scholarly journals ISOLATION AND STUDY OF MUTANTS LACKING A DEREPRESSIBLE PHOSPHATASE IN CHLAMYDOMONAS REINHARDI

Genetics ◽  
1975 ◽  
Vol 80 (2) ◽  
pp. 239-250
Author(s):  
R F Matagne ◽  
R Loppes
Keyword(s):  
Alkaline Phosphatase ◽  
Inorganic Phosphate ◽  
Double Mutant ◽  
Culture Medium ◽  
Acid Phosphatases ◽  
Wild Type ◽  
Optimal Activity ◽  
Wide Range ◽  
In The Wild ◽  
Definition Of

ABSTRACT In the green alga Chlamydomonas reinhardi, removal of inorganic phosphate from the culture medium results in the increase of phosphatase activity (derepression) in the wild-type (WT) strain as well as in a double mutant (P2Pa) lacking the two main constitutive acid phosphatases. Following treatment of WT and P2Pa with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), mutants were recovered which display very low phosphatase activities when grown in the absence of phosphate; as shown by electrophoresis, they lack one non-migrating phosphatase (PD mutants). This enzyme is active over a wide range of pH with an optimum at pH 7.5. The comparison of electropherograms from WT and mutants grown on media with or without phosphate allowed us to provide a tentative definition of the pool of derepressible phosphatases in Chlamydomonas : in addition to the neutral phosphatase lacking in PD mutants, Chlamydomonas produces two electrophoretic forms of alkaline phosphatase showing an optimal activity at pH 9.5.

scholarly journals Aspergillus nidulans Protein O-Mannosyltransferases Play Roles in Cell Wall Integrity and Developmental Patterning

2009 ◽  
Vol 8 (10) ◽  
pp. 1475-1485 ◽  
Author(s):  
Thanyanuch Kriangkripipat ◽  
Michelle Momany
Keyword(s):  
Cell Wall ◽  
Aspergillus Nidulans ◽  
Double Mutant ◽  
Elevated Temperatures ◽  
Cell Wall Integrity ◽  
Wild Type ◽  
Spatial Cues ◽  
Developmental Patterning ◽  
In The Wild ◽  
Increased Sensitivity

ABSTRACT Protein O-mannosyltransferases (Pmts) initiate O-mannosyl glycan biosynthesis from Ser and Thr residues of target proteins. Fungal Pmts are divided into three subfamilies, Pmt1, -2, and -4. Aspergillus nidulans possesses a single representative of each Pmt subfamily, pmtA (subfamily 2), pmtB (subfamily 1), and pmtC (subfamily 4). In this work, we show that single Δpmt mutants are viable and have unique phenotypes and that the ΔpmtA ΔpmtB double mutant is the only viable double mutant. This makes A. nidulans the first fungus in which all members of individual Pmt subfamilies can be deleted without loss of viability. At elevated temperatures, all A. nidulans Δpmt mutants show cell wall-associated defects and increased sensitivity to cell wall-perturbing agents. The Δpmt mutants also show defects in developmental patterning. Germ tube emergence is early in ΔpmtA and more frequent in ΔpmtC mutants than in the wild type. In ΔpmtB mutants, intrahyphal hyphae develop. All Δpmt mutants show distinct conidiophore defects. The ΔpmtA strain has swollen vesicles and conidiogenous cells, the ΔpmtB strain has swollen conidiophore stalks, and the ΔpmtC strain has dramatically elongated conidiophore stalks. We also show that AN5660, an ortholog of Saccharomyces cerevisiae Wsc1p, is modified by PmtA and PmtC. The Δpmt phenotypes at elevated temperatures, increased sensitivity to cell wall-perturbing agents and restoration to wild-type growth with osmoticum suggest that A. nidulans Pmts modify proteins in the cell wall integrity pathway. The altered developmental patterns in Δpmt mutants suggest that A. nidulans Pmts modify proteins that serve as spatial cues.

scholarly journals Deciphering the evolution of microbial interactions: in silico studies of two-member microbial communities

2022 ◽  
Author(s):  
Gayathri Sambamoorthy ◽  
Karthik Raman
Keyword(s):  
Microbial Communities ◽  
In Silico ◽  
Microbial Interactions ◽  
Interaction Patterns ◽  
Wild Type ◽  
Community Function ◽  
Evolutionary Forces ◽  
In Silico Studies ◽  
Wide Range ◽  
In The Wild

Microbes thrive in communities, embedded in a complex web of interactions. These interactions, particularly metabolic interactions, play a crucial role in maintaining the community structure and function. As the organisms thrive and evolve, a variety of evolutionary processes alter the interactions among the organisms in the community, although the community function remains intact. In this work, we simulate the evolution of two-member microbial communities in silico to study how evolutionary forces can shape the interactions between organisms. We employ genomescale metabolic models of organisms from the human gut, which exhibit a range of interaction patterns, from mutualism to parasitism. We observe that the evolution of microbial interactions varies depending upon the starting interaction and also on the metabolic capabilities of the organisms in the community. We find that evolutionary constraints play a significant role in shaping the dependencies of organisms in the community. Evolution of microbial communities yields fitness benefits in only a small fraction of the communities, and is also dependent on the interaction type of the wild-type communities. The metabolites cross-fed in the wild-type communities appear in only less than 50% of the evolved communities. A wide range of new metabolites are cross-fed as the communities evolve. Further, the dynamics of microbial interactions are not specific to the interaction of the wild-type community but vary depending on the organisms present in the community. Our approach of evolving microbial communities in silico provides an exciting glimpse of the dynamics of microbial interactions and offers several avenues for future investigations.

Production and control of extracellular enzymes in Micrococcus sodonensis

1974 ◽  
Vol 20 (1) ◽  
pp. 81-90 ◽  
Author(s):  
Cecily Mills ◽  
J. N. Campbell
Keyword(s):  
Alkaline Phosphatase ◽  
Inorganic Phosphate ◽  
Culture Medium ◽  
Extracellular Enzymes ◽  
Synthetic Medium ◽  
Culture Conditions ◽  
The Other ◽  
Organic Nutrients ◽  
And Control ◽  
Logarithmic Growth

Micrococcus sodonensis has been shown to produce several extracellular enzymes: an alkaline phosphatase, at least two forms of phosphodiesterase, a 5′-nucleotidase, and an alkaline proteinase. The quantitative release of these enzymes into the culture medium during logarithmic growth under all the various culture conditions tested indicates that these enzymes are truly extracellular in nature. Inorganic phosphate repressed the production of the alkaline phosphatase in synthetic as well as in complex media, whereas, the repression of the production of active diesterase and 5′-nucleotidase by inorganic phosphate was partly reversed by the addition of supplemental organic nutrients to the culture medium. Proteinase production was independent of the culture conditions used. A mutant strain of M. sodonensis with an altered production of diesterase was obtained; the other extracellular enzymes were unaffected. These results suggest that the extracellular enzymes of M. sodonensis are not produced in a pleiotropic fashion since the level of one of the enzymes can be changed without affecting a corresponding change in the levels of the other enzymes. An extracellular high molecular weight carbohydrate fraction was shown to be produced by M. sodonensis in synthetic medium. The fraction was also shown to contain glycoprotein.

A role for rhamnolipid in biofilm dispersion

Biofilms ◽  
2004 ◽  
Vol 1 (2) ◽  
pp. 91-99 ◽  
Author(s):  
S. R. Schooling ◽  
U. K. Charaf ◽  
D. G. Allison ◽  
P. Gilbert
Keyword(s):  
Surface Tension ◽  
Quorum Sensing ◽  
Biofilm Formation ◽  
Culture Medium ◽  
High Cell Density ◽  
Culture Media ◽  
Homoserine Lactone ◽  
Wild Type ◽  
In The Wild ◽  
Biofilm Dispersion

Biofilms are often considered as localized zones of high cell density. Quorum sensing provides a means for control of population processes and has been implicated in the regulation of biofilm activities. We present a role for quorum sensing in programmed detachment and dispersal processes. Biofilms of Pseudomonas aeruginosa PAO1 and its isogenic homoserine lactone (HSL) mutant P. aeruginosa PAO-JP2 were grown in batch culture on glass substrata; differences were found in the rate and extent of formation of biofilm. Climax communities were observed for PAO1 at 24 h. These were later accompanied by foaming, a drop in the surface tension of culture media and dispersal of the biofilm, after which no subsequent biofilm accretion occurred. PAO-JP2 cultures reformed biofilm post-detachment and did not foam. Prevention of biofilm reformation in the wild type was related to some component excreted into the culture medium. Rhamnolipid, a biosurfactant regulated by quorum sensing, was detected in PAO1 cultures. When rhamnolipid was added to freshly inoculated substrata, biofilm formation was inhibited. At 20 h, PAO1 biofilms were transferred to medium with added rhamnolipid: biofilm was relatively unaffected. Biofilm events were also studied in medium supplemented with N-butyryl-L-homoserine lactone, which is involved in the regulation of rhamnolipid synthesis. Both strains exhibited similar trends of rapid biofilm formation and dramatic changes in the rate and extent of biofilm accretion. In both cases, there was premature foaming, lowered surface tension and elevated rhamnolipid levels. A role for HSLs in maintenance of biofilm and events leading to dispersion of cells is proposed. This role would encompass dispersion but not necessarily detachment of cells from biofilm and supports a new function for rhamnolipid in pathogenesis, whereby rhamnolipid would promote the dissemination of cells from a nidus of infection.

Effects of sugars and polyols on basidiocarp formation in a phosphoglucose isomerase mutant of Coprinus cinereus

1984 ◽  
Vol 30 (1) ◽  
pp. 45-51 ◽  
Author(s):  
Hiroshi Nyunoya ◽  
Tsuneo Takemaru ◽  
Tatsuo Ishikawa
Keyword(s):  
Culture Medium ◽  
Complete Recovery ◽  
Biochemical Properties ◽  
Phosphoglucose Isomerase ◽  
Coprinus Cinereus ◽  
Partial Recovery ◽  
Wild Type ◽  
In The Wild ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Basidiocarp Formation

Several biochemical properties of a mutant deficient in phosphoglucose isomerase (pgi) of Coprinus cinereus were examined in connection with its ability to produce basidiocarps. Mycelium of the pgi mutant accumulated glucose-6-phosphate and showed higher glucose-6-phosphate dehydrogenase activity than wild type. A conventional fruiting medium did not support basidiocarp formation by the homozygous dikaryon pgi/pgi, but the addition of a reduced amount of glucose plus a compensatory amount of fructose to the culture medium resulted in partial recovery of fruiting ability. This modification of the culture medium decreased the intracellular glucose-6-phosphate to almost the same level as in the wild type. The addition of polyols such as mannitol resulted in complete recovery of fruiting ability by the dikaryon pgi/pgi without affecting the level of glucose-6-phosphate. Mycelium of the mutant showed an elevated activity of NAD-linked polyol dehydrogenase and an elevated intracellular NAD level, irrespective of whether the mycelium was grown in the presence or absence of polyol.

scholarly journals mrp, a Multigene, Multifunctional Locus inBacillus subtilis with Roles in Resistance to Cholate and to Na+ and in pH Homeostasis

1999 ◽  
Vol 181 (8) ◽  
pp. 2394-2402 ◽  
Author(s):  
Masahiro Ito ◽  
Arthur A. Guffanti ◽  
Bauke Oudega ◽  
Terry A. Krulwich
Keyword(s):  
Double Mutant ◽  
Ph Regulation ◽  
Inducible Promoter ◽  
Cytoplasmic Ph ◽  
Null Mutant ◽  
Wild Type ◽  
Ph Homeostasis ◽  
In The Wild ◽  
Single Transcript ◽  
Membrane Spanning

ABSTRACT A 5.9-kb region of the Bacillus subtilis chromosome is transcribed as a single transcript that is predicted to encode seven membrane-spanning proteins. Homologues of the first gene of this operon, for which the designation mrp (multiple resistance and pH adaptation) is proposed here, have been suggested to encode an Na+/H+ antiporter or a K+/H+ antiporter. In the present studies of theB. subtilis mrp operon, both polar and nonpolar mutations in mrpA were generated. Growth of these mutants was completely inhibited by concentrations of added Na+ as low as 0.3 M at pH 7.0 and 0.03 M at pH 8.3; there was no comparable inhibition by added K+. A null mutant that was constructed by full replacement of the mrp operon was even more Na+ sensitive. A double mutant with mutations in both mrpA and the multifunctional antiporter-encodingtetA(L) gene was no more sensitive than themrpA mutants to Na+, consistent with a major role for mrpA in Na+ resistance. Expression of mrpA from an inducible promoter, upon insertion into the amyE locus, restored significant Na+ resistance in both the polar and nonpolarmrpA mutants but did not restore resistance in the null mutant. The mrpA disruption also resulted in an impairment of cytoplasmic pH regulation upon a sudden shift in external pH from 7.5 to 8.5 in the presence of Na+ and, to some extent, K+ in the range from 10 to 25 mM. By contrast, themrpA tetA(L) double mutant, like the tetA(L) single mutant, completely lost its capacity for both Na+- and K+-dependent cytoplasmic pH regulation upon this kind of shift at cation concentrations ranging from 10 to 100 mM; thus, tetA(L) has a more pronounced involvement thanmrpA in pH regulation. Measurements of Na+efflux from the wild-type strain, the nonpolar mrpA mutant, and the complemented mutant indicated that inducible expression ofmrpA increased the rate of protonophore- and cyanide-sensitive Na+ efflux over that in the wild-type in cells preloaded with 5 mM Na+. The mrpA and null mutants showed no such efflux in that concentration range. This is consistent with MrpA encoding a secondary, proton motive force-energized Na+/H+ antiporter. Studies of a polar mutant that leads to loss of mrpFG and its complementation in trans by mrpF ormrpFG support a role for MrpF as an efflux system for Na+ and cholate. Part of the Na+ efflux capacity of the whole mrp operon products is attributable to mrpF. Neither mrpF nor mrpFGexpression in trans enhanced the cholate or Na+resistance of the null mutant. Thus, one or more other mrpgene products must be present, but not at stoichiometric levels, for stability, assembly, or function of both MrpF and MrpA expressed intrans. Also, phenotypic differences among themrp mutants suggest that functions in addition to Na+ and cholate resistance and pH homeostasis will be found among the remaining mrp genes.

scholarly journals REGULATION OF PHOSPHATE METABOLISM IN NEUROSPORA CRASSA. CHARACTERIZATION OF REGULATORY MUTANTS

Genetics ◽  
1973 ◽  
Vol 75 (1) ◽  
pp. 61-73
Author(s):  
John F Lehman ◽  
Mary K Gleason ◽  
Sandra K Ahlgren ◽  
Robert L Metzenberg
Keyword(s):  
Alkaline Phosphatase ◽  
Neurospora Crassa ◽  
Control Systems ◽  
Double Mutant ◽  
Phosphorus Metabolism ◽  
Phosphate Metabolism ◽  
Wild Type Allele ◽  
Wild Type ◽  
High Affinity

ABSTRACT A mutant of Neurospora crassa, called UW-6, differs from wild type in being partially constitutive for synthesis of a species of alkaline phosphatase, and also for a species of phosphate permease that has a high affinity for phosphate at high pH. UW-6 is possibly allelic with a mutant called nuc-2 that was previously isolated by Ishikawa. nuc-2 has the converse phenotype, in that it cannot be derepressed for either of these two activities. UW-6 is co-dominant with its wild-type allele in heterokaryons and in partial diploids. An unlinked mutant, nuc-1, is like nuc-2 in that it fails to make the alkaline phosphatase or the permease referred to above. nuc-1 is epistatic to UW-6 in the double mutant. The control of phosphorus metabolism is discussed, and is compared with some other control systems in filamentous fungi.

scholarly journals Genetic perturbation enhances functional heterogeneity in alkaline phosphatase

2021 ◽  
Author(s):  
Morito Sakuma ◽  
Shingo Honda ◽  
Hiroshi Ueno ◽  
Kentaro Miyazaki ◽  
Nobuhiko Tokuriki ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Catalytic Activity ◽  
Single Molecule ◽  
Single Point ◽  
Point Mutations ◽  
Clear Correlation ◽  
Wild Type ◽  
Functional Heterogeneity ◽  
Genetic Perturbations ◽  
In The Wild

Enzymes inherently exhibit molecule-to-molecule heterogeneity in catalytic activity or function, which underlies the acquisition of new functions in evolutionary processes. However, correlations between the functional heterogeneity of an enzyme and its multi-functionality or promiscuity remain elusive. In addition, the modulation of functional heterogeneity upon genetic perturbation is currently unexplored. Here, we quantitatively analyzed functional heterogeneity in the wild-type and 69 single-point mutants of Escherichia coli alkaline phosphatase (AP) by employing single-molecule assay with a femtoliter reactor array device. Most mutant enzymes exhibited higher functional heterogeneity than the wild-type enzyme, irrespective of catalytic activity. These results indicated that the wild-type AP minimizes functional heterogeneity, and single-point mutations can significantly expand the span of functional heterogeneity in AP. Moreover, we identified a clear correlation between functional heterogeneity and promiscuous activities. These findings suggest that enzymes can acquire greater functional heterogeneity following marginal genetic perturbations that concomitantly promote catalytic promiscuity.

scholarly journals Both galactosaminogalactan and α-1,3-glucan contribute to aggregation of Aspergillus oryzae hyphae in liquid culture

2019 ◽  
Author(s):  
Ken Miyazawa ◽  
Akira Yoshimi ◽  
Motoaki Sano ◽  
Fuka Tabata ◽  
Asumi Sugahara ◽  
...  
Keyword(s):  
Filamentous Fungi ◽  
Aspergillus Oryzae ◽  
Double Mutant ◽  
Liquid Culture ◽  
Bacterial Cells ◽  
Wild Type ◽  
Hydrogen Bond Formation ◽  
Relative Contribution ◽  
In The Wild ◽  
Increase Productivity

AbstractFilamentous fungi generally form aggregated hyphal pellets in liquid culture. We previously reported that α-1,3-glucan-deficient mutants of Aspergillus nidulans did not form hyphal pellets and their hyphae were fully dispersed, and we suggested that α-1,3-glucan functions in hyphal aggregation. Yet, Aspergillus oryzae α-1,3-glucan-deficient (AGΔ) mutants still form small pellets; therefore, we hypothesized that another factor responsible for forming hyphal pellets remains in these mutants. Here, we identified an extracellular matrix polysaccharide galactosaminogalactan (GAG) as such a factor. To produce a double mutant of A. oryzae (AG-GAGΔ), we disrupted the genes required for GAG biosynthesis in an AGΔ mutant. Hyphae of the double mutant were fully dispersed in liquid culture, suggesting that GAG is involved in hyphal aggregation in A. oryzae. Addition of partially purified GAG fraction to the hyphae of the AG-GAGΔ strain resulted in formation of mycelial pellets. Acetylation of the amino group in galactosamine of GAG weakened GAG aggregation, suggesting that hydrogen bond formation by this group is important for aggregation. Genome sequences suggest that α-1,3-glucan, GAG, or both are present in many filamentous fungi and thus may function in hyphal aggregation in these fungi. We also demonstrated that production of a recombinant polyesterase, CutL1, was higher in the AG-GAGΔ strain than in the wild-type and AGΔ strains. Thus, controlling hyphal aggregation factors of filamentous fungi may increase productivity in the fermentation industry.ImportanceProduction using filamentous fungi is an important part of the fermentation industry, but hyphal aggregation in these fungi in liquid culture limits productivity compared with that of yeast or bacterial cells. We found that galactosaminogalactan and α-1,3-glucan both function in hyphal aggregation in Aspergillus oryzae, and that the hyphae of a double mutant deficient in both polysaccharides become fully dispersed in liquid culture. We also revealed the relative contribution of α-1,3-glucan and galactosaminogalactan to hyphal aggregation. Recombinant protein production was higher in the double mutant than in the wild-type strain. Our research provides a potential technical innovation for the fermentation industry that uses filamentous fungi, as regulation of the growth characteristics of A. oryzae in liquid culture may increase productivity.

scholarly journals Osmotic stress induces phosphorylation of histone H3 at threonine 3 in pericentromeric regions of Arabidopsis thaliana

2015 ◽  
Vol 112 (27) ◽  
pp. 8487-8492 ◽  
Author(s):  
Zhen Wang ◽  
Juan Armando Casas-Mollano ◽  
Jianping Xu ◽  
Jean-Jack M. Riethoven ◽  
Chi Zhang ◽  
...  
Keyword(s):  
Osmotic Stress ◽  
Protein Kinases ◽  
Double Mutant ◽  
Histone H3 ◽  
Epigenetic Mark ◽  
Wild Type ◽  
Protein Coding ◽  
Transcriptional Reprogramming ◽  
Genome Wide ◽  
In The Wild

Histone phosphorylation plays key roles in stress-induced transcriptional reprogramming in metazoans but its function(s) in land plants has remained relatively unexplored. Here we report that an Arabidopsis mutant defective in At3g03940 and At5g18190, encoding closely related Ser/Thr protein kinases, shows pleiotropic phenotypes including dwarfism and hypersensitivity to osmotic/salt stress. The double mutant has reduced global levels of phosphorylated histone H3 threonine 3 (H3T3ph), which are not enhanced, unlike the response in the wild type, by drought-like treatments. Genome-wide analyses revealed increased H3T3ph, slight enhancement in trimethylated histone H3 lysine 4 (H3K4me3), and a modest decrease in histone H3 occupancy in pericentromeric/knob regions of wild-type plants under osmotic stress. However, despite these changes in heterochromatin, transposons and repeats remained transcriptionally repressed. In contrast, this reorganization of heterochromatin was mostly absent in the double mutant, which exhibited lower H3T3ph levels in pericentromeric regions even under normal environmental conditions. Interestingly, within actively transcribed protein-coding genes, H3T3ph density was minimal in 5′ genic regions, coincidental with a peak of H3K4me3 accumulation. This pattern was not affected in the double mutant, implying the existence of additional H3T3 protein kinases in Arabidopsis. Our results suggest that At3g03940 and At5g18190 are involved in the phosphorylation of H3T3 in pericentromeric/knob regions and that this repressive epigenetic mark may be important for maintaining proper heterochromatic organization and, possibly, chromosome function(s).

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