scholarly journals Electrical Phenotypes of Calcium Transport Mutant Strains of a Filamentous Fungus, Neurospora crassa

2012 ◽  
Vol 11 (5) ◽  
pp. 694-702 ◽  
Author(s):  
Ahmed Hamam ◽  
Roger R. Lew
Keyword(s):  
Atpase Activity ◽  
Calcium Transport ◽  
Tip Growth ◽  
Electrical Characterization ◽  
Mechanosensitive Channel ◽  
Wild Type ◽  
Current Voltage ◽  
Mutant Strains ◽  
Genes Encoding ◽  
Hyphal Tip

ABSTRACT We characterized the electrical phenotypes of mutants with mutations in genes encoding calcium transporters—a mechanosensitive channel homolog ( MscS ), a Ca 2+ /H + exchange protein ( cax ), and Ca 2+ -ATPases ( nca-1 , nca-2 , nca-3 )—as well as those of double mutants (the nca-2 cax , nca-2 nca-3 , and nca-3 cax mutants). The electrical characterization used dual impalements to obtain cable-corrected current-voltage measurements. Only two types of mutants (the MscS mutant; the nca-2 mutant and nca-2 -containing double mutants) exhibited lower resting potentials. For the nca-2 mutant, on the basis of unchanged conductance and cyanide-induced depolarization of the potential, the cause is attenuated H + -ATPase activity. The growth of the nca-2 mutant-containing strains was inhibited by elevated extracellular Ca 2+ levels, indicative of lesions in Ca 2+ homeostasis. However, the net Ca 2+ effluxes of the nca-2 mutant, measured noninvasively with a self-referencing Ca 2+ -selective microelectrode, were similar to those of the wild type. All of the mutants exhibited osmosensitivity similar to that of the wild type (the turgor of the nca-2 mutant was also similar to that of the wild type), suggesting that Ca 2+ signaling does not play a role in osmoregulation. The hyphal tip morphology and tip-localized mitochondria of the nca-2 mutant were similar to those of the wild type, even when the external [Ca 2+ ] was elevated. Thus, although Ca 2+ homeostasis is perturbed in the nca-2 mutant (B. J. Bowman et al., Eukaryot. Cell 10:654–661, 2011), the phenotype does not extend to tip growth or to osmoregulation but is revealed by lower H + -ATPase activity.

scholarly journals act Operon Control of Developmental Gene Expression in Myxococcus xanthus

2002 ◽  
Vol 184 (4) ◽  
pp. 1172-1179 ◽  
Author(s):  
Thomas M. A. Gronewold ◽  
Dale Kaiser
Keyword(s):  
Fruiting Body ◽  
Myxococcus Xanthus ◽  
The Other ◽  
Loss Of Function ◽  
Wild Type ◽  
Developmental Gene ◽  
Developmental Gene Expression ◽  
Mutant Strains ◽  
Genes Encoding ◽  
Signal Production

ABSTRACT Cell-bound C-signal guides the building of a fruiting body and triggers the differentiation of myxospores. Earlier work has shown that transcription of the csgA gene, which encodes the C-signal, is directed by four genes of the act operon. To see how expression of the genes encoding components of the aggregation and sporulation processes depends on C-signaling, mutants with loss-of-function mutations in each of the act genes were investigated. These mutations were found to have no effect on genes that are normally expressed up to 3 h into development and are C-signal independent. Neither the time of first expression nor the rate of expression increase was changed in actA, actB, actC, or actD mutant strains. Also, there was no effect on A-signal production, which normally starts before 3 h. By contrast, the null act mutants have striking defects in C-signal production. These mutations changed the expression of four gene reporters that are related to aggregation and sporulation and are expressed at 6 h or later in development. The actA and actB null mutations substantially decreased the expression of all these reporters. The other act null mutations caused either premature expression to wild-type levels (actC) or delayed expression (actD), which ultimately rose to wild-type levels. The pattern of effects on these reporters shows how the C-signal differentially regulates the steps that together build a fruiting body and differentiate spores within it.

EnteropathogenicEscherichia coliinhibits butyrate uptake in Caco-2 cells by altering the apical membrane MCT1 level

2006 ◽  
Vol 290 (1) ◽  
pp. G30-G35 ◽  
Author(s):  
Alip Borthakur ◽  
Ravinder K. Gill ◽  
Kim Hodges ◽  
Krishnamurthy Ramaswamy ◽  
Gail Hecht ◽  
...  
Keyword(s):  
Surface Expression ◽  
Monocarboxylate Transporter ◽  
Wild Type ◽  
Infantile Diarrhea ◽  
Monocarboxylate Transporter 1 ◽  
E Coli ◽  
Food Borne ◽  
Mutant Strains ◽  
Genes Encoding ◽  
Fatty Acid Absorption

Enteropathogenic Escherichia coli (EPEC), a food-borne human pathogen, is responsible for infantile diarrhea, especially in developing countries. The pathophysiology of EPEC-induced diarrhea, however, is not completely understood. Our recent studies showed modulation of Na+/H+and Cl−/HCO3−exchange activities in Caco-2 cells in response to EPEC infection. We hypothesized that intestinal short-chain fatty acid absorption mediated by monocarboxylate transporter 1 (MCT1) might also be altered by EPEC infection. The aim of the current studies was to examine the effect of EPEC infection on butyrate uptake. Caco-2 cells were infected with wild-type EPEC, various mutant strains, or nonpathogenic E. coli HS4, and [14C]butyrate uptake was determined. EPEC, but not nonpathogenic E. coli, significantly decreased butyrate uptake. Infection of cells with strains harboring mutations in escN, which encodes a putative ATPase for the EPEC type III secretion system (TTSS), or in the espA, espB, or espD genes encoding structural components of the TTSS, had no effect on butyrate uptake, indicating the TTSS dependence. On the other hand, strains with mutations in the effector protein genes espF, espG, espH, and map inhibited butyrate uptake, similar to the wild-type EPEC. Surface expression of MCT1 decreased considerably after EPEC but not after nonpathogenic E. coli infection. In conclusion, our studies demonstrate inhibition of MCT1-mediated butyrate uptake in Caco-2 cells in response to EPEC infection. This inhibition was dependent on a functional TTSS and the structural proteins EspA, -B, and -D of the translocation apparatus.

scholarly journals Parallel β-Helix Proteins Required for Accurate Capsule Polysaccharide Synthesis and Virulence in the Yeast Cryptococcus neoformans

2007 ◽  
Vol 6 (4) ◽  
pp. 630-640 ◽  
Author(s):  
Oliver W. Liu ◽  
Mark J. S. Kelly ◽  
Eric D. Chow ◽  
Hiten D. Madhani
Keyword(s):  
Cryptococcus Neoformans ◽  
Capsular Polysaccharide ◽  
Resonance Spectroscopy ◽  
Wild Type ◽  
Repeat Proteins ◽  
Knockout Mutants ◽  
Polysaccharide Synthesis ◽  
Mutant Strains ◽  
Genes Encoding ◽  
Opportunistic Fungal Pathogen

ABSTRACT The principal capsular polysaccharide of the opportunistic fungal pathogen Cryptococcus neoformans consists of an α-1,3-linked mannose backbone decorated with a repeating pattern of glucuronyl and xylosyl side groups. This structure is critical for virulence, yet little is known about how the polymer, called glucuronoxylomannan (GXM), is faithfully synthesized and assembled. We have generated deletions in two genes encoding predicted parallel β-helix repeat proteins, which we have designated PBX1 and PBX2. Deletion of either gene results in a dry-colony morphology, clumpy cells, and decreased capsule integrity. Two-dimensional nuclear magnetic resonance spectroscopy of purified GXM from the mutants indicated that both the wild-type GXM structure and novel, aberrant linkages were present. Carbohydrate composition and linkage analysis determined that these aberrant structures are correlated with the incorporation of terminal glucose residues that are not found in wild-type capsule polysaccharide. We conclude that Pbx1 and Pbx2 are required for the fidelity of GXM synthesis and may be involved in editing incorrectly added glucose residues. PBX1 and PBX2 knockout mutants showed severely attenuated virulence in a murine inhalation model of cryptococcosis. Unlike acapsular strains, these mutant strains induced delayed symptoms of cryptococcosis, though the infected animals eventually contained the infection and recovered.

scholarly journals Arsenite efflux limits microbial arsenic volatilization

2019 ◽  
Author(s):  
Changdong Ke ◽  
Qingyue Kuang ◽  
Chungui Zhao ◽  
Jiafu Luo ◽  
Wenzhang Wei ◽  
...  
Keyword(s):  
Gene Deletion ◽  
Rhodopseudomonas Palustris ◽  
Intrinsic Factor ◽  
Model Organism ◽  
Efflux Transporters ◽  
Wild Type ◽  
Efflux Rate ◽  
Ic50 Value ◽  
Mutant Strains ◽  
Genes Encoding

Abstract Background: Arsenic (As) methylation is regarded as a potential way to volatize and thereby remove As from the environment. However, most microorganisms conducting As methylation display low As volatilization efficiency as As methylation is limited by As efflux transporters as both processes compete for arsenite [As(III)]. In the study, we deleted arsB and acr3 from Rhodopseudomonas palustris CGA009, a good model organism for studying As detoxification, and further investigated the effect of As(III) efflux transporters on As methylation. Results: Two mutants were obtained by gene deletion. Compared to the growth inhibition rate (IC50) [1.57±0.11 mmol/L As(III) and 2.67±0.04 mmol/L arsenate [As(V)] of wildtype R. palustris CGA009, the As(III) and As(V) resistance of the mutants decreased, and IC50 value of the R. palustris CGA009 ∆arsB mutant was 1.47±0.02 mmol/L As(III) and 2.12±0.03 mmol/L As(V), respectively, and that of the R. palustris CGA009 ∆acr3 mutant was 1.21±0.07 mmol/L As(III) and 1.76±0.12 mmol/L As(V), respectively. The As volatilization rate of R. palustris CGA009 ∆arsB and R. palustris CGA009 ∆acr3 was 7.36 and 10.46 times higher than that of the R. palustris CGA009 at 100.0 µmol/L As(III) when incubated for 12 h, respectively, and 7.21 and 10.30 times higher than that of the R. palustris CGA009 when incubated with 25.0 mol/L As(V), respectively. At 25.0 mol/L As(III), low doses of methylarsonate [MAs(V)] and dimethylarsonate [DMAs(V)] were detected in both the wild type and in the deletion mutant strains. In addition, the content of As(III) in the medium changed significantly with the order being R. palustris CGA009 > R. palustris CGA009 ∆arsB > R. palustris CGA009 ∆acr3, indicating that Acr3 displayed the highest As(III) efflux rate. Conclusion: The results of this study showed that As efflux transporters were shown to be a remarkable intrinsic factor limiting As volatilization efficiency, and As volatilization rate could be significantly improved by deleting genes encoding microbial As efflux transporters. Our study provided an explanation for the often low rate of microbial As methylation and an effective strategy for screening microorganisms with high As volatilization.

scholarly journals Identification of Major Sporulation Proteins of Myxococcus xanthus Using a Proteomic Approach

2007 ◽  
Vol 189 (8) ◽  
pp. 3187-3197 ◽  
Author(s):  
John L. Dahl ◽  
Farah K. Tengra ◽  
David Dutton ◽  
Jinyuan Yan ◽  
Tracy M. Andacht ◽  
...  
Keyword(s):  
Fruiting Body ◽  
Myxococcus Xanthus ◽  
Sodium Dodecyl ◽  
Protein S ◽  
Differentially Expressed ◽  
Insertion Mutagenesis ◽  
Wild Type ◽  
Proteomic Approach ◽  
Mutant Strains ◽  
Genes Encoding

ABSTRACT Myxococcus xanthus is a soil-dwelling, gram-negative bacterium that during nutrient deprivation is capable of undergoing morphogenesis from a vegetative rod to a spherical, stress-resistant spore inside a domed-shaped, multicellular fruiting body. To identify proteins required for building stress-resistant M. xanthus spores, we compared the proteome of liquid-grown vegetative cells with the proteome of mature fruiting body spores. Two proteins, protein S and protein S1, were differentially expressed in spores, as has been reported previously. In addition, we identified three previously uncharacterized proteins that are differentially expressed in spores and that exhibit no homology to known proteins. The genes encoding these three novel major spore proteins (mspA, mspB, and mspC) were inactivated by insertion mutagenesis, and the development of the resulting mutant strains was characterized. All three mutants were capable of aggregating, but for two of the strains the resulting fruiting bodies remained flattened mounds of cells. The most pronounced structural defect of spores produced by all three mutants was an altered cortex layer. We found that mspA and mspB mutant spores were more sensitive specifically to heat and sodium dodecyl sulfate than wild-type spores, while mspC mutant spores were more sensitive to all stress treatments examined. Hence, the products of mspA, mspB, and mspC play significant roles in morphogenesis of M. xanthus spores and in the ability of spores to survive environmental stress.

Ca++-ATPase activity in the hepatopancreas cells of Oniscus asellus

1992 ◽  
Vol 50 (1) ◽  
pp. 820-821
Author(s):  
G.M. Vernon ◽  
A. Surace ◽  
R. Witkus
Keyword(s):  
B Cells ◽  
Epithelial Cell ◽  
Atpase Activity ◽  
Calcium Transport ◽  
Carcinus Maenas ◽  
Cell Types ◽  
Molt Cycle ◽  
Copper And Zinc

The hepatopancreas consists of a pair of bilobed tubules comprised of two epithelial cell types. S cells are absorptive and accumulate metals such as copper and zinc. Ca++ concentrations vary between the S and B cells and during the molt cycle. Roer and Dillaman implicated Ca++-ATPase in calcium transport during molting in Carcinus maenas. This study was undertaken to compare the localization of Ca++-ATPase activity in the S and B cells during intermolt.

Potent Chitin Synthase Inhibitors from Plants

2020 ◽  
Vol 16 (1) ◽  
pp. 58-63
Author(s):  
Amrutha Vijayakumar ◽  
Ajith Madhavan ◽  
Chinchu Bose ◽  
Pandurangan Nanjan ◽  
Sindhu S. Kokkal ◽  
...  
Keyword(s):  
Cell Wall ◽  
Candida Albicans ◽  
Caenorhabditis Elegans ◽  
Aspergillus Niger ◽  
Small Molecules ◽  
Tip Growth ◽  
Chitin Synthase ◽  
Chitin Synthesis ◽  
Hyphal Tip Growth ◽  
Hyphal Tip

Background: Chitin is the main component of fungal, protozoan and helminth cell wall. They help to maintain the structural and functional characteristics of these organisms. The chitin wall is dynamic and is repaired, rearranged and synthesized as the cells develop. Active synthesis can be noticed during cytokinesis, laying of primary septum, maintenance of lateral cell wall integrity and hyphal tip growth. Chitin synthesis involves coordinated action of two enzymes namely, chitin synthase (that lays new cell wall) and chitinase (that removes the older ones). Since chitin synthase is conserved in different eukaryotic microorganisms that can be a ‘soft target’ for inhibition with small molecules. When chitin synthase is inhibited, it leads to the loss of viability of cells owing to the self- disruption of the cell wall by existing chitinase. Methods: In the described study, small molecules from plant sources were screened for their ability to interfere with hyphal tip growth, by employing Hyphal Tip Burst assay (HTB). Aspergillus niger was used as the model organism. The specific role of these small molecules in interfering with chitin synthesis was established with an in-vitro method. The enzyme required was isolated from Aspergillus niger and its activity was deduced through a novel method involving non-radioactively labelled substrate. The activity of the potential lead molecules were also checked against Candida albicans and Caenorhabditis elegans. The latter was adopted as a surrogate for the pathogenic helminths as it shares similarity with regard to cell wall structure and biochemistry. Moreover, it is widely studied and the methodologies are well established. Results: Out of the 11 compounds and extracts screened, 8 were found to be prospective. They were also found to be effective against Candida albicans and Caenorhabditis elegans. Conclusion: Purified Methyl Ethyl Ketone (MEK) Fraction1 (F1) of Coconut (Cocos nucifera) Shell Extract (COSE) was found to be more effective against Candida albicans with an IC50 value of 3.04 μg/mL and on L4 stage of Caenorhabditis elegans with an IC50 of 77.8 μg/mL.

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