subunits while not affecting the expression of the regulatory operon csgDEFG. In addition we showed that YddV stimulating the transcription of PNAG biosynthetic operon pgaABCD affects PNAG-mediated biofilm formation. Thus, the yddV-dos operon constitutes a main regulatory element in adhesion factors production. Finally, I was able to show that PNAG production is controlled by polynucleotide phosphorylase (PNPase) at post transcriptional level. My results demonstrate the integration of signal molecules and regulatory protein in adhesion factor production, underling the complexity of biofilm regulation in E. coli