mRNA level of PKA-c gene in Setosphaeria turcica with different nutrition sources under metal ion or osmotic stress

ABSTRACT The standard method of producing recombinant proteins such as immunotoxins (rITs) in large quantities is to transform gram-negative bacteria and subsequently recover the desired protein from inclusion bodies by intensive de- and renaturing procedures. The major disadvantage of this technique is the low yield of active protein. Here we report the development of a novel strategy for the expression of functional rIT directed to the periplasmic space of Escherichia coli. rITs were recovered by freeze-thawing of pellets from shaking cultures of bacteria grown under osmotic stress (4% NaCl plus 0.5 M sorbitol) in the presence of compatible solutes. Compatible solutes, such as glycine betaine and hydroxyectoine, are low-molecular-weight osmolytes that occur naturally in halophilic bacteria and are known to protect proteins at high salt concentrations. Adding 10 mM glycine betaine for the cultivation of E. coliunder osmotic stress not only allowed the bacteria to grow under these otherwise inhibitory conditions but also produced a periplasmic microenvironment for the generation of high concentrations of correctly folded rITs. Protein purified by combinations of metal ion affinity and size exclusion chromatography was substantially stabilized in the presence of 1 M hydroxyecotine after several rounds of freeze-thawing, even at very low protein concentrations. The binding properties and cytotoxic potency of the rITs were confirmed by competitive experiments. This novel compatible-solute-guided expression and purification strategy might also be applicable for high-yield periplasmic production of recombinant proteins in different expression systems.

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Effect of Osmotic Stress on the Growth, Development and Pathogenicity of Setosphaeria turcica

Frontiers in Microbiology ◽

10.3389/fmicb.2021.706349 ◽

2021 ◽

Vol 12 ◽

Author(s):

Yuwei Liu ◽

Xiaodong Gong ◽

Moxiao Li ◽

Helong Si ◽

Qihui Zhou ◽

...

Keyword(s):

Osmotic Stress ◽

Mapk Pathway ◽

Pathogenic Fungus ◽

Germination Rate ◽

Phytopathogenic Fungi ◽

Severe Condition ◽

Potato Dextrose Agar Medium ◽

Knock Out ◽

Setosphaeria Turcica ◽

Growth Development

Osmotic stress is a severe condition frequently encountered by microorganisms; however, there is limited knowledge on the influence of hyperosmotic stress on the growth, development and pathogenicity of phytopathogenic fungi. Here, three osmotic conditions (0.4 M NaCl, 0.4 M KCl, and 0.6 M sorbitol supplemented in potato dextrose agar medium) were used to identify the effect of osmotic stress on the growth, development and pathogenicity of Setosphaeria turcica which is a plant pathogenic fungus and causes northern corn leaf blight disease in maize, sorghum, and related grasses. In osmotic stress, the growth rate of mycelium was decreased, and the number of vesicular structures and flocculent secretion outside the hypha cell wall were significantly increased. The qRT-PCR results showed that the osmotic stress quickly activated the HOG-MAPK pathway, up-regulated the expression of the downstream genes, and these genes were most highly expressed within 30 min of exposure to osmotic stress. Furthermore, the germination rate and the yield of conidia were significantly higher under osmotic stress than in the control. A pathogenicity analysis confirmed that pathogenicity of the conidia which were cultured under osmotic stress was significantly enhanced. By analyzing the knock-out mutants of an osmotic stress responsed gene StFPS1, an aquaglyceroporin downstream of the HOG-MAPK pathway, we found that StFPS1 was involved in the formation of appressorium and penetration peg, which affected the penetration ability of S. turcica. In summary, our work explained the correlation between osmotic stress and growth, development, and pathogenicity in S. turcica.

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Ptc1, a Type 2C Ser/Thr Phosphatase, Inactivates the HOG Pathway by Dephosphorylating the Mitogen-Activated Protein Kinase Hog1

Molecular and Cellular Biology ◽

10.1128/mcb.21.1.51-60.2001 ◽

2001 ◽

Vol 21 (1) ◽

pp. 51-60 ◽

Cited By ~ 136

Author(s):

Janel Warmka ◽

Jennifer Hanneman ◽

Ji Lee ◽

Dipesh Amin ◽

Irene Ota

Keyword(s):

Protein Kinase ◽

Osmotic Stress ◽

Metal Ion ◽

Mapk Pathway ◽

Negative Regulator ◽

Mitogen Activated Protein Kinase ◽

Hog Pathway ◽

Mitogen Activated Protein

ABSTRACT The HOG (high-osmolarity glycerol) mitogen-activated protein kinase (MAPK) pathway regulates the osmotic stress response in the yeast Saccharomyces cerevisiae. Three type 2C Ser/Thr phosphatases (PTCs), Ptc1, Ptc2, and Ptc3, have been isolated as negative regulators of this pathway. Previously, multicopy expression of PTC1 and PTC3 was shown to suppress lethality of the sln1Δ strain due to hyperactivation of the HOG pathway. In this work, we show thatPTC2 also suppresses sln1Δ lethality. Furthermore, the phosphatase activity of these PTCs was needed for suppression, as mutation of a conserved Asp residue, likely to coordinate a metal ion, inactivated PTCs. Further analysis of Ptc1 function in vivo showed that it inactivates the MAPK, Hog1, but not the MEK, Pbs2. In the wild type, Hog1 kinase activity increased transiently, ∼12-fold in response to osmotic stress, while overexpression of PTC1 limited activation to ∼3-fold. In contrast, overexpression of PTC1 did not inhibit phosphorylation of Hog1 Tyr in the phosphorylation lip, suggesting that Ptc1 does not act on Pbs2. Deletion of PTC1 also strongly affected Hog1, leading to high basal Hog1 activity and sustained Hog1 activity in response to osmotic stress, the latter being consistent with a role for Ptc1 in adaptation. In vitro, Ptc1 but not the metal binding site mutant, Ptc1D58N, inactivated Hog1 by dephosphorylating the phosphothreonine but not the phosphotyrosine residue in the phosphorylation lip. Consistent with its role as a negative regulator of Hog1, which accumulates in the nucleus upon activation, Ptc1 was found in both the nucleus and the cytoplasm. Thus, one function of Ptc1 is to inactivate Hog1.

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Aspects of high-resolution imaging with a scanning ion microprobe

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010014511x ◽

1986 ◽

Vol 44 ◽

pp. 750-751

Author(s):

R. Levi-Setti ◽

J. M. Chabala ◽

Y. L. Wang

Keyword(s):

Metal Ion ◽

Secondary Ion Mass Spectrometry ◽

Chromatic Aberration ◽

Ion Source ◽

Ion Microprobe ◽

Emission Pattern ◽

Intrinsic Resolution ◽

Resolution Imaging ◽

20 Nm ◽

Secondary Ion

We have shown the feasibility of 20 nm lateral resolution in both topographic and elemental imaging using probes of this size from a liquid metal ion source (LMIS) scanning ion microprobe (SIM). This performance, which approaches the intrinsic resolution limits of secondary ion mass spectrometry (SIMS), was attained by limiting the size of the beam defining aperture (5μm) to subtend a semiangle at the source of 0.16 mr. The ensuing probe current, in our chromatic-aberration limited optical system, was 1.6 pA with Ga+ or In+ sources. Although unique applications of such low current probes have been demonstrated,) the stringent alignment requirements which they imposed made their routine use impractical. For instance, the occasional tendency of the LMIS to shift its emission pattern caused severe misalignment problems.

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Scanning ion microscopy images

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010012583x ◽

1987 ◽

Vol 45 ◽

pp. 180-181

Author(s):

R. Levi-Setti ◽

J.M. Chabala ◽

Y.L. Wang

Keyword(s):

Metal Ion ◽

Secondary Emission ◽

Scanning Method ◽

Ion Channeling ◽

Secondary Ions ◽

High Resolution Images ◽

Ion Microscopy ◽

Z Contrast ◽

Focused Beams ◽

Microscopy Images

Finely focused beams extracted from liquid metal ion sources (LMIS) provide a wealth of secondary signals which can be exploited to create high resolution images by the scanning method. The images of scanning ion microscopy (SIM) encompass a variety of contrast mechanisms which we classify into two broad categories: a) Emission contrast and b) Analytical contrast.Emission contrast refers to those mechanisms inherent to the emission of secondaries by solids under ion bombardment. The contrast-carrying signals consist of ion-induced secondary electrons (ISE) and secondary ions (ISI). Both signals exhibit i) topographic emission contrast due to the existence of differential geometric emission and collection effects, ii) crystallographic emission contrast, due to primary ion channeling phenomena and differential oxidation of crystalline surfaces, iii) chemical emission or Z-contrast, related to the dependence of the secondary emission yields on the Z and surface chemical state of the target.

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Freeze-etch TEM of aqueous polymer gel network morphology

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100145327 ◽

1986 ◽

Vol 44 ◽

pp. 796-797

Author(s):

J. A. N. Zasadzinski ◽

R. K. Prud'homme

Keyword(s):

Metal Ion ◽

Polymer Network ◽

Polymer Gels ◽

Polymer Gel ◽

Deformation History ◽

Crosslinked Polymer ◽

Aqueous Polymer ◽

History Of ◽

Gel Network ◽

Network Morphology

The rheological and mechanical properties of crosslinked polymer gels arise from the structure of the gel network. In turn, the structure of the gel network results from: thermodynamically determined interactions between the polymer chain segments, the interactions of the crosslinking metal ion with the polymer, and the deformation history of the network. Interpretations of mechanical and rheological measurements on polymer gels invariably begin with a conceptual model of,the microstructure of the gel network derived from polymer kinetic theory. In the present work, we use freeze-etch replication TEM to image the polymer network morphology of titanium crosslinked hydroxypropyl guars in an attempt to directly relate macroscopic phenomena with network structure.

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Structures of c-di-GMP/cGAMP degrading phosphodiesterase VcEAL: identification of a novel conformational switch and its implication

Biochemical Journal ◽

10.1042/bcj20190399 ◽

2019 ◽

Vol 476 (21) ◽

pp. 3333-3353 ◽

Cited By ~ 3

Author(s):

Malti Yadav ◽

Kamalendu Pal ◽

Udayaditya Sen

Keyword(s):

Active Site ◽

Metal Binding ◽

Metal Ion ◽

Triosephosphate Isomerase ◽

Catalytic Mechanism ◽

Degradation Mechanism ◽

Structural Basis ◽

Conserved Residues ◽

Phosphodiester Bond ◽

Cyclic Dinucleotides

Cyclic dinucleotides (CDNs) have emerged as the central molecules that aid bacteria to adapt and thrive in changing environmental conditions. Therefore, tight regulation of intracellular CDN concentration by counteracting the action of dinucleotide cyclases and phosphodiesterases (PDEs) is critical. Here, we demonstrate that a putative stand-alone EAL domain PDE from Vibrio cholerae (VcEAL) is capable to degrade both the second messenger c-di-GMP and hybrid 3′3′-cyclic GMP–AMP (cGAMP). To unveil their degradation mechanism, we have determined high-resolution crystal structures of VcEAL with Ca2+, c-di-GMP-Ca2+, 5′-pGpG-Ca2+ and cGAMP-Ca2+, the latter provides the first structural basis of cGAMP hydrolysis. Structural studies reveal a typical triosephosphate isomerase barrel-fold with substrate c-di-GMP/cGAMP bound in an extended conformation. Highly conserved residues specifically bind the guanine base of c-di-GMP/cGAMP in the G2 site while the semi-conserved nature of residues at the G1 site could act as a specificity determinant. Two metal ions, co-ordinated with six stubbornly conserved residues and two non-bridging scissile phosphate oxygens of c-di-GMP/cGAMP, activate a water molecule for an in-line attack on the phosphodiester bond, supporting two-metal ion-based catalytic mechanism. PDE activity and biofilm assays of several prudently designed mutants collectively demonstrate that VcEAL active site is charge and size optimized. Intriguingly, in VcEAL-5′-pGpG-Ca2+ structure, β5–α5 loop adopts a novel conformation that along with conserved E131 creates a new metal-binding site. This novel conformation along with several subtle changes in the active site designate VcEAL-5′-pGpG-Ca2+ structure quite different from other 5′-pGpG bound structures reported earlier.

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