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Piszczek G Lee JC Tjandra N Lee CR Seok YJ Levine RL Peterkofsky A 《Archives of biochemistry and biophysics》2011,507(2):332-342
Enzyme I(Ntr) is the first protein in the nitrogen phosphotransferase pathway. Using an array of biochemical and biophysical tools, we characterized the protein, compared its properties to that of EI of the carbohydrate PTS and, in addition, examined the effect of substitution of all nonexchangeable protons by deuterium (perdeuteration) on the properties of EI(Ntr). Notably, we find that the catalytic function (autophosphorylation and phosphotransfer to NPr) remains unperturbed while its stability is modulated by deuteration. In particular, the deuterated form exhibits a reduction of approximately 4°C in thermal stability, enhanced oligomerization propensity, as well as increased sensitivity to proteolysis in vitro. We investigated tertiary, secondary, and local structural changes, both in the absence and presence of PEP, using near- and far-UV circular dichroism and Trp fluorescence spectroscopy. Our data demonstrate that the aromatic residues are particularly sensitive probes for detecting effects of deuteration with an enhanced quantum yield upon PEP binding and apparent decreases in tertiary contacts for Tyr and Trp side chains. Trp mutagenesis studies showed that the region around Trp522 responds to binding of both PEP and NPr. The significance of these results in the context of structural analysis of EI(Ntr) are evaluated. 相似文献
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Regulation of Escherichia coli starvation sigma factor (sigma s) by ClpXP protease. 总被引:21,自引:5,他引:16
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In Escherichia coli, starvation (stationary-phase)-mediated differentiation involves 50 or more genes and is triggered by an increase in cellular sigma s levels. Western immunoblot analysis showed that in mutants lacking the protease ClpP or its cognate ATPase-containing subunit ClpX, sigma s levels of exponential-phase cells increased to those of stationary-phase wild-type cells. Lack of other potential partners of ClpP, i.e., ClpA or ClpB, or of Lon protease had no effect. In ClpXP-proficient cells, the stability of sigma s increased markedly in stationary-phase compared with exponential-phase cells, but in ClpP-deficient cells, sigma s became virtually completely stable in both phases. There was no decrease in ClpXP levels in stationary-phase wild-type cells. Thus, sigma s probably becomes more resistant to this protease in stationary phase. The reported sigma s-stabilizing effect of the hns mutation also was not due to decreased protease levels. Studies with translational fusions containing different lengths of sigma s coding region suggest that amino acid residues 173 to 188 of this sigma factor may directly or indirectly serve as at least part of the target for ClpXP protease. 相似文献
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Mirel DB Estacio WF Mathieu M Olmsted E Ramirez J Márquez-Magaña LM 《Journal of bacteriology》2000,182(11):3055-3062
The sigma(D) regulon of Bacillus subtilis is composed of genes encoding proteins for flagellar synthesis, motility, and chemotaxis. Concurrent analyses of sigma(D) protein levels and flagellin mRNA demonstrate that sigD expression and sigma(D) activity are tightly coupled during growth in both complex and minimal media, although they exhibit different patterns of expression. We therefore used the sigma(D)-dependent flagellin gene (hag) as a model gene to study the effects of different nutritional environments on sigma(D)-dependent gene expression. In complex medium, the level of expression of a hag-lacZ fusion increased exponentially during the exponential growth phase and peaked early in the transition state. In contrast, the level of expression of this reporter remained constant and high throughout growth in minimal medium. These results suggest the existence of a nutritional signal(s) that affects sigD expression and/or sigma(D) activity. This signal(s) allows for nutritional repression early in growth and, based on reconstitution studies, resides in the complex components of sporulation medium, as well as in a mixture of mono-amino acids. However, the addition of Casamino Acids to minimal medium results in a dose-dependent decrease in hag-lacZ expression throughout growth and the postexponential growth phase. In work by others, CodY has been implicated in the nutritional repression of several genes. Analysis of a codY mutant bearing a hag-lacZ reporter revealed that flagellin expression is released from nutritional repression in this strain, whereas mutations in the transition state preventor genes abrB, hpr, and sinR failed to elicit a similar effect during growth in complex medium. Therefore, the CodY protein appears to be the physiologically relevant regulator of hag nutritional repression in B. subtilis. 相似文献
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Ades SE 《Current opinion in microbiology》2004,7(2):157-162
Signal transduction pathways that communicate information from the cell envelope to the cytoplasm of bacteria are crucial to maintain cell envelope homeostasis. In Escherichia coli, one of the key pathways that ensures the integrity of the cell envelope during stress and normal growth is controlled by the alternative sigma factor sigmaE. Recent studies have elucidated the signal transduction pathway that activates sigmaE in response to misfolded outer membrane porins. Unfolded porins trigger the degradation of the sigmaE-specific antisigma factor RseA by the sequential action of two inner membrane proteases, leading to release of sigmaE from RseA, and induction of the stress response. This mechanism of signal transduction, regulated intramembrane proteolysis, is used in transmembrane signaling pathways from bacteria to humans. 相似文献
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Transient regulation of enzyme synthesis in Escherichia coli 总被引:2,自引:0,他引:2
Summary After lysine addition to an exponentially growing culture of Escherichia coli K12, the kinetics of repression of aspartokinase III synthesis show a transient regulatory phenomenon: during one generation, enzyme synthesis is practically equal to zero (Fig. 1). A similar phenomenon appears to be involved during repression of aspartokinase I-homoserine dehydrogenase I synthesis by threonine and isoleucine (Fig. 2). This sort of phenomenon has been previously reported in another system and interpreted as an indication of regulation at the translational level. 相似文献
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SigmaE is an alternative sigma factor that controls the extracytoplasmic stress response in Escherichia coli. SigmaE is essential at high temperatures but was previously thought to be nonessential at temperatures below 37 degrees C. We present evidence that sigmaE is an essential sigma factor at all temperatures. Cells lacking sigmaE are able to grow at low temperatures because of the presence of a frequently arising, unlinked suppressor mutation. 相似文献
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The bacterial phosphoenolpyruvate-dependent sugar phosphotransferase system (PEP-PTS) is essential in the coupled transportation and phosphorylation of various types of carbohydrates. The CmtAB proteins of Escherichia coli are sequentially similar to the mannitol-specific phosphotransferase MtlA. The CmtB protein corresponds to the phosphotransferase enzyme IIA component. Here we report the solution structure of CmtB from E. coli at high resolution by NMR spectroscopy. The results show that CmtB adopts a globular fold consisting of a central mixed five-strand β-sheet flanked by seven helices at both sides. Structural comparison with the IIA domain of MtlA (IIAMtl) reveals high overall similarity, while notable conformational differences at the active site are observed. The active site pocket of CmtB appears to be wider, and the hydrophobic regions around it is larger compared to IIAMtl. Further, the essential arginine residue at the active site of IIAMtl is substituted by a serine in CmtB. Instead, the active pocket of CmtB contains another arginine at a distinct position, suggesting different molecular mechanisms for phosphoryl transfer. 相似文献
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Tang C Williams DC Ghirlando R Clore GM 《The Journal of biological chemistry》2005,280(12):11770-11780
The solution structure of trimeric Escherichia coli enzyme IIA(Chb) (34 kDa), a component of the N,N'-diacetylchitobiose/lactose branch of the phosphotransferase signal transduction system, has been determined by NMR spectroscopy. Backbone residual dipolar couplings were used to provide long range orientational restraints, and long range (|i - j| > or = 5 residues) nuclear Overhauser enhancement restraints were derived exclusively from samples in which at least one subunit was 15N/13C/2H/(Val-Leu-Ile)-methyl-protonated. Each subunit consists of a three-helix bundle. Hydrophobic residues lining helix 3 of each subunit are largely responsible for the formation of a parallel coiled-coil trimer. The active site histidines (His-89 from each subunit) are located in three symmetrically placed deep crevices located at the interface of two adjacent subunits (A and C, C and B, and B and A). Partially shielded from bulk solvent, structural modeling suggests that phosphorylated His-89 is stabilized by electrostatic interactions with the side chains of His-93 from the same subunit and Gln-91 from the adjacent subunit. Comparison with the x-ray structure of Lactobacillus lactis IIA(Lac) reveals some substantial structural differences, particularly in regard to helix 3, which exhibits a 40 degrees kink in IIA(Lac) versus a 7 degrees bend in IIA(Chb). This is associated with the presence of an unusually large (230-angstroms3) buried hydrophobic cavity at the trimer interface in IIA(Lac) that is reduced to only 45 angstroms3) in IIA(Chb). 相似文献
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