Local c-di-GMP Signaling in the Control of Synthesis of the E. coli Biofilm Exopolysaccharide pEtN-Cellulose.

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.

Genetic dissection of Escherichia coli's master diguanylate cyclase DgcE: Role of the N-terminal MASE1 domain and direct signal input from a GTPase partner system.

The ubiquitous second messenger c-di-GMP promotes bacterial biofilm formation by playing diverse roles in the underlying regulatory networks. This is reflected in the multiplicity of diguanylate cyclases (DGC) and phosphodiesterases (PDE) that synthesize and degrade c-di-GMP, respectively, in most bacterial species. One of the 12 DGCs of Escherichia coli, DgcE, serves as the top-level trigger for extracellular matrix production during macrocolony biofilm formation. Its multi-domain architecture-a N-terminal membrane-inserted MASE1 domain followed by three PAS, a GGDEF and a degenerate EAL domain-suggested complex signal integration and transmission through DgcE. Genetic dissection of DgcE revealed activating roles for the MASE1 domain and the dimerization-proficient PAS3 region, whereas the inhibitory EALdeg domain counteracts the formation of DgcE oligomers. The MASE1 domain is directly targeted by the GTPase RdcA (YjdA), a dimer or oligomer that together with its partner protein RdcB (YjcZ) activates DgcE, probably by aligning and promoting dimerization of the PAS3 and GGDEF domains. This activation and RdcA/DgcE interaction depend on GTP hydrolysis by RdcA, suggesting GTP as an inhibitor and the pronounced decrease of the cellular GTP pool during entry into stationary phase, which correlates with DgcE-dependent activation of matrix production, as a possible input signal sensed by RdcA. Furthermore, DgcE exhibits rapid, continuous and processive proteolytic turnover that also depends on the relatively disordered transmembrane MASE1 domain. Overall, our study reveals a novel GTP/c-di-GMP-connecting signaling pathway through the multi-domain DGC DgcE with a dual role for the previously uncharacterized MASE1 signaling domain.

Spatial organization of different sigma factor activities and c-di-GMP signalling within the three-dimensional landscape of a bacterial biofilm.

Bacterial biofilms are large aggregates of cells embedded in an extracellular matrix of self-produced polymers. In macrocolony biofilms of Escherichia coli, this matrix is generated in the upper biofilm layer only and shows a surprisingly complex supracellular architecture. Stratified matrix production follows the vertical nutrient gradient and requires the stationary phase σS (RpoS) subunit of RNA polymerase and the second messenger c-di-GMP. By visualizing global gene expression patterns with a newly designed fingerprint set of Gfp reporter fusions, our study reveals the spatial order of differential sigma factor activities, stringent control of ribosomal gene expression and c-di-GMP signalling in vertically cryosectioned macrocolony biofilms. Long-range physiological stratification shows a duplication of the growth-to-stationary phase pattern that integrates nutrient and oxygen gradients. In addition, distinct short-range heterogeneity occurs within specific biofilm strata and correlates with visually different zones of the refined matrix architecture. These results introduce a new conceptual framework for the control of biofilm formation and demonstrate that the intriguing extracellular matrix architecture, which determines the emergent physiological and biomechanical properties of biofilms, results from the spatial interplay of global gene regulation and microenvironmental conditions. Overall, mature bacterial macrocolony biofilms thus resemble the highly organized tissues of multicellular organisms.

Phosphoethanolamine cellulose: A naturally produced chemically modified cellulose.

Cellulose is a major contributor to the chemical and mechanical properties of plants and assumes structural roles in bacterial communities termed biofilms. We find that Escherichia coli produces chemically modified cellulose that is required for extracellular matrix assembly and biofilm architecture. Solid-state nuclear magnetic resonance spectroscopy of the intact and insoluble material elucidates the zwitterionic phosphoethanolamine modification that had evaded detection by conventional methods. Installation of the phosphoethanolamine group requires BcsG, a proposed phosphoethanolamine transferase, with biofilm-promoting cyclic diguanylate monophosphate input through a BcsE-BcsF-BcsG transmembrane signaling pathway. The bcsEFG operon is present in many bacteria, including Salmonella species, that also produce the modified cellulose. The discovery of phosphoethanolamine cellulose and the genetic and molecular basis for its production offers opportunities to modulate its production in bacteria and inspires efforts to biosynthetically engineer alternatively modified cellulosic materials.

Targeting of csgD by the small regulatory RNA RprA links stationary phase, biofilm formation and cell envelope stress in Escherichia coli.

RprA is a small regulatory RNA known to weakly affect the translation of σ(S) (RpoS) in Escherichia coli. Here we demonstrate that csgD, which encodes a stationary phase-induced biofilm regulator, as well as ydaM, which encodes a diguanylate cyclase involved in activating csgD transcription, are novel negatively controlled RprA targets. As shown by extensive mutational analysis, direct binding of RprA to the 5'-untranslated and translational initiation regions of csgD mRNA inhibits translation and reduces csgD mRNA levels. In the case of ydaM mRNA, RprA base-pairs directly downstream of the translational start codon. In a feedforward loop, RprA can thus downregulate > 30 YdaM/CsgD-activated genes including those for adhesive curli fimbriae. However, during early stationary phase, when csgD transcription is strongly activated, the synthesis of csgD mRNA exceeds that of RprA, which allows the accumulation of CsgD protein. This situation is reversed when csgD transcription is shut off - for instance, later in stationary phase or during biofilm formation - or by conditions that further activate RprA expression via the Rcs two-component system. Thus, antagonistic regulation of csgD and RprA at the mRNA level integrates cell envelope stress signals with global gene expression during stationary phase and biofilm formation.

Gene expression patterns and differential input into curli fimbriae regulation of all GGDEF/EAL domain proteins in Escherichia coli.

Switching from the motile planktonic bacterial lifestyle to a biofilm existence is stimulated by the signalling molecule bis-(3'-5')-cyclic-diguanosine monophosphate (cyclic-di-GMP), which is antagonistically controlled by diguanylate cyclases (DGCs; characterized by GGDEF domains) and specific phosphodiesterases (PDEs; mostly featuring EAL domains). Here, we present the expression patterns of all 28 genes that encode GGDEF/EAL domain proteins in Escherichia coli K-12. Twenty-one genes are expressed in Luria-Bertani medium, with 15 being under sigma(S) control. While a small subset of GGDEF/EAL proteins (YeaJ and YhjH) is dominant and modulates motility in post-exponentially growing cells, a diverse battery of GGDEF/EAL proteins is deployed during entry into stationary phase, especially in cells grown at reduced temperature (28 degrees C). This suggests that multiple signal input into cyclic-di-GMP control is particularly important in growth-restricted cells in an extra-host environment. Six GGDEF/EAL genes differentially control the expression of adhesive curli fimbriae. Besides the previously described ydaM, yciR, yegE and yhjH genes, these are yhdA (csrD), which stimulates the expression of the DGC YdaM and the major curli regulator CsgD, and yeaP, which contributes to expression of the curli structural operon csgBAC. Finally, we discuss why other GGDEF/EAL domain-encoding genes, despite being expressed, do not influence motility and/or curli formation.

Inverse regulatory coordination of motility and curli-mediated adhesion in Escherichia coli.

During the transition from post-exponential to stationary phase, Escherichia coli changes from the motile-planktonic to the adhesive-sedentary "lifestyle." We demonstrate this transition to be controlled by mutual inhibition of the FlhDC/motility and sigma(S)/adhesion control cascades at two distinct hierarchical levels. At the top level, motility gene expression and the general stress response are inversely coordinated by sigma(70)/sigma(FliA)/sigma(S) competition for core RNA polymerase and the FlhDC-controlled FliZ protein acting as a sigma(S) inhibitor. At a lower level, the signaling molecule bis-(3'-5')-cyclic-diguanosine monophosphate (c-di-GMP) reduces flagellar activity and stimulates transcription of csgD, which encodes an essential activator of adhesive curli fimbriae expression. This c-di-GMP is antagonistically controlled by sigma(S)-regulated GGDEF proteins (mainly YegE) and YhjH, an EAL protein and c-di-GMP phosphodiesterase under FlhDC/FliA control. The switch from motility-based foraging to the general stress response and curli expression requires sigma(S)-modulated down-regulation of expression of the flagellar regulatory cascade as well as proteolysis of the flagellar master regulator FlhDC. Control of YhjH by FlhDC and of YegE by sigma(S) produces a fine-tuned checkpoint system that "unlocks" curli expression only after down-regulation of flagellar gene expression. In summary, these data reveal the logic and sequence of molecular events underlying the motile-to-adhesive "lifestyle" switch in E. coli.

A role for Lon protease in the control of the acid resistance genes of Escherichia coli.

Lon protease is a major protease in cellular protein quality control, but also plays an important regulatory role by degrading various naturally unstable regulators. Here, we traced additional such regulators by identifying regulons with co-ordinately altered expression in a lon mutant by genome-wide transcriptional profiling. Besides many members of the RcsA regulon (which validates our approach as RcsA is a known Lon substrate), many genes of the sigmaS-dependent general stress response were upregulated in the lon mutant. However, the lon mutation did not affect sigmaS levels nor sigmaS activity in general, suggesting specific effects of Lon on secondary regulators involved in the control of subsets of sigmaS-controlled genes. Lon-affected genes also included the major acid resistance genes (gadA, gadBC, gadE, hdeAB and hdeD), which led to the discovery that the essential acid resistance regulator GadE (whose expression is sigmaS-controlled) is degraded in vivo in a Lon-dependent manner. GadE proteolysis is constitutive as it was observed even under conditions that induce the system (i.e. at low pH or during entry into stationary phase). GadE degradation was found to rapidly terminate the acid resistance response upon shift back to neutral pH and to avoid overexpression of acid resistance genes in stationary phase.

Stationary phase reorganisation of the Escherichia coli transcription machinery by Crl protein, a fine-tuner of sigmas activity and levels.

Upon environmental changes, bacteria reschedule gene expression by directing alternative sigma factors to core RNA polymerase (RNAP). This sigma factor switch is achieved by regulating relative amounts of alternative sigmas and by decreasing the competitiveness of the dominant housekeeping sigma(70). Here we report that during stationary phase, the unorthodox Crl regulator supports a specific sigma factor, sigma(S) (RpoS), in its competition with sigma(70) for core RNAP by increasing the formation of sigma(S)-containing RNAP holoenzyme, Esigma(S). Consistently, Crl has a global regulatory effect in stationary phase gene expression exclusively through sigma(S), that is, on sigma(S)-dependent genes only. Not a specific promoter motif, but sigma(S) availability determines the ability of Crl to exert its function, rendering it of major importance at low sigma(S) levels. By promoting the formation of Esigma(S), Crl also affects partitioning of sigma(S) between RNAP core and the proteolytic sigma(S)-targeting factor RssB, thereby playing a dual role in fine-tuning sigma(S) proteolysis. In conclusion, Crl has a key role in reorganising the Escherichia coli transcriptional machinery and global gene expression during entry into stationary phase.

Cyclic-di-GMP-mediated signalling within the sigma network of Escherichia coli.

Bis-(3'-5')-cyclic-di-guanosine monophosphate (c-di-GMP) is a bacterial signalling molecule produced by diguanylate cyclases (DGC, carrying GGDEF domains) and degraded by specific phosphodiesterases (PDE, carrying EAL domains). Neither its full physiological impact nor its effector mechanisms are currently understood. Also, the existence of multiple GGDEF/EAL genes in the genomes of most species raises questions about output specificity and robustness of c-di-GMP signalling. Using microarray and gene fusion analyses, we demonstrate that at least five of the 29 GGDEF/EAL genes in Escherichia coli are not only stationary phase-induced under the control of the general stress response master regulator sigma(S) (RpoS), but also exhibit differential control by additional environmental and temporal signals. Two of the corresponding proteins, YdaM (GGDEF only) and YciR (GGDEF + EAL), which in vitro show DGC and PDE activity, respectively, play an antagonistic role in the expression of the biofilm-associated curli fimbriae. This control occurs at the level of transcription of the curli and cellulose regulator CsgD. Moreover, we show that H-NS positively affects curli expression by inversely controlling the expression of ydaM and yciR. Furthermore, we demonstrate a temporally fine-tuned GGDEF cascade in which YdaM controls the expression of another GGDEF protein, YaiC. By genome-wide microarray analysis, evidence is provided that YdaM and YciR strongly and nearly exclusively control CsgD-regulated genes. We conclude that specific GGDEF/EAL proteins have very distinct expression patterns, and when present in physiological amounts, can act in a highly precise, non-global and perhaps microcompartmented manner on a few or even a single specific target(s).

High throughput identification of potential Arabidopsis mitogen-activated protein kinases substrates.

Mitogen-activated protein kinase (MAPK) cascades are universal and highly conserved signal transduction modules in eucaryotes, including plants. These protein phosphorylation cascades link extracellular stimuli to a wide range of cellular responses. However, the underlying mechanisms are so far unknown as information about phosphorylation substrates of plant MAPKs is lacking. In this study we addressed the challenging task of identifying potential substrates for Arabidopsis thaliana mitogen-activated protein kinases MPK3 and MPK6, which are activated by many environmental stress factors. For this purpose, we developed a novel protein microarray-based proteomic method allowing high throughput study of protein phosphorylation. We generated protein microarrays including 1,690 Arabidopsis proteins, which were obtained from the expression of an almost nonredundant uniclone set derived from an inflorescence meristem cDNA expression library. Microarrays were incubated with MAPKs in the presence of radioactive ATP. Using a threshold-based quantification method to evaluate the microarray results, we were able to identify 48 potential substrates of MPK3 and 39 of MPK6. 26 of them are common for both kinases. One of the identified MPK6 substrates, 1-aminocyclopropane-1-carboxylic acid synthase-6, was just recently shown as the first plant MAPK substrate in vivo, demonstrating the potential of our method to identify substrates with physiological relevance. Furthermore we revealed transcription factors, transcription regulators, splicing factors, receptors, histones, and others as candidate substrates indicating that regulation in response to MAPK signaling is very complex and not restricted to the transcriptional level. Nearly all of the 48 potential MPK3 substrates were confirmed by other in vitro methods. As a whole, our approach makes it possible to shortlist candidate substrates of mitogen-activated protein kinases as well as those of other protein kinases for further analysis. Follow-up in vivo experiments are essential to evaluate their physiological relevance.

Identification of barley CK2alpha targets by using the protein microarray technology.

We have successfully established a novel protein microarray-based kinase assay, which we applied to identify target proteins of the barley protein kinase CK2alpha. As a source of recombinant barley proteins we cloned cDNAs specific for filial tissues of developing barley seeds into an E. coli expression vector. By using robot technology, 21,500 library clones were arrayed in microtiter plates and gridded onto high-density filters. Protein expressing clones were detected using an anti-RGS-His6 antibody and rearrayed into a sublibrary of 4100 clones. All of these clones were sequenced from the 5'-end and the sequences were analysed by homology searches against protein databases. Based on these results we selected 768 clones expressing different barley proteins for protein purification. The purified proteins were robotically arrayed onto FAST slides. The generated protein microarrays were incubated with an expression library-derived barley CK2alpha in the presence of [gamma-33P]ATP, and signals were detected by X-ray film or phosphor imager. We were able to demonstrate the power of the protein microarray technology by identification of 21 potential targets out of 768 proteins including such well-known substrates of CK2alpha as high mobility group proteins and calreticulin.

Protein microarray technology and ultraviolet crosslinking combined with mass spectrometry for the analysis of protein-DNA interactions.

To gain insights into complex biological processes, such as transcription and replication, the analysis of protein-DNA interactions and the determination of their sequence requirements are of central importance. In this study, we probed protein microarray technology and ultraviolet crosslinking combined with mass spectrometry (MS) for their practicability to study protein-DNA interactions. We chose as a model system the well-characterized interaction of bacterial replication initiator DnaA with its cognate binding site, the DnaA box. Interactions of DnaA domain 4 with a high-affinity DnaA box (R4) and with a low-affinity DnaA box (R3) were compared. A mutant DnaA domain 4, A440V, was included in the study. DnaA domain 4, wt, spotted onto FAST slides, revealed a strong signal only with a Cy5-labeled, double-stranded, 21-mer oligonucleotide containing DnaA box R4. No signals were obtained when applying the mutant protein. Ultraviolet crosslinking combined with nanoLC/MALDI-TOF MS located the site of interaction to a peptide spanning amino acids 433- 442 of Escherichia coli DnaA. This fragment contains six residues that were identified as being involved in DNA binding by recently published crystal structure and nuclear magnetic resonance (NMR) analysis. In the future, the technologies applied in this study will become important tools for studying protein-DNA interactions.

A nonredundant human protein chip for antibody screening and serum profiling.

There is burgeoning interest in protein microarrays, but a source of thousands of nonredundant, purified proteins was not previously available. Here we show a glass chip containing 2413 nonredundant purified human fusion proteins on a polymer surface, where densities up to 1600 proteins/cm(2) on a microscope slide can be realized. In addition, the polymer coating of the glass slide enables screening of protein interactions under nondenaturing conditions. Such screenings require only 200-microl sample volumes, illustrating their potential for high-throughput applications. Here we demonstrate two applications: the characterization of antibody binding, specificity, and cross-reactivity; and profiling the antibody repertoire in body fluids, such as serum from patients with autoimmune diseases. For the first application, we have incubated these protein chips with anti-RGSHis(6), anti-GAPDH, and anti-HSP90beta antibodies. In an initial proof of principle study for the second application, we have screened serum from alopecia and arthritis patients. With analysis of large sample numbers, identification of disease-associated proteins to generate novel diagnostic markers may be possible.