Biomedical subjects

Ute Römling

Publications and source records attributed to Ute Römling.

At least 19 recordsLinked to original sources

PubMed2006

The PilZ domain is a receptor for the second messenger c-di-GMP: the PilZ domain protein YcgR controls motility in enterobacteria.

The ubiquitous bacterial second messenger c-di-GMP controls exopolysaccharide synthesis, flagella- and pili-based motility, gene expression, and interactions of bacteria with eukaryotic hosts. With the exception of bacterial cellulose synthases, the identities of c-di-GMP receptors and end targets have remained unknown. Recently, Amikam and Galperin (Amikam, D., and Galperin, M. (2006) Bioinformatics 22, 3-6) hypothesized that the PilZ domains present in the BcsA subunits of bacterial cellulose synthases function in c-di-GMP binding. This hypothesis has been tested here using the Escherichia coli PilZ domain protein YcgR, its individual PilZ domain and the PilZ domain from Gluconacetobacter xylinus BcsA. YcgR was purified and found to bind c-di-GMP tightly and specifically, Kd 0.84 microm. Individual PilZ domains from YcgR and BcsA also bound c-di-GMP, albeit with lesser affinity, indicating that PilZ is sufficient for binding. The site-directed mutagenesis performed on YcgR implicated the most conserved residues in the PilZ domain directly in c-di-GMP binding. It is suggested that c-di-GMP binding to PilZ brings about conformational changes in the protein that stabilize the bound ligand and initiate the downstream signal transduction cascade. While the identity of the downstream partner(s) of YcgR remains unknown, it is shown that YcgR regulates flagellum-based motility in a c-di-GMP-dependent manner. The inactivation of ycgR improves swimming and swarming motility of the poorly motile yhjH mutants of Salmonella enterica serovar Typhimurium UMR1. Therefore, biochemical and genetic evidence presented here establishes PilZ as a long sought after c-di-GMP-binding domain and YcgR as a c-di-GMP receptor affecting motility in enterobacteria.

Bacterial Proteins↗

PubMed2006

Flagellin in combination with curli fimbriae elicits an immune response in the gastrointestinal epithelial cell line HT-29.

Flagellin is the major cytokine-releasing factor when Salmonella enterica serovar Typhimurium (S. Typhimurium) infects intestinal epithelial cells. In this work it is shown that curli, an adhesive proteinaceous surface component of Enterobacteriaceae involved in biofilm formation of S. Typhimurium and Escherichia coli strains can bind flagellin and thus elicit an immune response by the intestinal epithelial cell line HT-29.

Bacterial Proteins↗

PubMed2006

Cyclic di-GMP as a second messenger.

In many bacteria bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP) signaling determines the timing and amplitude of complex biological processes from biofilm formation and virulence to photosynthesis. Thereby, the tightly regulated temporal and spatial activity patterns of GGDEF and EAL domain proteins, which synthesize and degrade c-di-GMP, respectively, are currently being resolved. Although details of the mechanisms of c-di-GMP signaling are not yet determined, the recent presentation of PilZ as a candidate c-di-GMP binding-domain opens the field for experimental investigations. Besides its role as an intracellular signaling molecule in bacteria, c-di-GMP also acts as an intercellular signaling molecule between prokaryotes and also has effects in eukaryotes that could provide a perspective in cancer treatment.

Animals↗

PubMed2006

ISPa20 advances the individual evolution of Pseudomonas aeruginosa clone C subclone C13 strains isolated from cystic fibrosis patients by insertional mutagenesis and genomic rearrangements.

Pseudomonas aeruginosa clone C strains, which chronically colonize the lungs of cystic fibrosis patients reorganize their genome structure. In this study, a novel member of the IS3 subfamily of IS elements, ISPa20, was detected which was specific for clone C subclone C13 strains. ISPa20, which was present in high copy number, mediated events of genomic reorganization. ISPa20 was inserted into P. aeruginosa backbone genes leading to adaptation to the cystic fibrosis lung habitat and into DNA acquired through horizontal gene transfer. Further on, large chromosomal inversions were mediated by ISPa20. In contrast to strains of other subclonal linages high rates of genomic rearrangements of subclone C13 strains were observed in vitro. The acquisition of mobile elements by P. aeruginosa clone C strains in the lungs of cystic fibrosis patients supports the chronic colonization by insertional mutagenesis and chromosome restructuring leading to microevolution within clone C that reflects macroevolution observed on the species level.

Chromosome Inversion↗

PubMed2006

Hierarchical involvement of various GGDEF domain proteins in rdar morphotype development of Salmonella enterica serovar Typhimurium.

GGDEF and EAL domain proteins are involved in the turnover of the novel secondary messenger cyclic-di(3'-->5')-guanylic acid (c-di-GMP) in many bacteria. In this work the role of the 12 GGDEF domain proteins encoded by the Salmonella enterica serovar Typhimurium (S. Typhimurium) chromosome in rdar morphotype development was investigated. Previously, it was shown that the GGDEF domain protein AdrA activated the biosynthesis of cellulose by production of c-di-GMP. Enhancement of the c-di-GMP levels by overexpression of the GGDEF domain protein AdrA did lead to the activation of curli fimbriae biosynthesis through the elevated expression of CsgD and CsgA. Although knock-out of the chromosomal copy of adrA influenced CsgA expression, CsgD expression was not altered, although more than half of the total cellular c-di-GMP was produced by AdrA at 16 h of growth. On the other hand, chromosomally encoded GGDEF-EAL domain proteins STM2123 and STM3388 were required to additively activate CsgD expression on a transcriptional and post-transcriptional level. Enhanced c-di-GMP levels did overcome temperature regulation of rdar morphotype expression by activation of curli fimbriae as well as cellulose biosynthesis through CsgD expression. Thus in the regulatory cascade leading to rdar morphotype expression c-di-GMP activates several subsequent steps in the network.

Amino Acid Motifs↗

PubMed2006

Regulatory components at the csgD promoter--additional roles for OmpR and integration host factor and role of the 5' untranslated region.

CsgD is a master regulator of multicellular behaviour in Salmonella enterica serovar Typhimurium. Expression of CsgD is highly regulated on the transcriptional level. A nucleo-protein complex had been defined where the global regulators OmpR and integration host factor (IHF) bind up- and downstream of the csgD core promoter. In this study, the nucleo-protein complex of PcsgD was extended through characterization of additional OmpR and IHF binding sites that influence the transcriptional activity of the csgD promoter. Furthermore, the role of the 174 bp long 5'-untranslated region on transcriptional activity was defined.

5' Untranslated Regions↗

PubMed2006

Identification of YhdA as a regulator of the Escherichia coli carbon storage regulation system.

The Escherichia coli BarA-UvrY two-component system, which controls adaptation via the CsrB and CsrC sRNAs, is induced at the entry of the stationary phase by an unknown stimulus. Using a csrB-lacZ fusion, we demonstrated that the factors RelA, SpoT and LuxS, previously suggested to act on orthologues of this system, have no role in BarA-UvrY induction. However, using a transposon screen, we identified the hypothetical protein YhdA as a new regulator of CsrB and CsrC expression. The YhdA protein is predicted to be membrane-bound and to harbor GGDEF and EAL domains, which, however, are highly divergent from the consensus motifs.

Carbon↗

PubMed2005

Microcolony formation: a novel biofilm model of Pseudomonas aeruginosa for the cystic fibrosis lung.

Pseudomonas aeruginosa colonizing the lung of cystic fibrosis patients is responsible for a decline in health and poor prognosis for these patients. Once established, growth of P. aeruginosa in microcolonies makes it very difficult to eradicate the organisms by antimicrobial treatment. An artificial sputum medium was developed to mimic growth of P. aeruginosa in the cystic fibrosis lung habitat and it was found that the organisms grew in tight microcolonies attached to sputum components. Several genes, such as algD, oprF and lasR but not fliC, were required for tight microcolony formation. Among the sputum components, amino acids, lecithin, DNA, salt and low iron were required for tight microcolony formation. Amino acids were also shown to be responsible for various other cystic-fibrosis-specific phenotypes of P. aeruginosa, such as diversification of colony morphology, alterations in LPS structure and hyperexpression of OprF. Since the amino acid content of sputum is elevated in severe lung disease, it is suggested that the tight microcolony biofilm is maintained in these conditions and that they contribute to the vicious cycle of disease severity and failure to eradicate the organism. Thus, growth of P. aeruginosa in artificial sputum medium is an appropriate model of chronic lung colonization and may be useful for evaluating therapeutic procedures and studying antibiotic-resistance mechanisms.

Amino Acids↗

PubMed2005

Expression of cellulose and curli fimbriae by Escherichia coli isolated from the gastrointestinal tract.

Escherichia coli colonizes the gastrointestinal tract of humans; however, little is known about the features of commensal strains. This study investigated whether expression of the biofilm extracellular matrix components cellulose and curli fimbriae is found among commensal isolates. Fifty-two E. coli strains were isolated from faecal samples and, as a control, 24 strains from urinary tract infections were also used. Faecal isolates were characterized by serotyping and phylogenetically grouped by PCR. The genotype was determined by PFGE and the presence of virulence factors was assessed. Co-expression of cellulose and curli fimbriae at 28 degrees C and 37 degrees C was typical for faecal isolates, while urinary tract infection strains typically expressed the extracellular matrix components at 28 degrees C only. Knockout studies in a representative faecal isolate revealed that the response regulator CsgD regulated cellulose and curli fimbriae, as found previously in Salmonella enterica. In contrast to S. enterica, at 37 degrees C pellicle formation occurred in the absence of cellulose and curli fimbriae. The gastrointestinal tract represents a source of biofilm-forming bacteria, which can spread to susceptible sites.

Adolescent↗

PubMed2005

C-di-GMP: the dawning of a novel bacterial signalling system.

Bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) has come to the limelight as a result of the recent advances in microbial genomics and increased interest in multicellular microbial behaviour. Known for more than 15 years as an activator of cellulose synthase in Gluconacetobacter xylinus, c-di-GMP is emerging as a novel global second messenger in bacteria. The GGDEF and EAL domain proteins involved in c-di-GMP synthesis and degradation, respectively, are (almost) ubiquitous in bacterial genomes. These proteins affect cell differentiation and multicellular behaviour as well as interactions between the microorganisms and their eukaryotic hosts and other phenotypes. While the role of GGDEF and EAL domain proteins in bacterial physiology and behaviour has gained appreciation, and significant progress has been achieved in understanding the enzymology of c-di-GMP turnover, many questions regarding c-di-GMP-dependent signalling remain unanswered. Among these, the key questions are the identity of targets of c-di-GMP action and mechanisms of c-di-GMP-dependent regulation. This review discusses phylogenetic distribution of the c-di-GMP signalling pathway in bacteria, recent developments in biochemical and structural characterization of proteins involved in its metabolism, and biological processes affected by c-di-GMP. The accumulated data clearly indicate that a novel ubiquitous signalling system in bacteria has been discovered.

Bacteria↗

PubMed2005

Worldwide distribution of Pseudomonas aeruginosa clone C strains in the aquatic environment and cystic fibrosis patients.

Highly successful bacterial clones have the ability to effectively colonize environmental niches and patients. However, the factors which determine the complex interplay between the colonization of environmental niches and patients are mainly unknown. In this study we show that Pseudomonas aeruginosa clone C strains are distributed worldwide and highly prone to infect cystic fibrosis (CF) patients in Canada, England, France and Germany. In Hanover, Germany and Vancouver, Canada, clone C strains are highly prevalent in the CF patient community, although the mechanisms of acquisition may have been different. All clone C strains showed highly related macrorestriction fragment pattern of the whole genome as visualized by pulsed-field gel electrophoresis and harboured the 102 kbp plasmid pKLC102. Comparison of three prevalent P. aeruginosa clones with different distribution between the environment and patients revealed that neither enhanced biofilm formation nor antibiotic resistance was responsible for the spread of clone C. Clone M, which was highly prevalent in the clinical environment such as sanitary facilities, lacked motility, which could explain its relatively low prevalence in CF patients. Elucidation of the mechanisms which lead to the prevalence of clone C strain in patients and the environment requires the investigation of additional phenotypes.

Biofilms↗

PubMed2005

Phenotypic convergence mediated by GGDEF-domain-containing proteins.

GGDEF domain-containing proteins have been implicated in bacterial signal transduction and synthesis of the second messenger molecule cyclic-di-GMP. A number of GGDEF proteins are involved in controlling the formation of extracellular matrices. AdrA (Salmonella enterica serovar Typhimurium) and HmsT (Yersinia pestis) contain GGDEF domains and are required for extracellular cellulose production and biofilm formation, respectively. Here we show that hmsT is able to restore cellulose synthesis to a Salmonella serovar Typhimurium adrA mutant and that adrA can replace hmsT in Y. pestis Hms-dependent biofilm formation. Like Y. pestis HmsT overproducers, Y. pestis cells carrying adrA under the control of an arabinose-inducible promoter produced substantial biofilms in the presence of arabinose. Finally, we demonstrate that HmsT is involved in the synthesis of cyclic di-GMP.

Bacterial Proteins↗

PubMed2004

GGDEF and EAL domains inversely regulate cyclic di-GMP levels and transition from sessility to motility.

Cyclic nucleotides represent second messenger molecules in all kingdoms of life. In bacteria, mass sequencing of genomes detected the highly abundant protein domains GGDEF and EAL. We show here that the GGDEF and EAL domains are involved in the turnover of cyclic-di-GMP (c-di-GMP) in vivo whereby the GGDEF domain stimulates c-di-GMP production and the EAL domain c-di-GMP degradation. Thus, most probably, GGDEF domains function as c-di-GMP cyclase and EAL domains as phosphdiesterase. We further show that, in the pathogenic organism Salmonella enterica serovar Typhimurium, the nosocomial pathogen Pseudomonas aeruginosa and the commensal species Escherichia coli, GGDEF and EAL domains mediate similar phenotypic changes related to the transition between sessility and motility. Thus, the data suggest that c-di-GMP is a novel global second messenger in bacteria the metabolism of which is controlled by GGDEF and EAL domain proteins.

Cyclic GMP↗

PubMed2003

The csgD promoter, a control unit for biofilm formation in Salmonella typhimurium.

Expression of cellulose and curli fimbriae in Salmonella typhimurium is dependent on the transcriptional regulator CsgD. Transcription of csgD itself is influenced by a variety of regulatory stimuli. Complex nucleoprotein arrangements modulate the transcriptional activity of csgD and trigger the transition between the planktonic status and biofilm formation.

Bacterial Proteins↗

PubMed2003

Impact of large chromosomal inversions on the adaptation and evolution of Pseudomonas aeruginosa chronically colonizing cystic fibrosis lungs.

Pseudomonas aeruginosa chronically colonizing the lungs of cystic fibrosis (CF) patients undergoes fast evolution leading to clonal divergence. More than half of the genotypes of P. aeruginosa clone C isolates exclusively from CF lung infection exhibit large chromosomal inversions (LCIs). To analyse the impact of LCIs, as a novel mechanism of bacterial adaptation, the underlying molecular mechanism was examined. Analysis of inversion breakpoints suggested an IS6100-induced coupled insertion-inversion mechanism. A selective advantage was created by insertion of IS6100 into wbpM, pilB and mutS which leads to common CF phenotypes such as O-antigen and type IV pili deficiency and hypermutability. Speciation in bacteria is accompanied by LCIs. Therefore adaptation by LCIs that allows persistence of P. aeruginosa in the CF lung and species diversification in that new ecological niche can serve as a model for bacterial genome evolution.

Adaptation, Physiological↗

PubMed2003

Complex regulation of csgD promoter activity by global regulatory proteins.

The starvation-induced csgD gene of Salmonella typhimurium encodes for the positive transcriptional regulator of extracellular matrix components curli fimbriae and cellulose. To analyse regulatory elements of csgD promoter (PcsgD) response genetic studies combined with in vitro experiments were performed. Six binding sites (D1 to D6) for OmpR, a transcriptional regulator, were identified by gel shifts and DNase I footprints. While ompR is required for PcsgD expression, binding of OmpR-P to D2 centred immediately upstream of D1 at position -70.5 is proposed to repress PcsgD activity. The elevated expression of regulated and semiconstitutive PcsgD in response to microaerophilic conditions required integration host factor (IHF). Subsequently, two IHF-binding sites were identified up- and downstream of PcsgD. IHF competes with OmpR-P for binding at its upstream site IHF1, which overlaps with D3-D6 and thereby modulates the response to microaerophilic conditions. A complex regulatory network involving IHF, H-NS and OmpR is proposed whereby the nucleo-complex composition in the csgD-csgBA intergenic region is altered in response to oxygen tension.

Aerobiosis↗

PubMed2003

Occurrence and regulation of the multicellular morphotype in Salmonella serovars important in human disease.

Multicellular behavior in Salmonella Typhimurium ATCC14028 called the rdar morphotype is characterized by the expression of the extracellular matrix components cellulose and curli fimbriae. Over 90% of S. Typhimurium and S. Enteritidis strains from human disease, food and animals expressed the rdar morphotype at 28 degrees C. Regulation of the rdar morphotype occurred via the response regulator ompR, which activated transcription of csgD required for production of cellulose and curli fimbriae. Serovar-specific regulation of csgD required rpoS in S. Typhimurium, but was partially independent of rpoS in S. Enteritidis. Rarely, strain-specific temperature-deregulated expression of the rdar morphotype was observed. The host-restricted serovars S. Typhimurium var. Copenhagen phage type DT2 and DT99, Salmonella Typhi and Salmonella Choleraesuis did not express the rdar morphotype, while in Salmonella Gallinarum cellulose expression at 37 degrees C was seen in some strains. Therefore, the expression pattern of the rdar morphotype is serovar specific and correlates with a disease phenotype breaching the intestinal epithelial cell lining.

Bacterial Proteins↗

PubMed2003

Production of cellulose and curli fimbriae by members of the family Enterobacteriaceae isolated from the human gastrointestinal tract.

Citrobacter spp., Enterobacter spp., and Klebsiella spp. isolated from the human gut were investigated for the biosynthesis of cellulose and curli fimbriae (csg). While Citrobacter spp. produced curli fimbriae and cellulose and Enterobacter spp. produced cellulose with various temperature-regulatory programs, Klebsiella spp. did not show pronounced expression of those extracellular matrix components. Investigation of multicellular behavior in two Citrobacter species and Enterobacter sakazakii showed an extracellular matrix, cell clumping, pellicle formation, and biofilm formation associated with the expression of cellulose and curli fimbriae. In those three strains, the csgD-csgBA region and the cellulose synthase gene bcsA were conserved. PCR screening for the presence of csgD, csgA and bcsA revealed that besides Klebsiella pneumoniae and Klebsiella oxytoca, all species investigated harbored the genetic information for expression of curli fimbriae and cellulose. Since Citrobacter spp., Enterobacter spp., and Klebsiella spp. are frequently found to cause biofilm-related infections such as catheter-associated urinary tract infections, the human gut could serve as a reservoir for dissemination of biofilm-forming isolates.

Amino Acid Sequence↗