Gerald Seidel

Interference of Components of the Phosphoenolpyruvate Phosphotransferase System with the Central Virulence Gene Regulator PrfA of Listeria monocytogenes

Analysis of Listeria monocytogenes ptsH, hprK, and ccpA mutants defective in carbon catabolite repression (CCR) control revealed significant alterations in the expression of PrfA-dependent genes. The hprK mutant showed high up-regulation of PrfA-dependent virulence genes upon growth in glucose-containing medium whereas expression of these genes was even slightly down-regulated in the ccpA mutant compared to the wild-type strain. The ptsH mutant could only grow in a rich culture medium, and here the PrfA-dependent genes were up-regulated as in the hprK mutant. As expected, HPr-Ser-P was not produced in the hprK and ptsH mutants and synthesized at a similar level in the ccpA mutant as in the wild-type strain. However, no direct correlation was found between the level of HPr-Ser-P or HPr-His-P and PrfA activity when L. monocytogenes was grown in minimal medium with different phosphotransferase system (PTS) carbohydrates. Comparison of the transcript profiles of the hprK and ccpA mutants with that of the wild-type strain indicates that the up-regulation of the PrfA-dependent virulence genes in the hprK mutant correlates with the down-regulation of genes known to be controlled by the efficiency of PTS-mediated glucose transport. Furthermore, growth in the presence of the non-PTS substrate glycerol results in high PrfA activity. These data suggest that it is not the component(s) of the CCR or the common PTS pathway but, rather, the component(s) of subsequent steps that seem to be involved in the modulation of PrfA activity.

Quantitative interdependence of coeffectors, CcpA and cre in carbon catabolite regulation of Bacillus subtilis.

The phosphoproteins HPrSerP and CrhP are the main effectors for CcpA‐mediated carbon catabolite regulation (CCR) in Bacillus subtilis. Complexes of CcpA with HPrSerP or CrhP regulate genes by binding to the catabolite responsive elements (cre). We present a quantitative analysis of HPrSerP and CrhP interaction with CcpA by surface plasmon resonance (SPR) revealing small and similar equilibrium constants of 4.8 ± 0.4 µm for HPrSerP–CcpA and 19.1 ± 2.5 µm for CrhP–CcpA complex dissociation. Forty millimolar fructose‐1,6‐bisphosphate (FBP) or glucose‐6‐phosphate (Glc6‐P) increases the affinity of HPrSerP to CcpA at least twofold, but have no effect on CrhP–CcpA binding. Saturation of binding of CcpA to cre as studied by fluorescence and SPR is dependent on 50 µm of HPrSerP or > 200 µm CrhP. The rate constants of HPrSerP–CcpA–cre complex formation are ka = 3 ± 1 × 106 m−1·s−1 and kd = 2.0 ± 0.4 × 10−3·s−1, resulting in a KD of 0.6 ± 0.3 nm. FBP and Glc6‐P stimulate CcpA–HPrSerP but not CcpA‐CrhP binding to cre. Maximal HPrSerP‐CcpA–cre complex formation in the presence of 10 mm FBP requires about 10‐fold less HPrSerP. These data suggest a specific role for FBP and Glc6‐P in enhancing only HPrSerP‐mediated CCR.

Quantification of the Influence of HPrSer46P on CcpA–cre Interaction

Carbon catabolite repression (CCR) of the Bacillus megateriumxyl operon is dependent on the catabolite responsive element cre, the catabolite control protein (CcpA) and the histidine-containing phosphocarrier protein phosphorylated at the serine 46 residue (HPrSer46P). The latter is formed in the presence of glucose and mediates CCR via CcpA. We present evidence for the presence of HPrSer46P in a ternary complex with CcpA and cre. We also demonstrate increased stability of this complex compared to the CcpA-cre complex by electrophoretic mobility shift analysis (EMSA). This stabilization by HPrSer46P is the same for the xyl cre and an improved cre. Thus, HPrSer46P is a co-repressor for CcpA. In addition, surface plasmon resonance (SPR) experiments yielded binding constants of CcpA and the CcpA-HPrSer46P complex with cre. HPrSer46P stimulated CcpA binding to cre 50-fold. The binding constant is 4.9(+/- 0.5) x 10(6) M(-1). Non-phosphorylated HPr did not affect the complex formation between CcpA and cre. Previously proposed effects by glucose-6-phosphate, fructose-1,6-diphosphate and NADP on CcpA-cre or CcpA-HPrSer46P-cre formation were not found in EMSA and SPR experiments.

Phosphoprotein Crh-Ser46-P displays altered binding to CcpA to effect carbon catabolite regulation.

In Gram-positive bacteria, the catabolite control protein A (CcpA) functions as the master transcriptional regulator of carbon catabolite repression/regulation (CCR). To effect CCR, CcpA binds a phosphoprotein, either HPr-Ser46-P or Crh-Ser46-P. Although Crh and histidine-containing protein (HPr) are structurally homologous, CcpA binds Crh-Ser46-P more weakly than HPr-Ser46-P. Moreover, Crh can form domain-swapped dimers, which have been hypothesized to be functionally relevant in CCR. To understand the molecular mechanism of Crh-Ser46-P regulation of CCR, we determined the structure of a CcpA-(Crh-Ser46-P)-DNA complex. The structure reveals that Crh-Ser46-P does not bind CcpA as a dimer but rather interacts with CcpA as a monomer in a manner similar to that of HPr-Ser46-P. The reduced affinity of Crh-Ser46-P for CcpA as compared with that of HPr-Ser46 P is explained by weaker Crh-Ser46-P interactions in its contact region I to CcpA, which causes this region to shift away from CcpA. Nonetheless, the interface between CcpA and helix α 2 of the second contact region (contact region II) of Crh-Ser46-P is maintained. This latter finding demonstrates that this contact region is necessary and sufficient to throw the allosteric switch to activate cre binding by CcpA.

High- and low-affinity cre boxes for CcpA binding in Bacillus subtilis revealed by genome-wide analysis

BackgroundIn Bacillus subtilis and its relatives carbon catabolite control, a mechanism enabling to reach maximal efficiency of carbon and energy sources metabolism, is achieved by the global regulator CcpA (carbon catabolite protein A). CcpA in a complex with HPr-Ser-P (seryl-phosphorylated form of histidine-containing protein, HPr) binds to operator sites called catabolite responsive elements, cre. Depending on the cre box position relative to the promoter, the CcpA/HPr-Ser-P complex can either act as a positive or a negative regulator. The cre boxes are highly degenerate semi-palindromes with a lowly conserved consensus sequence. So far, studies aimed at revealing how CcpA can bind such diverse sites were focused on the analysis of single cre boxes. In this study, a genome-wide analysis of cre sites was performed in order to identify differences in cre sequence and position, which determine their binding affinity.ResultsThe transcriptomes of B. subtilis cultures with three different CcpA expression levels were compared. The higher the amount of CcpA in the cells, the more operons possessing cre sites were differentially regulated. The cre boxes that mediated regulation at low CcpA levels were designated as strong (high affinity) and those which responded only to high amounts of CcpA, as weak (low affinity). Differences in the sequence and position in relation to the transcription start site between strong and weak cre boxes were revealed.ConclusionsCertain residues at specific positions in the cre box as well as, to a certain extent, a more palindromic nature of cre sequences and the location of cre in close vicinity to the transcription start site contribute to the strength of CcpA-dependent regulation. The main factors contributing to cre regulatory efficiencies, enabling subtle differential control of various subregulons of the CcpA regulon, are identified.

CcpA forms complexes with CodY and RpoA in Bacillus subtilis.

The Bacillus subtilis catabolite control protein A (CcpA) is a global transcriptional regulator that is controlled by interactions with the phosphoproteins histidine‐containing protein (HPr)Ser46P and the catabolite responsive HPr (Crh)Ser46P and with low molecular weight effectors, depending on the availability of preferred carbon sources such as glucose. Distinct point mutations in CcpA abolish the regulation of some but not all target genes, suggesting additional interactions of CcpA. Therefore, in vivo crosslinking and MS were applied to identify CcpA complexes active in repression and activation. To compensate for an excess of promoters only repressed by CcpA, this experiment was accomplished with cells using multiple copies of the activated ackA promoter. Among the identified proteins HPr, RNA polymerase subunits and the global regulator transcriptional pleiotropic repressor (CodY) were observed. Bacterial two‐hybrid assays combining each RNA polymerase subunit with CcpA localized CcpA binding at the α‐subunit of the RNA polymerase (RpoA). In vivo crosslinking combined with immunoblot analyses revealed CcpA–RpoA complexes in cultures with or without glucose, whereas CcpA–HPr and CcpA–CodY complexes occurred only or predominantly in cultures with glucose. Surface plasmon resonance analyses confirmed the binding of CcpA to the N‐terminal domain (αNTD) and C‐terminal domain (αCTD) of RpoA, as well as to CodY. Furthermore, interactions of CodY with the αNTD and the αCTD were detected by surface plasmon resonance. The KD values of complexes of CcpA or CodY with the αNTD or the αCTD are in the range 5–8 μm. CcpA and CodY form a loose complex with a KD of 60 μm. These data were combined to propose a model for a transcription initiation complex at the ackA promoter.

Species-Specific Differences in the Activity of PrfA, the Key Regulator of Listerial Virulence Genes

PrfA, the master regulator of LIPI-1, is indispensable for the pathogenesis of the human pathogen Listeria monocytogenes and the animal pathogen Listeria ivanovii. PrfA is also present in the apathogenic species Listeria seeligeri, and in this study, we elucidate the differences between PrfA proteins from the pathogenic and apathogenic species of the genus Listeria. PrfA proteins of L. monocytogenes (PrfA(Lm) and PrfA*(Lm)), L. ivanovii (PrfA(Li)), and L. seeligeri (PrfA(Ls)) were purified, and their equilibrium constants for binding to the PrfA box of the hly promoter (Phly(Lm)) were determined by surface plasmon resonance. In addition, the capacities of these PrfA proteins to bind to the PrfA-dependent promoters Phly and PactA and to form ternary complexes together with RNA polymerase were analyzed in electrophoretic mobility shift assays, and their abilities to initiate transcription in vitro starting at these promoters were compared. The results show that PrfA(Li) resembled the constitutively active mutant PrfA*(Lm) more than the wild-type PrfA(Lm), whereas PrfA(Ls) showed a drastically reduced capacity to bind to the PrfA-dependent promoters Phly and PactA. In contrast, the efficiencies of PrfA(Lm), PrfA*(Lm), and PrfA(Li) forming ternary complexes and initiating transcription at Phly and PactA were rather similar, while those of PrfA(Ls) were also much lower. The low binding and transcriptional activation capacities of PrfA(Ls) seem to be in part due to amino acid exchanges in its C-terminal domain (compared to PrfA(Lm) and PrfA(Li)). In contrast to the significant differences in the biochemical properties of PrfA(Lm), PrfA(Li), and PrfA(Ls), the PrfA-dependent promoters of hly (Phly(Lm), Phly(L)(i), and Phly(L)(s)) and actA (PactA(Lm), PactA(L)(i), and PactA(L)(s)) of the three Listeria species did not significantly differ in their binding affinities to the various PrfA proteins and in their strengths to promote transcription in vitro. The allelic replacement of prfA(Lm) with prfA(Ls) in L. monocytogenes leads to low expression of PrfA-dependent genes and to reduced in vivo virulence of L. monocytogenes, suggesting that the altered properties of PrfA(Ls) protein are a major cause for the low virulence of L. seeligeri.

Complex formation between malate dehydrogenase and isocitrate dehydrogenase from Bacillus subtilis is regulated by tricarboxylic acid cycle metabolites

In Bacillus subtilis, recent in vivo studies revealed that particular enzymes of the tricarboxylic acid cycle form complexes that allow an efficient transfer of metabolites. Remarkably, a complex of the malate dehydrogenase (Mdh) (EC 1.1.1.37) with isocitrate dehydrogenase (Icd) (EC 1.1.1.42) was identified, although both enzymes do not catalyze subsequent reactions. In the present study, the interactions between these enzymes were characterized in vitro by surface plasmon resonance in the absence and presence of their substrates and cofactors. These analyses revealed a weak but specific interaction between Mdh and Icd, which was specifically stimulated by a mixture of substrates and cofactors of Icd: isocitrate, NADP+ and Mg2+. Wild‐type Icd converted these substrates too fast, preventing any valid quantitative analysis of the interaction with Mdh. Therefore, binding of the IcdS104P mutant to Mdh was quantified because the mutation reduced the enzymatic activity by 174‐fold but did not affect the stimulatory effect of substrates and cofactors on Icd–Mdh complex formation. The analysis of the unstimulated Mdh–IcdS104P interaction revealed kinetic constants of ka = 2.0 ± 0.2 × 102 m−1·s−1 and kd = 1.0 ± 0.1 × 10−3·s−1 and a KD value of 5.0 ± 0.1 μm. Addition of isocitrate, NADP+ and Mg2+ stimulated the affinity of IcdS104P to Mdh by 33‐fold (KD = 0.15 ± 0.01 μm, ka = 1.7 ± 0.7 × 103 m−1·s−1, kd = 2.6 ± 0.6 × 10−4·s−1). Analyses of the enzymatic activities of wild‐type Icd and Mdh showed that Icd activity doubles in the presence of Mdh, whereas Mdh activity was slightly reduced by Icd. In summary, these data indicate substrate control of complex formation in the tricarboxylic acid cycle metabolon assembly and maintenance of the α‐ketoglutarate supply for amino acid anabolism in vivo.

CcpA Mutants with Differential Activities in Bacillus subtilis

CcpA is the master regulator for carbon catabolite regulation in Bacillus subtilis and regulates more than 300 genes by repression or activation. To revealthe effects of different functional domains of CcpA on various regulatory modes, we compared the activities of CcpA point mutants in activation (alsS, ackA) and repression (xynP, gntR). CcpA variants mutated at residues in the HPrSerP-binding region without allosteric functions are inactive. On the other hand, CcpA variants mutated at residues that change their conformation upon HPrSerP or CrhP binding regulate only ackA. Another set of mutants with alterations in the corepressor-binding region show glucose-independent regulation of xynP. The data presented here demonstrate the involvement of HPrSerP and/or CrhP in activation of ackA and alsS by CcpA. Furthermore, these data also indicatethat activation and repression mediated by CcpA may utilize different conformational changes of the protein.

Residues His-15 and Arg-17 of HPr Participate Differently in Catabolite Signal Processing via CcpA

The carbon catabolite control protein A (CcpA) senses the physiological state of the cell by binding several effectors and responds with differential regulation of many genes in Bacilli. HPr-Ser46-P or Crh-Ser46-P interact with CcpA and stimulate binding to catabolite responsive elements. In addition, the glycolytic intermediates fructose 1,6-bisphosphate (FBP) and glucose 6-phosphate (Glc-6-P) stimulate HPr-Ser46-P but not Crh-Ser46-P binding to CcpA. The mechanisms by which coeffector binding to CcpA is linked to differential gene expression are unclear. To address this question we mutated residues participating in the interaction between HPr-Ser46-P or Crh-Ser46-P and CcpA and analyzed their effects on CcpA binding and stimulation of cre binding by surface plasmon resonance. The HPrH15A and CcpAD297A mutations do not affect complex formation but abolish FBP and Glc-6-P stimulation. Likewise, the CrhQ15H mutant becomes sensitive to these glycolytic intermediates. Hence, the contact of HPrHis-15 to Asp-297 in CcpA is a determinant for HPr specific FBP and Glc-6-P stimulation. The HPrR17A and -K mutants are both strongly impaired in stimulation of CcpA binding to cre, but only HPrR17A is defect in binding to CcpA indicating that these residues affect allostery of CcpA. Mutations of the residues of CcpA, which contact Arg-17 of HPr, exhibit differential effects on regulation of catabolic genes. Taken together, His-15 of HPr processes sensing information, while Arg-17 is involved in determining the genetic output.