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Jörg Stülke

Publications and source records attributed to Jörg Stülke.

At least 19 recordsLinked to original sources
PubMed2007

Tools for the genetic analysis of Mycoplasma.

The mycoplasmas have attracted much scientific attention since they contain the smallest known genomes of any independently viable bacterial species. However, the detailed genetic analysis of these bacteria has lagged behind the well-analyzed bacterial model organisms for a long time. This is due to the use of the UGA codon for tryptophan rather than as stop codon, which had often prevented the expression of full-length Mycoplasma proteins in heterologous hosts. Additionally, insufficient efficiency of homologous recombination prevented the targeted disruption of genes in some species such as M. pneumoniae whereas homologous recombination is operative in other mycoplasmas. Only recently, efficient screening systems for the use of transposon-based mutagenesis have been developed to circumvent this problem and to allow the targeted isolation of desired transposon insertion mutants. With the availability of several Mycoplasma genome sequences, artificial plasmids based on the chromosomal origin of replication were constructed that can now be used for complementation studies and for the stable introduction of foreign genetic material. In this review, we give an overview on recent developments in Mycoplasma genetics that facilitate the genetic manipulation of these interesting organisms.

Codon↗
PubMed2007

Characterization of Bacillus subtilis mutants with carbon source-independent glutamate biosynthesis.

Bacillus subtilis synthesizes glutamate from 2-oxoglutarate and glutamine using the glutamate synthase, encoded by the gltAB operon. Glutamate degradation involves the catabolic glutamate dehydrogenase (GDH) RocG. Expression of both gltAB and rocG is controlled by the carbon and nitrogen sources. In the absence of glucose or other well-metabolizable carbon sources, B. subtilis is unable to grow unless provided with external glutamate. In this work, we isolated mutations that suppressed this growth defect of B. subtilis on minimal media (sgd mutants). All mutations enabled the cells to express the gltAB operon even in the absence of glucose. The mutations were all identified in the rocG gene suggesting that the catabolic GDH is essential for controlling gltAB expression in response to the availability of sugars.

Bacillus subtilis↗
PubMed2007

Regulation of carbon metabolism in the mollicutes and its relation to virulence.

The mollicutes are cell wall-less bacteria that live in close association with their eukaryotic hosts. Their genomes are strongly reduced and so are their metabolic capabilities. A survey of the available genome sequences reveals that the mollicutes are capable of utilizing sugars as source of carbon and energy via glycolysis. The pentose phosphate pathway is incomplete in these bacteria, and genes encoding enzymes of the tricarboxylic acid cycle are absent from the genomes. Sugars are transported by the phosphotransferase system. As in related bacteria, the phosphotransferase system does also seem to play a regulatory role in the mollicutes as can be concluded from the functionality of the regulatory HPr kinase/phosphorylase. In Mycoplasma pneumoniae, the activity of HPr kinase is triggered in the presence of glycerol. This carbon source may be important for the mollicutes since it is available in epithelial tissues and its metabolism results in the formation of hydrogen peroxide, the major virulence factor of several mollicutes. In plant-pathogenic mollicutes such as Spiroplasma citri, the regulation of carbon metabolism is crucial in the adaptation to life in plant tissues or the insect vectors. Thus, carbon metabolism seems to be intimately linked to pathogenicity in the mollicutes.

Bacterial Proteins↗
PubMed2006

Transcriptional and metabolic responses of Bacillus subtilis to the availability of organic acids: transcription regulation is important but not sufficient to account for metabolic adaptation.

The soil bacterium Bacillus subtilis can use sugars or organic acids as sources of carbon and energy. These nutrients are metabolized by glycolysis, the pentose phosphate pathway, and the Krebs citric acid cycle. While the response of B. subtilis to the availability of sugars is well understood, much less is known about the changes in metabolism if organic acids feeding into the Krebs cycle are provided. If B. subtilis is supplied with succinate and glutamate in addition to glucose, the cells readjust their metabolism as determined by transcriptome and metabolic flux analyses. The portion of glucose-6-phosphate that feeds into the pentose phosphate pathway is significantly increased in the presence of organic acids. Similarly, important changes were detected at the level of pyruvate and acetyl coenzyme A (acetyl-CoA). In the presence of organic acids, oxaloacetate formation is strongly reduced, whereas the formation of lactate is significantly increased. The alsSD operon required for acetoin formation is strongly induced in the presence of organic acids; however, no acetoin formation was observed. The recently discovered phosphorylation of acetolactate decarboxylase may provide an additional level of control of metabolism. In the presence of organic acids, both types of analyses suggest that acetyl-CoA was catabolized to acetate rather than used for feeding the Krebs cycle. Our results suggest that future work has to concentrate on the posttranslational mechanisms of metabolic regulation.

Adaptation, Physiological↗
PubMed2006

Keeping signals straight in transcription regulation: specificity determinants for the interaction of a family of conserved bacterial RNA-protein couples.

Regulatory systems often evolve by duplication of ancestral systems and subsequent specialization of the components of the novel signal transduction systems. In the Gram-positive soil bacterium Bacillus subtilis, four homologous antitermination systems control the expression of genes involved in the metabolism of glucose, sucrose and beta-glucosides. Each of these systems is made up of a sensory sugar permease that does also act as phosphotransferase, an antitermination protein, and a RNA switch that is composed of two mutually exclusive structures, a RNA antiterminator (RAT) and a transcriptional terminator. We have studied the contributions of sugar specificity of the permeases, carbon catabolite repression, and protein-RAT recognition for the straightness of the signalling chains. We found that the beta-glucoside permease BglP does also have a minor activity in glucose transport. However, this activity is irrelevant under physiological conditions since carbon catabolite repression in the presence of glucose prevents the synthesis of the beta-glucoside permease. Reporter gene studies, in vitro RNA-protein interaction analyzes and northern blot transcript analyzes revealed that the interactions between the antiterminator proteins and their RNA targets are the major factor contributing to regulatory specificity. Both structural features in the RATs and individual bases are important specificity determinants. Our study revealed that the specificity of protein-RNA interactions, substrate specificity of the permeases as well as the general mechanism of carbon catabolite repression together allow to keep the signalling chains straight and to avoid excessive cross-talk between the systems.

Bacillus subtilis↗
PubMed2006

Interaction of the membrane-bound GlnK-AmtB complex with the master regulator of nitrogen metabolism TnrA in Bacillus subtilis.

PII proteins are widespread and highly conserved signal transduction proteins occurring in bacteria, Archaea, and plants and play pivotal roles in controlling nitrogen assimilatory metabolism. This study reports on biochemical properties of the PII-homologue GlnK (originally termed NrgB) in Bacillus subtilis (BsGlnK). Like other PII proteins, the native BsGlnK protein has a trimeric structure and readily binds ATP in the absence of divalent cations, whereas 2-oxoglutarate is only weakly bound. In contrast to other PII-like proteins, Mg2+ severely affects its ATP-binding properties. BsGlnK forms a tight complex with the membrane-bound ammonium transporter AmtB (NrgA), from which it can be relieved by millimolar concentrations of ATP. Immunoprecipitation and co-localization experiments identified a novel interaction between the BsGlnK-AmtB complex and the major transcription factor of nitrogen metabolism, TnrA. In vitro in the absence of ATP, TnrA is completely tethered to membrane (AmtB)-bound GlnK, whereas in extracts from BsGlnK- or AmtB-deficient cells, TnrA is entirely soluble. The presence of 4 mm ATP leads to concomitant solubilization of BsGlnK and TnrA. This ATP-dependent membrane re-localization of TnrA by BsGlnK/AmtB may present a novel mechanism to control the global nitrogen-responsive transcription regulator TnrA in B. subtilis under certain physiological conditions.

Bacillus subtilis↗
PubMed2006

Regulatory protein phosphorylation in Mycoplasma pneumoniae. A PP2C-type phosphatase serves to dephosphorylate HPr(Ser-P).

Among the few regulatory events in the minimal bacterium Mycoplasma pneumoniae is the phosphorylation of the HPr phosphocarrier protein of the phosphotransferase system. In the presence of glycerol, HPr is phosphorylated in an ATP-dependent manner by the HPr kinase/phosphorylase. The role of the latter enzyme was studied by constructing a M. pneumoniae hprK mutant defective in HPr kinase/phosphorylase. This mutant strain no longer exhibited HPr kinase activity but, surprisingly, still had phosphatase activity toward serine-phosphorylated HPr (HPr(Ser-P)). An inspection of the genome sequence revealed the presence of a gene (prpC) encoding a presumptive protein serine/threonine phosphatase of the PP2C family. The phosphatase PrpC was purified and its biochemical activity in HPr(Ser-P) dephosphorylation demonstrated. Moreover, a prpC mutant strain was isolated and found to be impaired in HPr(Ser-P) dephosphorylation. Homologues of PrpC are present in many bacteria possessing HPr(Ser-P), suggesting that PrpC may play an important role in adjusting the cellular HPr phosphorylation state and thus controlling the diverse regulatory functions exerted by the different forms of HPr.

Allosteric Regulation↗
PubMed2006

Expression of CD83 is regulated by HuR via a novel cis-active coding region RNA element.

Dendritic cells are the most potent of the antigen-presenting cells and are characterized by surface expression of CD83. Here, we show that the coding region of CD83 mRNA contains a novel cis-acting structured RNA element that binds to HuR, a member of the ELAV family of AU-rich element RNA-binding proteins. Transient transfection of mammalian cells demonstrated that this CD83 mRNA-derived element acts as a post-transcriptional regulatory element in cells overexpressing HuR. Notably, binding of HuR to the CD83 post-transcriptional regulatory element did not affect mRNA stability. Using RNA interference, we show that HuR mediated efficient expression of CD83. In particular, HuR was required for cytoplasmic accumulation of CD83 transcripts. Likewise, inhibition of the CRM1 nuclear export pathway by leptomycin B or overexpression of a defective form of the nucleoporin Nup214/CAN diminished cytoplasmic CD83 mRNA levels. In summary, the data presented demonstrate that the HuR-CRM1 axis affects the nucleocytoplasmic translocation of CD83 mRNA under regular physiological conditions.

Active Transport, Cell Nucleus↗
PubMed2006

Regulatory links between carbon and nitrogen metabolism.

The metabolism of carbon- and nitrogen-containing compounds is fundamental to all forms of life. To cope with changing environmental conditions, bacteria have to sense the nutrient supply and adapt their metabolism accordingly. In addition to nutrient- and pathway-specific responses, they integrate information from the different branches of metabolism to coordinate the control of the expression of many metabolic genes. Two major players interconnecting carbon and nitrogen regulation are the PII proteins and the phosphotransferase system. Moreover, several DNA-binding transcription regulators sense signals are derived from both carbon and nitrogen metabolism. The regulatory networks enable the bacteria to make the appropriate metabolic responses to changing nutrient availabilities in the environment.

Antigens, Bacterial↗
PubMed2006

Regulation of citB expression in Bacillus subtilis: integration of multiple metabolic signals in the citrate pool and by the general nitrogen regulatory system.

The tricarboxylic acid (TCA) cycle is one of the major routes of carbon catabolism in Bacillus subtilis. The syntheses of the enzymes performing the initial reactions of the cycle, citrate synthase, and aconitase, are synergistically repressed by rapidly metabolizable carbon sources and glutamine. This regulation involves the general transcription factor CcpA and the specific repressor CcpC. In this study, we analyzed the expression and intracellular localization of CcpC. The synthesis of citrate, the effector of CcpC, requires acetyl-CoA. This metabolite is located at a branching point in metabolism. It can be converted to acetate in overflow metabolism or to citrate. Manipulations of the fate of acetyl-CoA revealed that efficient citrate synthesis is required for the expression of the citB gene encoding aconitase and that control of the two pathways utilizing acetyl-CoA converges in the control of citrate synthesis for the induction of the TCA cycle. The citrate pool seems also to be controlled by arginine catabolism. The presence of arginine results in a severe CcpC-dependent repression of citB. In addition to regulators involved in sensing the carbon status of the cell, the pleiotropic nitrogen-related transcription factor, TnrA, activates citB transcription in the absence of glutamine.

Aconitate Hydratase↗
PubMed2005

Dual phosphorylation of Mycoplasma pneumoniae HPr by Enzyme I and HPr kinase suggests an extended phosphoryl group susceptibility of HPr.

In Gram-positive bacteria, the HPr protein of the phosphoenolpyruvate:sugar phosphotransferase system can be phosphorylated at two distinct sites, His-15 and Ser-46. While the former phosphorylation is implicated in phosphoryl transfer to the incoming sugars, the latter serves regulatory purposes. In Bacillus subtilis, the two phosphorylation events are mutually exclusive. In contrast, doubly phosphorylated HPr is present in cell extracts of Mycoplasma pneumoniae. In this work, we studied the ability of the two single phosphorylated HPr species to accept a second phosphoryl group. Indeed, both Enzyme I and the HPr kinase/phosphorylase from M. pneumoniae are able to use phosphorylated HPr as a substrate. The formation of doubly phosphorylated HPr is substantially slower as compared to the phosphorylation of free HPr. However, the rate of formation of doubly phosphorylated HPr is sufficient to account for the amount of HPr(His approximately P)(Ser-P) detected in M. pneumoniae cells.

Bacterial Proteins↗
PubMed2005

Multiple-mutation reaction: a method for simultaneous introduction of multiple mutations into the glpK gene of Mycoplasma pneumoniae.

In Mycoplasma pneumoniae, the UGA opal codon specifies tryptophan rather than a translation stop site. This often makes it difficult to express Mycoplasma proteins in E. coli isolates. In this work, we developed a strategy for the one-step introduction of several mutations. This method, the multiple-mutation reaction, is used to simultaneously replace nine opal codons in the M. pneumoniae glpK gene.

Codon↗
PubMed2005

The RsbRST stress module in bacteria: a signalling system that may interact with different output modules.

In the Gram-positive bacterium Bacillus subtilis, the activity of the alternative sigma factor sigma(B) is triggered upon exposure of the bacteria to environmental stress conditions or to nutrient limitation. sigma(B) activity is controlled by protein-phosphorylation-dependent interactions of anti-sigma with anti-anti-sigma factors. Under stress conditions, the phosphatase RsbU triggers release of sigma(B) and thus induces the expression of stress genes. RsbU activity is controlled by three proteins, RsbR, RsbS and RsbT which form a supramolecular complex called the stressosome. Here we review the occurrence of the genes encoding the stressosome proteins (called the RsbRST module) in a wide variety of bacteria. While this module is linked to the gene encoding sigma(B) and its direct regulators in B. subtilis and its close relatives, genes encoding two-component regulatory systems and more complex phosphorelays are clustered with the RsbRST module in bacteria as diverse as cyanobacteria, bacteroidetes, proteobacteria, and deinococci. The conservation of the RsbRST module and its clustering with different types of regulatory systems suggest that the stressosome proteins form a signal sensing and transduction unit that relays information to very different output modules.

Bacillus subtilis↗
PubMed2004

A protein-dependent riboswitch controlling ptsGHI operon expression in Bacillus subtilis: RNA structure rather than sequence provides interaction specificity.

The Gram-positive soil bacterium Bacillus subtilis transports glucose by the phosphotransferase system. The genes for this system are encoded in the ptsGHI operon. The expression of this operon is controlled at the level of transcript elongation by a protein-dependent riboswitch. In the absence of glucose a transcriptional terminator prevents elongation into the structural genes. In the presence of glucose, the GlcT protein is activated and binds and stabilizes an alternative RNA structure that overlaps the terminator and prevents termination. In this work, we have studied the structural and sequence requirements for the two mutually exclusive RNA structures, the terminator and the RNA antiterminator (the RAT sequence). In both cases, the structure seems to be more important than the actual sequence. The number of paired and unpaired bases in the RAT sequence is essential for recognition by the antiterminator protein GlcT. In contrast, mutations of individual bases are well tolerated as long as the general structure of the RAT is not impaired. The introduction of one additional base in the RAT changed its structure and resulted in complete loss of interaction with GlcT. In contrast, this mutant RAT was efficiently recognized by a different B.subtilis antitermination protein, LicT.

Bacillus subtilis↗
PubMed2004

Mycoplasma pneumoniae HPr kinase/phosphorylase.

HPr kinase/phosphorylase (HPrK/P) is the key regulator of carbon metabolism in many Gram-positive bacteria. It phosphorylates/dephosphorylates the HPr protein of the bacterial phosphotransferase system on a regulatory serine residue in response to the nutrient status of the cell. In Mycoplasma pneumoniae, HPrK/P is one of the very few regulatory proteins encoded in the genome. The regulation of this enzyme by metabolites is unique among HPrK/P proteins studied so far: it is active as a kinase at low ATP concentrations, whereas the proteins from other bacteria need high ATP concentrations as an indicator of a good nutrient supply for kinase activity. We studied the interaction of M. pneumoniae HPrK/P with ATP, Fru1,6P2 and Pi by fluorescence spectroscopy. In agreement with the previously observed unique regulation, we found a very high affinity for ATP (K(d)=5.4 microM) compared with the HPrK/P proteins from other bacteria. The Kd for Fru1,6P2 was three orders of magnitude higher, which explains why Fru1,6P2 has only a weak regulatory effect on M. pneumoniae HPrK/P. Mutations of two important regions in the active site of HPrK/P, the nucleotide binding P-loop and the HPrK/P family signature sequence, had different effects. P-loop region mutations strongly affect ATP binding and thus all enzymatic functions, whereas the signature sequence motif seems to be important for the catalytic mechanism rather than for nucleotide binding.

Adenosine Triphosphate↗
PubMed2004

In vivo activity of enzymatic and regulatory components of the phosphoenolpyruvate:sugar phosphotransferase system in Mycoplasma pneumoniae.

Mycoplasma pneumoniae is a pathogenic bacterium that is highly adapted to life on mucosal surfaces. This adaptation is reflected by the very compact genome and the small number of regulatory proteins. However, M. pneumoniae possesses the HPr kinase/phosphorylase (HPrK/P), the key regulator of carbon metabolism in the Firmicutes. In contrast to the enzymes of other bacteria, the HPrK/P of M. pneumoniae is already active at very low ATP concentrations, suggesting a different mode of regulation. In this work, we studied the ability of M. pneumoniae to utilize different carbohydrates and their effects on the activity of the different phosphotransferase system (PTS) components. Glucose served as the best carbon source, with a generation time of about 30 h. Fructose and glycerol were also used but at lower rates and with lower yields. In contrast, M. pneumoniae is unable to use mannitol even though the bacterium is apparently equipped with all the genes required for mannitol catabolism. This observation is probably a reflection of the continuing and ongoing reduction of the M. pneumoniae genome. The general enzymatic and regulatory components of the PTS, i.e., enzyme I, HPr, and HPrK/P, were present under all growth conditions tested in this study. However, HPrK/P activity is strongly increased if the medium contains glycerol. Thus, the control of HPrK/P in vivo differs strongly between M. pneumoniae and the other Firmicutes. This difference may relate to the specific conditions on lipid-rich cell surfaces.

Bacterial Proteins↗
PubMed2003

Control of the Bacillus subtilis antiterminator protein GlcT by phosphorylation. Elucidation of the phosphorylation chain leading to inactivation of GlcT.

Bacillus subtilis transports glucose by the phosphotransferase system (PTS). The genes for this system are encoded in the ptsGHI operon, which is induced by glucose and depends on a termination/antitermination mechanism involving a riboswitch and the RNA-binding antitermination protein GlcT. In the absence of glucose, GlcT is inactive, and a terminator is formed in the leader region of the ptsG mRNA. If glucose is present, GlcT can bind to its RNA target and prevent transcription termination. The GlcT protein is composed of three domains, an N-terminal RNA binding domain and two PTS regulation domains, PTS regulation domain (PRD) I and PRD-II. In this work, we demonstrate that GlcT can be phosphorylated by two PTS proteins, HPr and the glucose-specific enzyme II (EIIGlc). HPr-dependent phosphorylation occurs on PRD-II and has a slight stimulatory effect on GlcT activity. In contrast, EIIGlc phosphorylates the PRD-I of GlcT, and this phosphorylation inactivates GlcT. This latter phosphorylation event links the availability of glucose to the expression of the ptsGHI operon via the phosphorylation state of EIIGlc and GlcT. This is the first in vitro demonstration of a direct phosphorylation of an antiterminator of the BglG family by the corresponding PTS permease.

Bacillus subtilis↗