[Show abstract][Hide abstract] ABSTRACT: In 2011, nearly 4,000 people in Germany were infected by Shiga toxin (Stx)-producing Escherichia coli O104:H4 with > 22% of patients developing haemolytic uraemic syndrome (HUS). Genome sequencing showed the outbreak strain to be related to enteroaggregative E. coli (EAEC), suggesting its high virulence results from EAEC-typical strong adherence and biofilm formation combined to Stx production. Here, we report that the outbreak strain contains a novel diguanylate cyclase (DgcX)—producing the biofilm-promoting second messenger c-di-GMP—that shows higher expression than any other known E. coli diguanylate cyclase. Unlike closely related E. coli, the outbreak strain expresses the c-di-GMP-controlled biofilm regulator CsgD and amyloid curli fibres at 37°C, but is cellulose-negative. Moreover, it constantly generates derivatives with further increased and deregulated production of CsgD and curli. Since curli fibres are strongly proinflammatory, with cellulose counteracting this effect, high c-di-GMP and curli production by the outbreak O104:H4 strain may enhance not only adherence but may also contribute to inflammation, thereby facilitating entry of Stx into the bloodstream and to the kidneys where Stx causes HUS.
EMBO Molecular Medicine 10/2014; · 7.80 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Amyloid curli fibers and cellulose are extracellular matrix components produced in the stationary phase top layer of E. coli macrocolonies, which confer physical protection, strong cohesion, elasticity, and wrinkled morphology to these biofilms. Curli and cellulose synthesis is controlled by a three-level transcription factor (TF) cascade with the RpoS sigma subunit of RNA polymerase at the top, the MerR-like TF MlrA, and the biofilm regulator CsgD, with two c-di-GMP control modules acting as key switching devices. Additional signal input and fine-tuning is provided by an entire series of small RNAs-ArcZ, DsrA, RprA, McaS, OmrA/OmrB, GcvB, and RydC-that differentially control all three TF modules by direct mRNA interaction. This review not only summarizes the mechanisms of action of these sRNAs, but also addresses the question of how these sRNAs and the regulators they target contribute to building the intriguing three-dimensional microarchitecture and macromorphology of these biofilms.
[Show abstract][Hide abstract] ABSTRACT: In natural habitats bacteria often occur in multicellular communities characterized by a robust extracellular matrix of proteins, amyloid fibres, exopolysaccharides and extracellular DNA. These biofilms show pronounced stress resistance including a resilience against antibiotics, which causes serious medical and technical problems. This review summarizes recent studies that have revealed clear spatial physiological differentiation, complex supracellular architecture and striking morphology in macrocolony biofilms. By responding to gradients of nutrients, oxygen, waste products and signaling compounds that build up in growing biofilms, various stress responses determine whether bacteria grow and proliferate or whether they enter into stationary phase and use their remaining resources for maintenance and survival. As a consequence, biofilms differentiate into at least two distinct layers of vegetatively growing and stationary phase cells that exhibit very different cellular physiology. This includes a stratification of matrix production with a major impact on microscopic architecture, biophysical properties and directly visible morphology of macrocolony biofilms. Using E. coli as a model system, this review also describes our detailed current knowledge about the underlying molecular control networks – prominently featuring sigma factors, transcriptional cascades and second messengers – that drive this spatial differentiation and points out directions for future research.
[Show abstract][Hide abstract] ABSTRACT: Morphological form in multicellular aggregates emerges from the interplay of genetic constitution and environmental signals. Bacterial macrocolony biofilms, which form intricate three-dimensional structures such as large and often radially oriented ridges, concentric rings and elaborate wrinkles, provide a unique opportunity to understand this interplay of 'nature and nurture' in morphogenesis at the molecular level. Macrocolony morphology depends on self-produced extracellular matrix components. In Escherichia coli, these are stationary phase-induced amyloid curli fibres and cellulose. While the widely used 'domesticated' E. coli K-12 laboratory strains are unable to generate cellulose, we could restore cellulose production and macrocolony morphology of E. coli K-12 strain W3110 by 'repairing' a single chromosomal SNP in the bcs operon. Using scanning electron and fluorescence microscopy, cellulose filaments, sheets and nano-composites with curli fibres were localized in situ at cellular resolution within the physiologically two-layered macrocolony biofilms of this 'de-domesticated' strain. As an architectural element, cellulose confers cohesion and elasticity, i.e. tissue-like properties that - together with the cell-encasing curli fibre network and geometrical constraints in a growing colony - explain the formation of long and high ridges and elaborate wrinkles of wildtype macrocolonies. By contrast, a biofilm matrix consisting of the curli fibre network only is brittle and breaks into a pattern of concentric dome-shaped rings separated by deep crevices. These studies now set the stage for clarifying how regulatory networks and in particular c-di-GMP signaling operate in the three-dimensional space of highly structured and 'tissue-like' bacterial biofilms.
Journal of bacteriology 10/2013; · 3.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The ESF-EMBO Conference on 'Bacterial Networks' (BacNet13) was held in March 2013, in Pultusk, Poland. It brought together 164 molecular microbiologists, bacterial systems biologists and synthetic biologists to discuss the architecture, function and dynamics of regulatory networks in bacteria.
[Show abstract][Hide abstract] ABSTRACT: C-di-GMP-which is produced by diguanylate cyclases (DGC) and degraded by specific phosphodiesterases (PDEs)-is a ubiquitous second messenger in bacterial biofilm formation. In Escherichia coli, several DGCs (YegE, YdaM) and PDEs (YhjH, YciR) and the MerR-like transcription factor MlrA regulate the transcription of csgD, which encodes a biofilm regulator essential for producing amyloid curli fibres of the biofilm matrix. Here, we demonstrate that this system operates as a signalling cascade, in which c-di-GMP controlled by the DGC/PDE pair YegE/YhjH (module I) regulates the activity of the YdaM/YciR pair (module II). Via multiple direct interactions, the two module II proteins form a signalling complex with MlrA. YciR acts as a connector between modules I and II and functions as a trigger enzyme: its direct inhibition of the DGC YdaM is relieved when it binds and degrades c-di-GMP generated by module I. As a consequence, YdaM then generates c-di-GMP and-by direct and specific interaction-activates MlrA to stimulate csgD transcription. Trigger enzymes may represent a general principle in local c-di-GMP signalling.
[Show abstract][Hide abstract] ABSTRACT: Biofilm formation in Escherichia coli and other enteric bacteria involves the inverse regulation of the synthesis of flagella and biofilm matrix components such as amyloid curli fibres, cellulose, colanic acid and poly-N-acetylglucosamine (PGA). Physiologically, these processes reflect the transition from growth to stationary phase. At the molecular level, they are tightly controlled by various sigma factors competing for RNA polymerase, a series of transcription factors acting in hierarchical regulatory cascades and several nucleotide messengers, including cyclic-di-GMP. In addition, a surprisingly large number of small regulatory RNAs (sRNAs) have been shown to directly or indirectly modulate motility and/or biofilm formation. This review aims at giving an overview of these sRNA regulators and their impact in biofilm formation in E. coli and Salmonella. Special emphasis will be put on sRNAs, that have known targets such as the mRNAs of the flagellar master regulator FlhDC, the stationary phase sigma factor σS (RpoS) and the key biofilm regulator CsgD that have recently been shown to act as major hubs for regulation by multiple sRNAs.
International Journal of Molecular Sciences 01/2013; 14(3):4560-79. · 2.46 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: ABSTRACT Bacterial biofilms are highly structured multicellular communities whose formation involves flagella and an extracellular matrix of adhesins, amyloid fibers, and exopolysaccharides. Flagella are produced by still-dividing rod-shaped Escherichia coli cells during postexponential growth when nutrients become suboptimal. Upon entry into stationary phase, however, cells stop producing flagella, become ovoid, and generate amyloid curli fibers. These morphological changes, as well as accompanying global changes in gene expression and cellular physiology, depend on the induction of the stationary-phase sigma subunit of RNA polymerase, σ(S) (RpoS), the nucleotide second messengers cyclic AMP (cAMP), ppGpp, and cyclic-di-GMP, and a biofilm-controlling transcription factor, CsgD. Using flagella, curli fibers, a CsgD::GFP reporter, and cell morphology as "anatomical" hallmarks in fluorescence and scanning electron microscopy, different physiological zones in macrocolony biofilms of E. coli K-12 can be distinguished at cellular resolution. Small ovoid cells encased in a network of curli fibers form the outer biofilm layer. Inner regions are characterized by heterogeneous CsgD::GFP and curli expression. The bottom zone of the macrocolonies features elongated dividing cells and a tight mesh of entangled flagella, the formation of which requires flagellar motor function. Also, the cells in the outer-rim growth zone produce flagella, which wrap around and tether cells together. Adjacent to this growth zone, small chains and patches of shorter curli-surrounded cells appear side by side with flagellated curli-free cells before curli coverage finally becomes confluent, with essentially all cells in the surface layer being encased in "curli baskets." IMPORTANCE Heterogeneity or cellular differentiation in biofilms is a commonly accepted concept, but direct evidence at the microscale has been difficult to obtain. Our study reveals the microanatomy and microphysiology of an Escherichia coli macrocolony biofilm at an unprecedented cellular resolution, with physiologically different zones and strata forming as a function of known global regulatory networks that respond to biofilm-intrinsic gradients of nutrient supply. In addition, this study identifies zones of heterogeneous and potentially bistable CsgD and curli expression, shows bacterial curli networks to strikingly resemble Alzheimer plaques, and suggests a new role of flagella as an architectural element in biofilms.
[Show abstract][Hide abstract] ABSTRACT: In response to the 2011 European health alert caused by a pathogenic Escherichia coli O104:H4 outbreak, the European Academy of Microbiology (EAM), established by the Federation of European Microbiological Societies (FEMS), convened a meeting in Paris on November 30th, 2011 on 'EHEC infection and control' attended by world renowned experts in pathogenic E. coli. The major aims of this group were to review the scientific issues raised by the outbreak, to assess the handling of the crisis at the scientific and political levels, and to propose future actions. Several conclusions, which will have impact on future potential E. coli outbreaks, are outlined here.
EMBO Molecular Medicine 08/2012; 4(9):841-8. · 7.80 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Escherichia coli senses blue light via the BLUF-EAL protein BluF (YcgF). The degenerate EAL domain of BluF does not have cyclic-di-GMP phosphodiesterase activity, but BluF directly antagonizes the MerR-like repressor BluR (YcgE), which leads to expression of the ycgZ-ymgABC operon and activation of the Rcs system (Tschowri et al., 2009; Genes Dev 23: 522-534). While bluR, bluF and ycgZ have individual transcriptional start sites, comparative genome analysis indicates that the bluR-bluF-ycgZ-ymgAB region represents a functional unit in various enteric bacteria that is characterized by bluF alleles encoding degenerate EAL domains. Re-introducing conserved amino acids involved in phosphodiesterase activity of EAL domains did not restore enzymatic activity or c-di-GMP binding of BluF, but weakened its ability to antagonize BluR and improved a residual interaction with the BluR paralogue MlrA, which controls expression of the biofilm regulator CsgD and curli fibres. We identified the BluR binding site in the ycgZ promoter and observed that BluR also has residual affinity for the MlrA-dependent csgD promoter. Altogether, we propose that BluF evolved from a blue light-regulated PDE into a specific antagonist of a duplicate of MlrA that became BluR, which controls not only curli but various biofilm functions via the Ymg/Rcs pathway.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: FliZ, a global regulatory protein under the control of the flagellar master regulator FlhDC, was shown to antagonize σ(S)-dependent gene expression in Escherichia coli. Thereby it plays a pivotal role in the decision between alternative life-styles, i.e. FlhDC-controlled flagellum-based motility or σ(S)-dependent curli fimbriae-mediated adhesion and biofilm formation. Here, we show that FliZ is an abundant DNA-binding protein that inhibits gene expression mediated by σ(S) by recognizing operator sequences that resemble the -10 region of σ(S)-dependent promoters. FliZ does so with a structural element that is similar to region 3.0 of σ(S). Within this element, R108 in FliZ corresponds to K173 in σ(S), which contacts a conserved cytosine at the -13 promoter position that is specific for σ(S)-dependent promoters. R108 as well as C(-13) are also crucial for DNA binding by FliZ. However, while a number of FliZ binding sites correspond to known σ(S)-dependent promoters, promoter activity is not a prerequisite for FliZ binding and repressor function. Thus, we demonstrate that FliZ also feedback-controls flagellar gene expression by binding to a site in the flhDC control region that shows similarity only to a -10 element of a σ(S)-dependent promoter, but does not function as a promoter.
Nucleic Acids Research 02/2012; 40(11):4783-93. · 8.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In response to the 2011 European health alert caused by a pathogenic Escherichia coli O104:H4 outbreak, the European Academy of Microbiology (EAM), established by the Federation of European Microbiological Societies (FEMS), convened a meeting in Paris on November 30th, 2011 on ‘EHEC infection and control’ attended by world renowned experts in pathogenic E. coli. The major aims of this group were to review the scientific issues raised by the outbreak, to assess the handling of the crisis at the scientific and political levels, and to propose future actions. Several conclusions, which will have impact on future potential E. coli outbreaks, are outlined here.
EMBO Molecular Medicine 01/2012; 4(9). · 7.80 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Most bacteria can exist in either a planktonic-motile single-cell state or an adhesive multicellular state known as a biofilm. Biofilms cause medical problems and technical damage since they are resistant against antibiotics, disinfectants or the attacks of the immune system. In recent years it has become clear that most bacteria use cyclic diguanylate (c-di-GMP) as a biofilm-promoting second messenger molecule. C-di-GMP is produced by GGDEF-domain-containing diguanylate cyclases and is degraded by phosphodiesterases featuring EAL or HD-GYP domains. Many bacterial species possess multiple proteins with GGDEF and EAL domains, which actually belong to the most abundant protein families in genomic data bases. Via an unprecedented variety of effector components, which include c-di-GMP-binding proteins as well as RNAs, c-di-GMP controls a wide range of targets that down-regulate motility, stimulate adhesin and biofilm matrix formation or even control virulence gene expression. Moreover, local c-di-GMP signaling in macromolecular complexes seems to allow the independent and parallel control of different output reactions. In this review, we use Escherichia coli as a paradigm for c-di-GMP signaling. Despite the huge diversity of components and molecular processes involved in biofilm formation throughout the bacterial kingdom, c-di-GMP signaling represents a unifying principle, which suggests that the enzymes that make and break c-di-GMP may be promising targets for anti-biofilm drugs.
Journal of Biotechnology 12/2011; 160(1-2):10-6. · 3.18 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Rare codons can influence the stability of messenger RNAs, promote regular spacing of ribosomes on a transcript, or modulate stability and proper folding of nascent proteins. The mRNA specifying the stationary phase master regulator RpoS, which belongs to the RpoD family of sigma factors, contains a high number of rare codons, including many codons at positions corresponding to more frequent codons encoding the same amino acids in the homologous RpoD sequence. Substituting these rare codons in rpoS by the more frequent synonymous rpoD codons resulted in decreased transcript and protein levels compared to the natural rare-codon wildtype version of rpoS. The frequent-codon mutant rpoS transcript exhibited faster turnover than the rare-codon wildtype mRNA. Studies with endoribonuclease-deficient strains revealed RNase E to be crucial for this accelerated mRNA degradation. Thus, in the case of RpoS expression, "less is obviously more", as our data suggest a model, in which slowing down translational speed by ribosomal pausing at many rare codons along a transcript could reduce ribosome spacing and thereby protect the transcript against ribonucleolytic attack by RNase E. Such a mechanism may be especially important for translationally controlled genes like rpoS where the formation of secondary structure in the translational initiation region competes with (therefore relatively inefficient) ribosome loading. Moreover, strong codon differences in genes encoding isoenzymes expressed in exponential and stationary phase suggest that transcript protection by repetitive ribosome pausing at multiple rare codons in stationary phase-expressed transcripts may be a general principle to save resources under nutrient-limited conditions.
[Show abstract][Hide abstract] ABSTRACT: In bacteria, promoter identification by RNA polymerase is mediated by a dissociable σ factor. The housekeeping σ(70) factor of Escherichia coli recognizes two well characterized DNA sequence elements, known as the '-10' and '-35' hexamers. These elements are separated by 'spacer' DNA, the sequence of which is generally considered unimportant. Here, we use a combination of bioinformatics, genetics and biochemistry to show that σ(70) can sense the sequence and conformation of the promoter spacer region. Our data illustrate how alterations in spacer region sequence can increase promoter activity. This stimulatory effect requires σ(70) side chain R451, which is located in close proximity to the non-template strand at promoter position -18. Conversely, R451 is not required to mediate transcriptional stimulation by improvement of the -10 element. Mutation of σ(70) residue R451, which is highly conserved, results in reduced growth rate, consistent with a central role in promoter recognition.
Nucleic Acids Research 03/2011; 39(12):5109-18. · 8.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The ubiquitous bacterial signaling molecule bis-(3'-5')-cyclic guanosine monophosphate (c-di-GMP) has brought second messenger signaling back onto the agenda of molecular microbiologists. This is due not only to its general role in promoting biofilm formation, but also to the increasingly diverse array of effector molecules bound by c-di-GMP and of the target processes affected. Effectors include diverse transcription factors and proteins that directly interact with complex cellular machineries, as well as RNA molecules that act as riboswitches to regulate transcriptional elongation or translation. This flexibility in c-di-GMP action enables it to control diverse molecular processes in bacterial cells. New evidence further extends this range to include a c-di-GMP riboswitch linked to a self-splicing intron that has been "domesticated" by its carrier, the pathogenic bacterium Clostridium difficile, to serve in the control of expression of a downstream gene.
[Show abstract][Hide abstract] ABSTRACT: OxyS is one of at least three small non-coding RNAs, which affect rpoS expression. It is induced under oxidative stress and reduces the levels of the stationary phase sigma factor RpoS. We analyzed the turn-over of OxyS and rpoS mRNA in early exponential and in stationary growth phase in different E. coli strains to learn more about the mechanisms of processing and about a possible impact of processing on growth-dependent regulation. We could not attribute a major role of RNase E, RNase III, PNPase or RNase II on OxyS turn-over in exponential growth phase. Only the simultaneous lack of RNase E, PNPase and RNase II activity resulted in some stabilization of OxyS in exponential growth phase, implying the action of multiple ribonucleases on OxyS turn-over. A major role of RNase E on OxyS stability was observed in stationary phase and was dependent on the presence of the RNA binding protein Hfq and of DsrA, one of the other small RNAs binding to rpoS mRNA. Our data also confirm a role of RNase III in rpoS turn-over, however, only in exponential growth phase.We conclude that OxyS and rpoS mRNA processing is influenced by different RNases and additional factors like Hfq and DsrA and that the impact of these factors is strongly dependent on growth phase.