Biomedical subjects

M Hecker

Publications and source records attributed to M Hecker.

At least 73 records · Page 4Linked to original sources

PubMed2000

Human antibody response during sepsis against targets expressed by methicillin resistant Staphylococcus aureus.

The identification of target structures is a prerequisite for the development of new treatment options, like antibody based therapy, against methicillin resistant Staphylococcus aureus (MRSA). In this study we identified immunodominant structures which were expressed in vivo during sepsis caused by MRSA. Using human sera we compared the immune response of humans with MRSA sepsis with the immune response of normal individuals and asymptomatically colonized individuals. We identified and characterized four staphylococcal specific antigenic structures. One target is a staphylococcal protein of 29 kDa that exhibited 29% identity to secreted protein SceA precursor of Staphylococcus carnosus. The putative function of this protein, which was designated IsaA (immunodominant staphylococcal antigen), is unknown. The second target is an immunodominant protein of 17 kDa that showed no homology to any currently known protein. This immunodominant protein was designated IsaB. The third and fourth antigens are both immunodominant proteins of 10 kDa. One of these proteins showed 100% identity to major cold shock protein CspA of S. aureus and the other protein was identified as the phosphocarrier protein Hpr of S. aureus. The identified immunodominant proteins may serve as potential targets for the development of antibody based therapy against MRSA.

Amino Acid Sequence↗

PubMed2000

Decoy oligodeoxynucleotide characterization of transcription factors controlling endothelin-B receptor expression in vascular smooth muscle cells.

Endothelin-1 is not only a powerful vasoconstrictor but also a potent mitogen for vascular smooth muscle cells (SMC), acting through both the endothelin-A and endothelin-B receptor (ET(B)-R). Although vascular SMC are known to express the ET(B)-R, its transcriptional regulation has not been studied thus far. Here we demonstrate that the potent inhibitor of nuclear factor kappaB activation, pyrrolidine dithiocarbamate (PDTC; 30-100 microM), induces de novo ET(B)-R expression in rat aortic and mesenteric cultured SMC. Electrophoretic mobility shift analyses revealed that besides inhibition of nuclear factor kappaB, PDTC enhances activator protein-1 (AP-1), CCAAT/enhancer-binding protein (C/EBP), and GATA-2 activity in these cells. Preincubation of PDTC-stimulated cells with appropriate decoy oligodeoxynucleotides confirmed the involvement of these three transcription factors, namely that of AP-1, in ET(B)-R expression. The stimulatory effect of PDTC on ET(B)-R expression was also confirmed functionally by monitoring an enhanced ET-1-induced apoptosis in PDTC-treated cells that was sensitive to the ET(B)-R antagonist, BQ788. Taken together, these findings demonstrate that C/EBP, GATA-2, and in particular AP-1 can control ET(B)-R expression in vascular SMC. They further support the notion that ET(B)-R expression in these cells may play an important role in cardiovascular complications, such as restenosis following angioplasty that in the early phase is characterized by prominent SMC apoptosis.

Animals↗

PubMed2000

The clp proteases of Bacillus subtilis are directly involved in degradation of misfolded proteins.

The presence of the heat stress response-related ATPases ClpC and ClpX or the peptidase ClpP in the cell is crucial for tolerance of many forms of stress in Bacillus subtilis. Assays for detection of defects in protein degradation suggest that ClpC, ClpP, and ClpX participate directly in overall proteolysis of misfolded proteins. Turnover rates for abnormal puromycyl peptides are significantly decreased in clpC, clpP, and clpX mutant cells. Electron-dense aggregates, most likely due to the accumulation of misfolded proteins, were noticed in studies of ultrathin cryosections in clpC and clpP mutant cells even under nonstress conditions. In contrast, in the wild type or clpX mutants such aggregates could only be observed after heat shock. This phenomenon supports the assumption that clpC and clpP mutants are deficient in the ability to solubilize or degrade damaged and aggregated proteins, the accumulation of which is toxic for the cell. By using immunogold labeling with antibodies raised against ClpC, ClpP, and ClpX, the Clp proteins were localized in these aggregates, showing that the Clp proteins act at this level in vivo.

ATPases Associated with Diverse Cellular Activitie↗

PubMed2000

Phosphate starvation-inducible proteins of Bacillus subtilis: proteomics and transcriptional analysis.

The phosphate starvation response in Bacillus subtilis was analyzed using two-dimensional (2D) polyacrylamide gel electrophoresis of cell extracts and supernatants from phosphate-starved cells. Most of the phosphate starvation-induced proteins are under the control of sigma(B), the activity of which is increased by energy depletion. In order to define the proteins belonging to the Pho regulon, which is regulated by the two-component regulatory proteins PhoP and PhoR, the 2D protein pattern of the wild type was compared with those of a sigB mutant and a phoR mutant. By matrix-assisted laser desorption ionization-time of flight mass spectrometry, two alkaline phosphatases (APases) (PhoA and PhoB), an APase-alkaline phosphodiesterase (PhoD), a glycerophosphoryl diester phosphodiesterase (GlpQ), and the lipoprotein YdhF were identified as very strongly induced PhoPR-dependent proteins secreted into the extracellular medium. In the cytoplasmic fraction, PstB1, PstB2, and TuaD were identified as already known PhoPR-dependent proteins, in addition to PhoB, PhoD, and the previously described PstS. Transcriptional studies of glpQ and ydhF confirmed the strong PhoPR dependence. Northern hybridization and primer extension experiments showed that glpQ is transcribed monocistronically from a sigma(A) promoter which is overlapped by four putative TT(A/T)ACA-like PhoP binding sites. Furthermore, ydhF might be cotranscribed with phoB initiating from the phoB promoter. Only a small group of proteins remained phosphate starvation inducible in both phoR and sigB mutant and did not form a unique regulation group. Among these, YfhM and YjbC were controlled by sigma(B)-dependent and unknown PhoPR-independent mechanisms. Furthermore, YtxH and YvyD seemed to be induced after phosphate starvation in the wild type in a sigma(B)-dependent manner and in the sigB mutant probably via sigma(H). YxiE was induced by phosphate starvation independently of sigma(B) and PhoPR.

Alkaline Phosphatase↗

PubMed2000

Alternative transcription factor sigma(B) is involved in regulation of biofilm expression in a Staphylococcus aureus mucosal isolate.

Osmotic stress was found to induce biofilm formation in a Staphylococcus aureus mucosal isolate. Inactivation of a global regulator of the bacterial stress response, the alternative transcription factor sigma(B), resulted in a biofilm-negative phenotype and loss of salt-induced biofilm production. Complementation of the mutant strain with an expression plasmid encoding sigma(B) completely restored the wild-type phenotype. The combined data suggest a critical role of sigma(B) in S. aureus biofilm regulation under environmental stress conditions.

Bacterial Proteins↗

PubMed2000

Characterization of the sigma(B) regulon in Staphylococcus aureus.

The sigma(B)-dependent stress regulon in gram-positive bacteria might fulfill a physiological role in stress response and virulence similar to that of the sigma(S) regulon in Escherichia coli and other gram-negative bacteria. In order to obtain evidence for the function of the sigma(B) regulon of Staphylococcus aureus, especially in virulence control, sigma(B)-dependent stress genes were identified. The two-dimensional protein pattern of wild-type cells of S. aureus COL was compared with that of an isogenic sigB mutant. By this approach, we found that the synthesis of about 27 cytoplasmic proteins seemed to be under the positive control of sigma(B). N-terminal sequencing of 18 proteins allowed the identification of their genes on the almost finished genome sequence of S. aureus COL and the analysis of the promoter structure. Transcriptional analyses of 11 of these genes confirmed their sigma(B) dependency, and moreover, about 7 additional sigma(B)-dependent genes were found which are cotranscribed with the newly detected genes, forming operons. Altogether, we identified 23 sigma(B)-dependent genes and their corresponding proteins. Among them are proteins probably involved in the generation of NADH or in membrane transport mechanisms. Furthermore, at least one clpC-homologous gene was localized on the S. aureus sequence solely transcribed by sigma(B). In contrast, a second clpC-homologous gene in S. aureus forming an operon with ctsR, yacH, and yacI was sigma(B) independently expressed.

Bacterial Proteins↗

PubMed2000

Improvement of nitric oxide-dependent vasodilatation by HMG-CoA reductase inhibitors through attenuation of endothelial superoxide anion formation.

Three 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (HCRIs), atorvastatin, pravastatin, and cerivastatin, inhibited phorbol ester-stimulated superoxide anion (O(2)(-)) formation in endothelium-intact segments of the rat aorta in a time- and concentration-dependent manner (maximum inhibition of 70% after 18 hours at 1 to 10 micromol/L). The HMG-CoA reductase product mevalonic acid (400 micromol/L) reversed the inhibitory effect of the HCRIs, which, conversely, was mimicked by inactivation of p21 Rac with Clostridium sordellii lethal toxin but not by inactivation of p21 Rho with Clostridium botulinum exoenzyme (C3). A mevalonate-sensitive inhibition of phorbol ester-stimulated O(2)(-) formation by atorvastatin was also observed in porcine cultured endothelial cells and in a murine macrophage cell line. In the rat aorta, no effect of the HCRIs on protein kinase C, NADPH oxidase, or superoxide dismutase (SOD) activity and expression was detected, whereas that of endothelial nitric oxide (NO) synthase was enhanced approximately 2-fold. Moreover, exposure of the segments to atorvastatin resulted in a significant improvement of endothelium-dependent NO-mediated relaxation, and this effect was abolished in the presence of SOD. Taken together, these findings suggest that in addition to augmenting endothelial NO synthesis, HCRIs inhibit endothelial O(2)(-) formation by preventing the isoprenylation of p21 Rac, which is critical for the assembly of NADPH oxidase after activation of protein kinase C. The resulting shift in the balance between NO and O(2)(-) in the endothelium improves endothelial function even in healthy blood vessels and therefore may provide a reasonable explanation for the beneficial effects of HCRIs in patients with coronary heart disease in addition to or as an alternative to the reduction in serum LDL cholesterol.

Animals↗

PubMed2000

Pressure-induced upregulation of preproendothelin-1 and endothelin B receptor expression in rabbit jugular vein in situ : implications for vein graft failure?

Upregulation of endothelin-1 (ET-1) synthesis in venous bypass grafts in response to arterial levels of blood pressure may play a major role in graft failure. To investigate this hypothesis, isolated segments of the rabbit jugular vein were perfused at physiological (0 to 5 mm Hg) and nonphysiological (20 mm Hg) levels of intraluminal pressure. As judged by reverse transcription-polymerase chain reaction analysis (mRNA level), neither endothelin-converting enzyme nor endothelin A receptor expression appeared to be pressure sensitive. In contrast, there was a profound and time-dependent increase in endothelial prepro-ET-1 mRNA and intravascular ET-1 abundance (by ELISA) as well as in smooth muscle endothelin B receptor mRNA and functional protein (by superfusion bioassay) on raising the perfusion pressure from 5 to 20 mm Hg, but not from 0 to 5 mm Hg, for up to 12 hours. Video microscopy analysis revealed that the segments were distended by 75% at 5 mm Hg and near maximally at 20 mm Hg compared with the resting diameter at 0 to 1 mm Hg. Treatment of the segments with actinomycin D (1 micromol/L), the specific protein kinase C inhibitor, Ro 31-8220 (0.1 micromol/L), or the c-Src family-specific tyrosine kinase inhibitor, herbimycin A (0.1 micromol/L), demonstrated that the pressure-induced expression of these gene products occurs at the level of transcription and requires activation of protein kinase C, but not c-Src. In venous bypass grafts such deformation-induced changes in gene expression may contribute not only to acute graft failure through ET-1-induced vasospasm but also to endothelin A receptor- and/or endothelin B receptor-mediated smooth muscle cell hyperplasia and graft occlusion.

Animals↗

PubMed2000

Elevated perfusion pressure upregulates endothelin-1 and endothelin B receptor expression in the rabbit carotid artery.

To investigate the hypothesis that high blood pressure activates the endothelin system in the vessel wall, isolated segments of the rabbit carotid artery were subjected to different levels of perfusion pressure. Both preproendothelin-1 (ppET-1) mRNA abundance and intravascular ET-1 peptide content were strongly upregulated on raising the intraluminal pressure from 90 to 160 mm Hg for 3 to 12 hours, and this increase in ppET-1 mRNA occurred predominantly in the endothelial cells. Endothelin-converting enzyme-1 and endothelin A receptor (ET(A)-R) expression were pressure-insensitive, whereas that of the ET(B)-R in the smooth muscle cells was also significantly enhanced. Both the pressure-induced increase in ppET-1 and ET(B)-R expression required RNA synthesis because they were abolished by actinomycin D. The nuclear signaling mechanisms involved therein, however, appeared to be different. Thus, the pressure-induced expression of ppET-1 and activation of CCAAT-enhancer binding proteins beta and delta were blocked by the tyrosine kinase inhibitor herbimycin A, whereas ET(B)-R expression and the nuclear translocation of activator protein-1 were abolished by the protein kinase C inhibitor Ro 31-8220. One consequence of these presumably deformation-induced changes in gene expression was an increased rate of apoptosis of the smooth muscle cells in the media that if transferable to the situation in human blood vessels may contribute to hypertension-induced arterial remodeling.

Animals↗

PubMed2000

L-Arginine-nitric oxide pathway-related metabolites in the aqueous humor of diabetic patients.

PURPOSE: Nitric oxide (NO) is an important signal-transduction molecule that plays a significant role in the regulation of cardiovascular functions. In the L-arginine-NO pathway, NO synthase (NOS) converts L-arginine (L-Arg), the only known biologic substrate for NO formation, to NO and L-citrulline (L-Cit). Excessive NO production mediated by the inducible isoform of NOS has been implicated in the pathogenesis of various diseases. In the present study it was hypothesized that in vitreoretinal disorders such as diabetic retinopathy the production of L-Arg-NO pathway-related metabolites may be upregulated as a result of increased NO generation. METHODS: From 20 eyes of nondiabetic subjects and 22 eyes of diabetic patients with (n = 14) and without (n = 8) diabetic retinopathy, undiluted samples of aqueous humor were drawn before cataract surgery. Levels of L-Arg, L-Cit, and the specific NOS by-product N(G)-hydroxy-L-arginine (HOArg) were measured by high-performance liquid chromatography. RESULTS: L-Arg, L-Cit, and HOArg were detected in all aqueous humor samples from diabetic and nondiabetic patients (n = 42). Comparison of HOArg levels in nondiabetic and diabetic subjects showed significantly higher levels in diabetic patients (P = 0.002). Concentrations of HOArg were higher in samples from patients with (P = 0.005) and without diabetic retinopathy (P = 0.033) than in control subjects. No statistically significant differences were observed in L-Arg or L-Cit levels. CONCLUSIONS: Elevated levels of HOArg in the aqueous humor of diabetic patients reflect the possible role of NO as a significant factor in the regulation of retinal vascular functions and intraocular proliferative changes in diabetes mellitus in vivo. The control of intraocular NO production may constitute a potential therapeutic approach in diabetic retinopathy.

Aged↗

PubMed1999

Monitoring of genes that respond to process-related stress in large-scale bioprocesses.

In large-scale aerobic fed-batch processes, cells are exposed to local zones of high glucose concentrations that can also cause local oxygen limitations at high cell densities. The mRNA levels of four stress genes (clpB, dnaK, uspA, and proU) and three genes responding to oxygen limitation or glucose excess (pfl, frd, and ackA) were investigated in an industrial 20-m(3) Escherichia coli process and in a scale-down reactor with defined high-glucose and low-oxygen zones. The mRNA levels of ackA and proU were high during the batch growth phase, but declined drastically when glucose became limited, whereas the mRNA levels of the other stress genes were relatively constant throughout the process. In the industrial-scale reactor, the stress gene mRNA levels were, in most cases, highest in the middle part and at the top of the reactor, where the substrate was fed. Cells passing through the high glucose zone of the scale-down reactor had elevated mRNA levels for the oxygen limitation genes and had also elevated heat-shock gene mRNA levels. Both responses to stress occurred within seconds. The approach presented in this study offers a tool for monitoring process-related changes in the transcriptional regulation of genes.

Animals↗

PubMed1999

Cytokine-inducible CD40 gene expression in vascular smooth muscle cells is mediated by nuclear factor kappaB and signal transducer and activation of transcription-1.

The interaction of T-lymphocytes expressing the CD40 ligand (CD154) and cells of the vessel wall expressing the corresponding receptor protein (CD40) may play an important role in chronic inflammation including arteriosclerosis. One way of interfering with CD40-CD154 signalling is to prevent CD40 expression, the regulation of which, however, has yet to be elucidated. Therefore, we studied CD40 expression in rat aortic cultured smooth muscle cells. Both CD40 mRNA and protein expression in these cells was markedly enhanced as early as 6 h after exposure to different pro-inflammatory cytokines. Experiments with actinomycin D and subsequent run-on analyses revealed that CD40 expression in response to these cytokines was regulated at the level of transcription. Moreover, electrophoretic mobility shift analyses along with the employment of transcription factor decoy oligodeoxynucleotides demonstrated that tumor necrosis factor alpha via nuclear kappaB and interferon-gamma via signal transducer and activator of transcription-1 up-regulate CD40 gene expression in rat aortic cultured smooth muscle cells.

Animals↗

PubMed1999

Dual channel imaging of two-dimensional electropherograms in Bacillus subtilis.

The allocation of proteins to stimulons and regulons is an essential step towards the understanding of the global regulation of the expression of entire genomes. The computer-aided evaluation and matching of two-dimensional protein gels loaded with radioactively labeled proteins from exponentially growing or stressed cells is a useful but time-consuming procedure for the description of stimulons and regulons. This paper describes the dual-channel image analysis that offers the opportunity to visualize the content and synthesis rate of a whole set of bacterial proteins on a single electropherogram. By pulse-labeling with L-[35S]methionine, the protein synthesis pattern (red color) can be directly compared with the protein level pattern (green color). Because matching of other gels can be avoided, this new technique is useful for the rapid search for proteins that belong to different stimulons or regulons. This approach was tested for the identification of proteins of heat stress or oxidative stress stimulons. Proteins that were induced by heat or oxidative stress colored red while proteins whose synthesis was switched off by the stress factor colored green. Proteins that were continuously synthesized before and after the imposition of stress retained their yellow color. The advantages and possible pitfalls of the technique are discussed.

Bacillus subtilis↗

PubMed1999

Endothelium-derived hyperpolarizing factor, but not nitric oxide or prostacyclin release, is resistant to menadione-induced oxidative stress in the bovine coronary artery.

The interaction of superoxide anions (O2-) generated by menadione with the synthesis and/or action of nitric oxide (NO), prostacyclin (PGI2) and endothelium-derived hyperpolarizing factor (EDHF) was investigated in segments of the left anterior descending coronary artery (LAD) isolated from bovine hearts. EDHF and NO release were monitored by superfusion bioassay in segments pre-constricted with the thromboxane mimetic, U46619, PGI2 release in addition by enzyme immunoassay for 6-keto-prostaglandin F1alpha, and generation of O2- by lucigenin-enhanced chemiluminescence. Bradykinin (1-1,000 pmol) elicited a prominent, endothelium-dependent, relaxant response, 50-60% of which was insensitive to combined blockade of cyclooxygenase with diclofenac (1 microM) and NO synthase with NG-nitro-L-arginine (50 microM). This diclofenac/NG-nitro-L-arginine-insensitive relaxant response was completely abrogated in the presence of tetrabutylammonium (3 mM), a non-selective inhibitor of Ca2+-dependent K+ channels, or when the segments were pre-constricted with potassium chloride (60 mM) instead of U46619, and thus most likely mediated by EDHF. Despite causing a two- to fourfold increase in the concentration of O2- in or at the vessel wall, menadione (30 microM) did not affect the diclofenac/NG-nitro-L-arginine-insensitive relaxant response to bradykinin. When administered in the absence of NG-nitro-L-arginine, however, menadione significantly inhibited the relaxant response to bradykinin, presumably by attenuating the NO-mediated relaxation. Menadione also abolished the bradykinin-stimulated release of PGI2 from luminally perfused segments of the LAD. This effect was more pronounced in the absence of NG-nitro-L-arginine, indicating that PGI2 release in this preparation may be more sensitive to inhibition by peroxynitrite, the reaction product of NO and O2-, than to O2- alone. These findings suggest that, in contrast to NO and PGI2, the release, and presumably also the mechanism of action, of EDHF in the bovine LAD is resistant to an increase in the local concentration of O2- or peroxynitrite which is thought to play an important role in coronary heart disease.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

PubMed1999

Regulation of sigmaB-dependent transcription of sigB and asp23 in two different Staphylococcus aureus strains.

The alkaline shock protein Asp23 was identified as a sigmaB-dependent protein in Staphylococcus aureus. In Bacillus subtilis, the asp23 promoter from S. aureus is regulated like other sigmaB-dependent promoters, which are strongly induced by heat and ethanol stress. However, almost no induction of asp23 expression was found after heat or ethanol stress in S. aureus MA13 grown in a synthetic medium, where the basal expression level of asp23 is high. Under the same experimental conditions the sigmaB gene itself showed a similar expression pattern: it was highly expressed in synthetic medium but not induced by heat or ethanol stress. In contrast, sigmaB activity was increased by heat stress when the cells were grown in a complex medium. The constitutive expression of sigB and sigmaB-dependent stress genes in S. aureus MA13 grown in a synthetic medium is in a sharp contrast to the regulation of sigmaB activity in B. subtilis, and needs further investigation. A deletion of 11 bp in the rsbU gene, which encodes the phosphatase that acts on RsbV (the anti-anti-sigma factor), in S. aureus NCTC 8325-4 might be responsible for the failure of heat stress to activate sigmaB in complex medium, and thus reduce the initiation of transcription at sigmaB-dependent promoters in this strain.

Bacillus subtilis↗

PubMed1999

The katX gene of Bacillus subtilis is under dual control of sigmaB and sigmaF.

The gene katX, which encodes a catalase in Bacillus subtilis, is transcribed by EsigmaF in the pre-spore. This catalase is responsible for the resistance to hydrogen peroxide shown by germinating and outgrowing spores. We demonstrated that katX is also a sigmaB-dependent general stress gene, since it is strongly induced by heat, salt and ethanol stress, as well as by energy depletion. The -10 and -35 sequences of the sigmaB- and sigmaF-dependent promoters of katX overlap, and the transcriptional start points used by EsigmaB and EsigmaF differ by only one nucleotide. Our results indicate that the level of KatX level in outgrowing spores depends mainly on EsigmaF, because sigB mutants show normal KatX activity in dormant and outgrowing spores. katX mutants also develop the non-specific resistance to oxidative stress that is typical of glucose-starved cells. Therefore, the physiological role of sigmaB-dependent katX expression remains obscure. The results indicate an overlap between the sigmaB regulon and the sigmaF regulon, and the physiological implications of this overlap are discussed.

Arabidopsis Proteins↗

PubMed1999

Regulation of inducible nitric oxide synthase gene expression in vascular smooth muscle cells.

1. Formation of nitric oxide (NO) by the constitutive calcium-dependent NO synthase expressed in endothelial cells plays an important role in the control of local blood flow and vascular homeostasis. Expression of the inducible calcium-independent NO synthase (iNOS) in vascular smooth muscle cells (VSMC), on the other hand, is thought to play a potentially detrimental role in the pathogenesis of chronic inflammation or septic shock. In vascular injury, however, iNOS expression in VSMC may be beneficial as a compensatory mechanism for the lack of endothelial NO synthesis, e.g., by preventing restenosis following angioplasty or heart transplant vasculopathy. 2. Because iNOS activity does not seem to be controlled once the enzyme is expressed, regulation of NO release from iNOS-expressing cells predominantly occurs at the transcriptional and/or posttranscriptional level. 3. This review summarizes what is currently known about the regulation of expression of this enzyme in VSMC, details some of the transcription factors involved therein as well as their mode of activation, and highlights some pharmacological strategies based on these findings that may be employed for the control of iNOS expression in VSMC in the clinical arena.

Animals↗

PubMed1999

Regulation of the activity of the Bacillus subtilis antiterminator LicT by multiple PEP-dependent, enzyme I- and HPr-catalysed phosphorylation.

The transcriptional antiterminator LicT regulates the induction and carbon catabolite repression of the Bacillus subtilis bglPH operon. LicT is inactive in mutants affected in one of the two general components of the phosphoenolpyruvate (PEP):glycose phosphotransferase system, enzyme I or histidine-containing protein (HPr). We demonstrate that LicT becomes phosphorylated in the presence of PEP, enzyme I and HPr. The phosphoryl group transfer between HPr and LicT is reversible. Phosphorylation of LicT with PEP, enzyme I and HPr led to the appearance of three additional LicT bands on polyacrylamide-urea gels. These bands probably correspond to one-, two- and threefold phosphorylated LicT. After phosphorylation of LicT with [32P]-PEP, enzyme I and HPr, proteolytic digestion of [32P]-P-LicT, separation of the peptides by reverse-phase chromatography, mass spectrometry and N-terminal sequencing of radiolabelled peptides, three histidyl residues were found to be phosphorylated in LicT. These three histidyl residues (His-159, His-207 and His-269) are conserved in most members of the BglG/SacY family of transcriptional antiterminators. Phosphorylation of LicT in the presence of serylphosphorylated HPr (P-Ser-HPr) was much slower compared with its phosphorylation in the presence of HPr. The slower phosphorylation in the presence of P-Ser-HPr leading to reduced LicT activity is presumed to play a role in a recently described LicT-mediated CcpA-independent carbon catabolite repression mechanism operative for the bglPH operon.

Amino Acid Sequence↗