Regulatory Networks in Prokaryotes Chapter Abstracts
Chapter 1
Regulatory Networks in Prokaryotes: Variations on a Theme
Bärbel Friedrich
An overview of the subject and an introduction to the book.
Chapter 2
Regulation of Gas Vesicle Formation in Halophilic Archaea
Felicitas Pfeifer, Dagmar Gregor, Annette Hofacker, Petra Plösser and Peter Zimmermann
The halophilic archaea Halobacterium salinarum and Haloferax mediterranei produce gas vesicles depending on the growth phase and on environmental factors such as light, salt, or oxygen. Fourteen different gvp genes (gvpACNO and gvpDEFGHIJKLM) are involved in their formation, and the regulation of gvp gene expression occurs at the transcriptional and translational level. Haloferax volcanii offers a clean genetic background for the functional analysis of gas vesicle genes by transformation experiments. Such experiments show that the promoter of the gvpA gene encoding the major gas vesicle structural protein is activated by the endogenous basic leucine-zipper protein GvpE. On the other hand, the GvpD protein, which contains a p-loop motif, is involved either directly or indirectly in the repression of the gvpA promoter activity. Eight of the fourteen p-gvp genes (p-gvpAO and p-gvpFGJKLM) enable gas vesicle formation in Hf. volcanii transformants and thus constitute the minimal p-vac region.
Chapter 3
Sensory Transduction and Motion Control in Sinorhizobium meliloti
Birgit Scharf and Rüdiger Schmitt
Molecular mechanisms that govern chemotaxis and motility in the nitrogen-fixing soil bacterium, Sinorhizobium meliloti, are distinguished from the well-studied taxis systems of enterobacteria by new features. (i) In addition to six transmembrane chemotaxis receptors, S. meliloti has two cytoplasmic receptor proteins, McpY (methyl-accepting protein Y) and IcpA (internal chemotaxis protein A). (ii) The tactic response is mediated by two response regulators, CheY1 and CheY2, but no phosphatase, CheZ. Phosphorylated CheY2 (CheY2-P) is the main regulator of motor function, whereas CheY1 assumes the role of a sink for phosphate that is shuttled from CheY2-P back to CheA. This phospho-transfer from surplus CheY2-P to CheA to CheY1 replaces CheZ phosphatase. (iii) S. meliloti flagella have a complex structure with three helical ribbons that render the filaments rigid and unable to undergo polymorphic transitions from right- to left-handedness. Flagella rotate only clockwise and their motors can increase and decrease rotary speeds. Hence, directional changes of a swimming cell occur during slow-down, when several flagella rotate at different speed. Two novel motility proteins, the periplasmic MotC and the cytoplasmic MotD, are essential for motility and rotary speed variation. Our data suggest that MotC, by binding to MotB, stabilizes the energizing MotA-MotB proton channel, while MotD (increasing speed) and CheY2-P (decreasing speed) compete for binding to the cytoplasmic surface of the motor (FliM) in an antagonistic fashion, which leads to variations in flagellar rotary speed.
Chapter 4
Regulation of Succinoglycan and Galactoglucan Biosynthesis in Sinorhizobium meliloti
Anke Becker, Silvia Rüberg, Birgit Baumgarth, Peter Alexander Bertram-Drogatz, Ingmar Quester, and Alfred Pühler
Sinorhizobium meliloti (Rhizobium meliloti) 2011 has the ability to produce the two acidic exopolysaccharides succinoglycan (EPS I) and galactoglucan (EPS II). EPS I is a branched heteropolysaccharide composed of octasaccharide repeating units, whereas EPS II is a linear heteropolysaccharide consisting of disaccharide sub-units. The exoexs and exp gene clusters are involved in the biosynthesis of EPS I and EPS II, respectively. EPS I and EPS II biosynthesis genes are differentially expressed resulting in a complex regulation of EPS production in S. meliloti. The phosphate concentration was identified as an important factor affecting the expression of exp genes.-
Chapter 5
Control of Temperature-Responsive Synthesis of the Phytotoxin Coronatine in Pseudomonas syringae by the Unconventional Two-Component System CorRPS
Angela V. Smirnova, Ling Wang, Bettina Rohde, Ina Budde, Helge Weingart, and Matthias S. Ullrich
The phytopathogen Pseudomonas syringae produces the phytotoxin coronatine (COR) as a major virulence factor. COR and its precursor, coronafacic acid, function as molecular mimics of the plant signaling molecule jasmonate. A 32.8-kb plasmid-borne gene cluster mediates COR biosynthesis, which is optimal at 18°C and non-detectable at 28°C, the optimal growth temperature for P. syringae. The thermoregulation is mediated at the transcriptional level by an unconventional two-component regulatory system consisting of a histidine protein kinase, CorS, and two transcriptional activators, CorR and CorP. Dissection of this regulatory triad revealed that CorR binds to its target sequences in a thermoresponsive manner and that its DNA-binding activity is controlled by CorS. A Preliminary model for thermo-sensing by CorS is proposed based on its membrane topology and the analysis of translational fusions of CorS to reporter enzymes at different temperatures. CorP lacks a typical helix-turn-helix motif but possibly functions as a modulator of CorR or CorS activity. The thermoregulation of COR biosynthetic genes is widespread among various COR-producing P. syringae strains. Post-translational processes also contribute to the thermo-responsiveness of COR production. Additionally, COR synthesis in P. syringae is influenced by nutrient availability, rpoN encoding the alternative sigma factor s54, and HrpV, a negative regulator of hrp gene expression, suggesting a complex regulatory network governing phytotoxin synthesis.
Chapter 6
Virulence Gene Regulation in Bordetella pertussis
Jochen König, Andreas Bock, Anne-Laure Perraud, Thilo M. Fuchs, Dagmar Beier, and Roy Gross
Most pathogenic bacteria encounter changing growth conditions during their infectious cycle and, accordingly, have to modulate gene expression to enable the efficient colonization of different environments outside or within their host organisms. In Bordetella pertussis the transcription of most virulence factors including several toxins and adhesins is regulated coordinately by the BvgAS two-component system. The molecular characterization of the BvgAS system revealed that it belongs to the small group of unorthodox two-component systems applying an obligate multistep phosphorelay. Moreover, despite the coordinated control of the virulence regulon, subtle differences in the regulation of individual virulence genes were observed which led to the identification of sophisticated mechanisms possibly engaged in fine tuning of virulence gene expression.
Chapter 7
Influence of the leuX-Encoded tRNA
Ulrich Dobrindt, Katharine Piechaczek, Angelika Schierhorn, Gunter Fischer, Michael Hecker and Jörg Hacker
The leuX gene encoding the minor tRNA5Leu is important for the expression of several virulence factors of pathogenic Escherichia coli strains. The differential usage of minor codons to control the expression of specialized genes has been proposed to be a general mechanism of bacteria to regulate gene expression at the posttranscriptional level. The minor codon usage theory foots on the biased codon usage of bacterial genes and the selective availability of tRNA isoacceptors. We aimed at the further investigation of the regulatory role of the tRNA5Leu for gene expression in pathogenic E. coli. For this purpose, the molecular mechanism underlying the tRNA5Leu-dependent regulation of different virulenceassociated genes of pathogenic E. coli as well as the regulation of leuX transcription under various growth conditions were investigated in detail. The global impact of the presence or absence of the leuX-encoded tRNA on gene expression of the uropathogenic E. coli strain 536 was studied by proteome analysis. The obtained results argue for a general importance of the tRNA5Leu for gene expression of E. coli and the involvement of this tRNA in global regulatory networks.-
Chapter 8
The ciaR/ciaH System of Streptococcus pneumoniae is Involved in Beta-Lactam Resistance and Genetic Competence
Dorothea Zähner, Kristina Kaminski, Mark van der Linden, Thorsten Mascher, Michelle Merai, and Regine Hakenbeck
New mechanisms for b-lactam resistance independent on the target penicillin-binding proteins were detected in b-lactam-resistant laboratory mutants of Streptococcus pneumoniae. The link between mutations in the histidine protein kinase CiaH and phenotypic expression of cefotaxime resistance suggests that the cell is able to monitor the integrity of the cell wall and in emergency cases such as during the action of b-lactams can counteract such danger. At least one ciaH mutation Thr230 > Pro is likely to affect its phosphatase activity resulting in elevated phosphorylation of CiaR, the cognate response regulator, but other CiaH-independent signaling pathways may also result in CiaR phosphorylation. Mutants in CiaH, either alone or in combination with a mutated penicillin-binding protein 2x(PBP2x) fail to develop genetic competence. In all cases complementation of this phenotype was observed upon addition of the competence inducing pheromone peptide CSP, the processed product of the comC gene. This indicates that the cia system is part of a regulatory network that includes another two component system comDE. The DNA binding property of CiaR and ComE were exploited to isolate specifically interacting DNA fragments as a first step to identify genes targeted by individual response regulators.
Chapter 9
Members of the Fur Protein Family Regulate Iron and Zinc Transport in E. coli and Characteristics of the Fur-Regulated FhuF Protein
Klaus Hantke
The regulator Fur represses with Fe2+ as cofactor iron uptake genes. The fhuF gene reacts very sensitive to minor changes of Fe2+ and Fur. It is assumed that FhuF helps in the mobilisation of iron out of the hydroxamate siderophores transported into the cell. Analysis of the protein revealed an unusual [2Fe-2S] cluster bound to a Cys-CysX10-Cys-X2-Cys motif in FhuF. suf genes responsible for the synthesis of the iron sulfur center were identified. The Zur protein shows 27% identity to the Fur protein of E. coli. It regulates as a repressor the high affinity uptake system znuACB. Only two additional Zur binding sites in the promoter region of genes with unknown function were found. Properties of Zur and Fur proteins from different bacteria are compared.-
Chapter 10
Stimulus Perception and Signal Transduction by the KdpD/KdpE System of Escherichia coli
Kirsten Jung and Karlheinz Altendorf
The membrane-bound histidine kinase KdpD is a putative turgor sensor that regulates, together with the response regulator KdpE, expression of the kdpFABC operon. This operon encodes the high affinity K+-uptake system KdpFABC of Escherichia coli. Expression of kdpFABC is induced under K+-limiting growth conditions and in response to an osmotic upshift. Various structural features of KdpD and KdpE, which are important for stimulus perception and/or signal transduction were identified and are described here. Furthermore, various studies undertaken to elucidate the nature of the stimulus for KdpD result in a new model for KdpD stimulus perception. According to this, autophosphorylation activity of KdpD is not a result of changes in turgor per se. Instead, various -- mainly intracellular parameters -- that are related to changes of environmental conditions influence the activities of KdpD.
Chapter 11
Mechanism of Regulation of the Bifunctional Histidine Kinase NtrB in Escherichia coli
Verena Weiss, Günter Kramer, Thomas Dünnebier, and Annette Flotho
NtrB is the bifunctional histidine kinase for nitrogen regulation. Dependent on the availability of nitrogen, it either autophosphorylates and serves as the phosphodonor for its cognate response regulator, NtrC, or, it promotes the rapid dephosphorylation of NtrC-P. The activity of NtrB depends on the interaction of two subdomains within its transmitter domain, the H-domain and the kinase domain. Both phosphotransfer activity and phosphatase activity reside in the H-domain. When separately expressed, this domain acts as a phosphatase. Interaction with the kinase domain results in the inhibition of the phosphatase activity and the phosphorylation of the conserved histidine of the H-domain.
Chapter 12
Regulation of Nitrogen Fixation in Klebsiella pneumoniae and Azotobacter vinelandii: NifL, Transducing Two Environmental Signals to the nif Transcriptional Activator NifA
Ruth A. Schmitz, Kai Klopprogge, Roman Grabbe, and Jessica Stips
The enzymatic reduction of molecular nitrogen to ammonia requires high amounts of energy, and the presence of oxygen causes the catalyzing nitrogenase complex to be irreversible inactivated. Thus nitrogen-fixing microorganisms tightly control both the synthesis and activity of nitrogenase to avoid the unnecessary consumption of energy. In the free-living diazotrophs Klebsiella pneumoniae and Azotobacter vinelandii, products of the nitrogen fixation nifLA operon regulate transcription of the other nif operons. NifA activates transcription of nif genes by the alternative form of RNA-polymerase, s54-holoenzyme; NifL modulates the activity of the transcriptional activator NifA in response to the presence of combined nitrogen and molecular oxygen. The translationally-coupled synthesis of the two regulatory proteins, in addition to evidence from studies of NifL/NifA complex formation, imply that the inhibition of NifA activity by NifL occurs via direct protein-protein interaction in vivo. The inhibitory function of the negative regulator NifL appears to lie in the C-terminal domain, whereas the N-terminal domain binds FAD as a redox-sensitive cofactor, which is required for signal transduction of the internal oxygen status. Recently it was shown, that NifL acts as a redox-sensitive regulatory protein, which modulates NifA activity in response to the redox-state of its FAD cofactor, and allows NifA activity only in the absence of oxygen. In K. pneumoniae, the primary oxygen sensor appears to be Fnr (fumarate nitrate reduction regulator), which is presumed to transduce the signal of anaerobiosis towards NifL by activating the transcription of gene(s) whose product(s) function to relieve NifL inhibition through reduction of the FAD cofactor. In contrast, the reduction of A. vinelandii-NifL appears to occur unspecifically in response to the availability of reducing equivalents in the cell. Nitrogen status of the cells is transduced towards the NifL/NifA regulatory system by the GlnK protein, a paralogue PII-protein, which appears to interact with the NifL/NifA regulatory system via direct protein-protein interaction. It is not currently known whether GlnK interacts with NifL alone or affects the NifL/NifA-complex; moreover the effects appear to be the opposite in K. pneumoniae and A. vinelandii. In addition to these environmental signals, adenine nucleotides also affect the inhibitory function of NifL; in the presence of ATP or ADP the inhibitory effect on NifA activity in vitro is increased. The NifL proteins from the two organisms differ, however, in that stimulation of K. pneumoniae-NifL occurs only when synthesized under nitrogen excess, and is correlated with the ability to hydrolyze ATP. In general, transduction of environmental signals to the nif regulatory system appears to involve a conformational change of NifL or the NifL/NifA complex. However, experimental data suggest that K. pneumoniae and A. vinelandii employ significantly different species-specific mechanisms of signal transduction.
Chapter 13
Regulation of Synthesis and Activity of Molybdenum Nitrogenase and the Alternative Nitrogenase in Rhodobacter capsulatus
Thomas Drepper, Bernd Masepohl, Annette Paschen, Silke Gross, Alice Pawlowski, Karsten Raabe, Kai-Uwe Riedel, and Werner Klipp
In R. capsulatus synthesis and activity of both molybdenum nitrogenase and the alternative nitrogenase is controlled at three levels by the environmental factors ammonium, molybdenum, light, and oxygen. At the first level, transcription of the nifA1, nifA2, and anfA genes - which encode the transcriptional activators of all other nif and anf genes, respectively - is controlled by the Ntr system in dependence on ammonium availability. Mutations in glnB (coding for the signal transduction protein PII) result in significant expression of nifA and anfA in the presence of ammonium. In contrast to GlnB, the PII-paralogue GlnK is not involved in the Ntr signal transduction mechanism. In addition to ammonium control, transcription of anfA is inhibited by traces of molybdenum via the molybdate-dependent repressor proteins MopA and MopB. At the second level of regulation, activity of NifA1, NifA2, and AnfA is inhibited by ammonium in an NtrC-independent manner. The post-translational ammonium control of NifA activity is partially released in the absence of GlnK, and completely abolished in a glnB/glnK double mutant. In contrast, AnfA activity is still inhibited by ammonium in the glnB/glnK mutant background. At the third level of regulation, both GlnB and GlnK as well as the (methyl)-ammonium transporter AmtB are involved in ammonium control of the DraT/DraG system, which mediates reversible ADPribosylation of both nitrogenase reductases (NifH and AnfH) in response to changes in ammonium availability or light intensity. Most remarkably, in a glnB/glnK double mutant ammonium control of the molybdenum (but not of the alternative) nitrogenase is completely relieved, leading to synthesis of active nitrogenase in the presence of high concentrations of ammonium.-
Chapter 14
Oxygen-Regulated Expression of Genes for Pigment Binding Proteins in Rhodobacter capsulatus
Jutta Gregor and Gabriele Klug
Oxygen is the major external factor affecting the expression of photosynthesis genes in facultatively photosynthetic bacteria. Many investigations over the last years mainly carried out on the closely related species Rhodobacter capsulatus and Rhodobacter sphaeroides have identified a number of proteins involved in the oxygen-regulated signal pathway, in which the RegB/RegA two component system plays a central role. While the RegB/RegA system activates photosynthesis genes under low oxygen tension other proteins like CrtJ and PPBP have a repressing function under high oxygen tension. Additional DNA binding proteins like the integration host factor can modulate the expression of photosynthesis genes. The role of alternative sigma factors in this signal pathway is still unclear.
Chapter 15
The Hydrogen-Sensing Apparatus in Ralstonia eutropha
Oliver Lenz, Michael Bernhard, Thorsten Buhrke, Edward Schwartz, and Bärbel Friedrich
Molecular hydrogen is widely used by microorganisms as a source of energy. One of the best studied aerobic hydrogen oxidizers, the b-proteobacterium Ralstonia eutropha (formerly Alcaligenes eutrophus), harbors two distinct [NiFe]-hydrogenases which catalyze the heterolytic cleavage of H
Chapter 16
Control of the O2 Sensor/Regulator FNR of Escherichia coli by O2 and Reducing Agents In Vivo and In Vitro
S. Achebach, Y. Zeuner, and G. Unden
The synthesis of the enzymes constituting the electron transport chain of Escherichia coli is controlled by electron acceptors in order to achieve high ATP yields and high metabolic rates as well. High ATP yields (or efficiency) are obtained by the use of electron acceptors for respiration which allow high ATP yields, preferentially O2, and nitrate in the absence of O2. The rate of metabolism is adjusted by use of respiratory isoenzymes which differ in the rate and the efficiency of energy conservation, such as the non-coupling NADH dehydrogenase II (ndh gene) and the coupling NADH dehydrogenase I (nuo genes). By combination of the contrary principles (rate versus efficiency), growth is optimized for growth yields and rates. One of the major transcriptional regulators controlling the switch from aerobic to anaerobic respiration is FNR (fumarate nitrate reductase regulator). FNR is located in the cytoplasm and contains a [4Fe-4S] cluster in the active (anaerobic) state. By reaction with O2 the cluster is converted to a [2Fe-2S] cluster and finally to apoFNR. O2 diffuses into the cytoplasm even at very low O2-tensions (1 µM) where it inactivates [4Fe-4S].FNR. In vitro, glutathione or other thiol compounds can be used as reducing agents for the reconstitution of [4Fe4S].FNR. A model for the control of the functional state of FNR by O2 and glutathione or other reducing agents is discussed. According to this model the functional state of FNR is determined by a (rapid) inactivation of FNR by O2, and a slow (constant) reactivation or reconstitution of [4Fe-4S].FNR by reducing agents like glutathione.-
Chapter 17
The Molecular Biology of Formate Metabolism in Enterobacteria
Susanne Leonhartsberger, Ingrid Korsa, and August Böck
Formate is the signature compound in the anaerobic metabolism of Escherichia coli and other enterobacteria. Its synthesis and degradation is integrated in a network of metabolic routes which are elegantly regulated to adjust the carbon flux to the metabolic needs. This review summarises the information on the biochemistry of synthesis and degradation of formate, on the genetics of the members of the regulon and on the mechanism underlying the regulation.-
Chapter 18
Nitric Oxide Signaling and NO Dependent Transcriptional Control in Bacterial Denitrification by Members of the FNR-CRP Regulator Family
Walter G. Zumft
Bacterial denitrification transforms nitrate to dinitrogen. The process is expressed facultatively in response to environmental conditions. Around 50 components make up the denitrification apparatus and its assembly pathways. We are beginning to understand how exogenous signals provided by oxygen and N oxides are processed for activating the underlying gene programs. Key signals are provided by nitrate, nitric oxide, and a low oxygen tension. In the genus Pseudomonas the nitrate signal is processed by a two component regulatory system which activates the nar operon encoding respiratory nitrate reductase. Nitric oxide is not only an essential respiratory substrate of the denitrifying cell but constitutes in nanomolar concentrations also a key signal for the expression of nitrite reductase and NO reductase which control cellular NO homeostasis. The signal pathway in the genera Pseudomonas, Paracoccus and Rhodobacter involves regulators of the FNR family of transcription factors, which cluster phylogenetically in a separate subgroup. In contrast, Ralstonia eutropha requires a sigma-54 dependent regulator of the NtrC family for the expression of its quinol-dependent NO reductase. Important questions are directed currently at the mechanism(s) of activation of these transcription factors by NO, and avoidance of crosstalk with FNR factors at target promoters operating with identical recognition motifs.
Chapter 19
Regulation of Heme Biosynthesis in Non-Phototrophic Bacteria
Max Schobert and Dieter Jahn
The biosynthesis of tetrapyrroles like hemes and chlorophylls is essential for most living organisms. In bacteria hemes are integral parts of energy conserving electron transport chains and cofactors of various enzymes. Changes of environmental conditions usually lead to an adaption of the bacterial energy metabolism and often coincide with significant changes of cellular heme levels. This review focuses on the known regulatory mechanisms in non-phototrophic bacteria involved in the control of the formation of the heme biosynthetic apparatus. Species specific differences in the mode of energy generation result in various regulatory strategies. Focusing on the well investigated bacteria Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, and Salmonella typhimurium the involved environmental stimuli, employed transcriptional regulators and promoter structures as well as the role of protein stability are described. Broad variations of the used regulatory principles were observed.
Chapter 20
Multiple Levels of Regulation of Solventogenesis in Clostridium acetobutylicum
Brigitte Zickner, Michael Böhringer, Stephan Nakotte, Steffen Schaffer, Kai Thormann, and Peter Dürre
Solvent synthesis in Clostridium acetobutylicum is induced in concert with sporulation to counteract the dangerous effects of produced butyric and acetic acids and to provide the cell with sufficient time to complete endospore formation. Cardinal transcription units for butanol and acetone production are the sol and adc operons encoding butyraldehyde/butanol dehydrogenase and coenzyme A transferase as well as acetoacetate decarboxylase. Induction is achieved by a decreased level of DNA supercoiling and the transcription factor Spo0A, possibly in cooperation with other regulatory proteins. A number of other operons is also turned on during this metabolic switch, whose physiological relevance, however, is only partly understood. The recent completion of C. acetobutylicum genome sequencing will pave the way for transcriptional profiling and thus allow comprehension of the coherent regulatory networks of solventogenesis and sporulation.
Chapter 21
Network Regulation of the Escherichia coli Maltose System
Anja Schlegel, Alex Böhm, Sung-Jae Lee, Ralf Peist, Katja Decker and Winfried Boos
The genes of the Escherichia coli maltose regulon are controlled by MalT, the specific transcriptional activator which, together with the inducer maltotriose and ATP, is essential for mal gene transcription. Network regulation in this system affects the function of MalT and occurs on two levels. The first concerns the expression of malT. It has long been known that malT is under catabolite repression and thus under the control of the cAMP/CAP complex. We found that, in addition, the global regulator Mlc is a repressor for malT transcription. The repressor activity of Mlc is controlled by the transport status of the glucosespecific enzyme EIICB of the PTS that causes sequestration (and inactivation as a repressor) of Mlc when glucose is transported. The second level of MalT regulation affects its activity. MalT is activated by maltotriose which is not only formed when the cells are growing on any maltodextrin but also, in low amounts, endogenously when the cells grow on non-maltodextrin carbon sources. Thus, cellular metabolism, for instance degradation of galactose or trehalose, can cause mal gene induction. It was found that unphosphorylated internal glucose takes part in endogenous maltodextrin biosynthesis and is therefore a key element in endogenous mal gene expression. In addition to the maltotriose-dependent activation, MalT can interact with three different enzymes that lead to its inactivation as a transcriptional activator. The first is MalK, the energy transducing ABC subunit of the maltodextrin transport system. Transport controls the interaction of MalK and MalT thus affecting gene expression. The second enzyme is MalY, a pyridoxal phosphate containing enzyme exhibiting cystathionase activity. The crystal structure of MalY was established and mutations in MalY that reduce mal gene repression map in a hydrophobic MalT interaction patch on the surface of the enzyme. The last enzyme is a soluble esterase of as yet unknown function. When overproduced, this enzyme specifically reduces mal gene expression and affects the activity of MalT in an in vitro transcription assay.-
Chapter 22
Catabolite Control Protein CcpA-Dependent Glucose Repression in Staphylococcus xylosus: Efficient Activation of CcpA by Glucose Transported Independently from the Phosphotransferase System
Ivana Jankovic, Jess Meyer, and Reinhold Brückner
Carbon catabolic repression (CR) by the catabolite control protein CcpA has been analyzed in Staphylococcus xylosus. Genes encoding components needed to utilize lactose, sucrose, and maltose were found to be repressed by CcpA. In addition, the ccpA gene is under negative autogenous control. Among several tested sugars, glucose caused strongest CcpA-dependent repression. Glucose can enter S. xylosus in nonphosphorylated form via the glucose uptake protein GlcU. Internal glucose is then phosphorylated by the glucose kinase GlkA. Alternatively, glucose can be transported and concomitantly phosphorylated by glucose-specific permease(s) of the phosphotransferase system (PTS). S. xylosus mutant strains deficient in GlcU or GlkA showed partial relief of glucosespecific, CcpA-dependent repression. Likewise, blocking PTS activity completely by inactivation of the gene encoding the general PTS protein enzyme I resulted in diminished glucose-mediated repression. Thus, both glucose entry routes contribute to glucose-specific CR in S. xylosus. The sugar transport activity of the PTS is not required to trigger glucose-specific repression. The phosphocarrier protein HPr however, is absolutely essential for CcpA activity. Inactivation of the HPr gene led to a complete loss of CR. Repression is also abolished upon inactivation of the HPr kinase gene or by replacing serine at position 46 of HPr by alanine. These results clearly show that HPr kinase provides the signal, seryl-phosphorylated HPr, to activate CcpA in S. xylosus.-
Chapter 23
Carbon Catabolite Repression in Bacillus subtilis: Mechanisms Beside the Main Regulator CcpA
Michael K. Dahl
The past decade has witnessed an exiting unveiling of numerous molecular mechanisms that characterize signal transduction by protein-protein interaction. The recent findings encouraged an increasing effort to understand the sequential metabolism of different sugars available as energy sources at the same time. It seems probable that at least three principle mechanisms which act together or separately, mediate carbon catabolite repression (CCR) depending on the system which is under metabolic control: i) by the main signal transducing chain via the ATP-dependent HPr-kinase, HPr(Ser46~P) or alternatively Crh via the central component CcpA and its interaction with cre, ii) by signals sensed from the specific regulators directly or via phosphorylation by HPr, iii) by inducer exclusion based on the concurrence of the enzyme IIAGlc domain of the glucose permease, and other PTS-dependent permeases composed only of the B and C domains and lacking the enzyme IIA domain.
Chapter 24
Structure-Function Relationship and Regulation of Two Bacillus subtilis DNA-Binding Proteins, HBsu and AbrB
Wolfgang Klein, and Mohamed A. Marahiel
Microorganisms use a number of small basic proteins for organization and compaction of their DNA. By their interaction with the genome, these proteins do have a profound effect on gene expression, growth behavior, and viability. It has to be distinguished between indirect effects as a consequence of the state of chromosome condensation and relaxation that influence the rate of RNA polymerase action as represented by the histone-like proteins, and direct effects by specific binding of proteins to defined DNA segments predominantly located around promoter sequences. This latter class is represented by the transition-state regulators that are involved in integrating various global stimuli and orchestrating expression of the genes under their regulation for a better adaptation to changes in growth rate. In this article we will focus on two different but abundant DNA binding proteins of the gram-positive model organism Bacillus subtilis, the histone-like HBsu as a member of the unspecific and the transition state regulator AbrB as a member of specific classes of DNA binding proteins.
Chapter 25
Regulation of Ribosomal RNA Synthesis in E. coli: Effects of the Global Regulator Guanosine Tetraphosphate (ppGpp)
Rolf Wagner
The global regulatory nucleotides (p)ppGpp are major effectors for the control of ribosomal RNA in bacteria. The effector molecules accumulate to different cellular levels at amino acid deprivation or during different growth rates. They change the activity of RNA polymerase to transcribe from sensitive promoters (e.g. ribosomal RNA promoters). Sensitive promoters are characterized by a GC-rich discriminator element in addition to further structural requirements not completely understood. ppGpp must also be regarded as a mediator for growth rate control although it appears that ppGppindependent regulatory mechanisms exist. Inhibition occurs at various steps during initiation but also during elongation where RNA polymerase pausing is observed. From the existing data a mechanistic model for the action of ppGpp is suggested considering structural details of RNA polymerase obtained at high resolution.-
Chapter 26
The General Stress Response Regulatory Network in Escherichia coli
Regine Hengge-Aronis
Many bacterial species exhibit a general stress response that can be induced by numerous very different stress conditions and, phenotypically, renders the cells broadly stress resistant. In Escherichia coli, this response is dependent on the sS (RpoS) subunit of RNA polymerase. sS is a close relative of the vegetative sigma factor s70 (RpoD) and recognizes very similar promoter sequences. In recent years, significant progress has been made with respect to elucidating (i) the molecular mechanisms that control the cellular sS level, which include translational regulation as well as intricate control of sS proteolysis, and (ii) the molecular function of sS as a transcription initiation factor, where a number of sS-dependent promoters have been studied in great detail, and the mechanisms that generate sS selectivity are now becoming apparent.
Chapter 27
Positive Regulation of Gene Expression by the Catabolite Control Protein CcpA in Bacillus subtilis
Holger Ludwig, Hans-Matti Blencke, Matthias Schmalisch, Christian Detsch, Matthias Merzbacher and Jörg Stülke
In Bacillus subtilis and other Gram-positive bacteria, carbon catabolite control is mediated by the pleiotropic regulatory protein CcpA. In addition to loss of catabolite repression, ccpA mutants exhibit a severe growth defect. This growth defect may result from loss of expression of several genes that are activated by CcpA. Gene activation by CcpA has been studied at different levels such as proteome and transcriptome analysis and by investigation of the regulation of individual genes in wild type and ccpA mutant strains. Important cellular functions such as glycolysis, overflow metabolism to excrete excess carbon from the cell, and ammonium assimilation depend on a functional CcpA. While CcpA can act directly as a transcriptional activator to allow expression of ackA and pta genes, its role is indirect for genes of glycolysis. In this case, the accumulation of an intracellular inducer cannot occur in ccpA mutants due to a defect in sugar transport by the phosphoenolpyruvate:sugar phosphotransferase system. Several mutations were isolated that exhibit loss of catabolite repression due to the ccpA mutation but that do not cause a growth defect. These mutations were identified within the ccpA gene or are extragenic suppressors.
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