scholarly journals Impact of Multiple β-Ketothiolase Deletion Mutations in Ralstonia eutropha H16 on the Composition of 3-Mercaptopropionic Acid-Containing Copolymers

2010 ◽  
Vol 76 (16) ◽  
pp. 5373-5382 ◽  
Author(s):  
Nicole Lindenkamp ◽  
Katja Peplinski ◽  
Elena Volodina ◽  
Armin Ehrenreich ◽  
Alexander Steinbüchel
Keyword(s):  
Ralstonia Eutropha ◽  
Dry Weight ◽  
Deletion Mutants ◽  
Transcriptome Data ◽  
Wild Type ◽  
Acetyl Coenzyme A ◽  
Deletion Mutations ◽  
Knockout Mutants ◽  
In The Wild ◽  
Percentage Ratio

ABSTRACT β-Ketothiolases catalyze the first step of poly(3-hydroxybutyrate) [poly(3HB)] synthesis in bacteria by condensing two molecules of acetyl coenzyme A (acetyl-CoA) to acetoacetyl-CoA. Analyses of the genome sequence of Ralstonia eutropha H16 revealed 15 isoenzymes of PhaA in this bacterium. In this study, we generated knockout mutants of various phaA homologues to investigate their role in and contributions to poly(3HB) metabolism and to suppress biosynthesis of 3HB-CoA for obtaining enhanced molar 3-mercaptopriopionate (3MP) contents in poly(3HB-co-3MP) copolymers when cells were grown on gluconate plus 3-mercaptopropionate or 3,3′-dithiodipropionate. In silico sequence analysis of PhaA homologues, transcriptome data, and other aspects recommended the homologues phaA, bktB, H16_A1713/H16_B1771, H16_A1528, H16_B1369, H16_B0381, and H16_A0170 for further analysis. Single- and multiple-deletion mutants were generated to investigate the influence of these β-ketothiolases on growth and polymer accumulation. The deletion of single genes resulted in no significant differences from the wild type regarding growth and polymer accumulation during cultivation on gluconate or gluconate plus 3MP. Deletion of phaA plus bktB (H16Δ2 mutant) resulted in approximately 30% less polymer accumulation than in the wild type. Deletion of H16_A1713/H16_B1771, H16_A1528, H16_B0381, and H16_B1369 in addition to phaA and bktB gave no differences in comparison to the H16Δ2 mutant. In contrast, deletion of H16_A0170 additionally to phaA and bktB yielded a mutant which accumulated about 30% poly(3HB) (wt/wt of the cell dry weight [CDW]). Although we were not able to suppress poly(3HB) biosynthesis completely, the copolymer compositions could be altered significantly with a lowered percentage ratio of 3HB constituents (from 85 to 52 mol%) and an increased percentage ratio of 3MP constituents (from 15 to 48 mol%), respectively. In this study, we demonstrated that PhaA, BktB, and H16_A0170 are majorly involved in poly(3HB) synthesis in R. eutropha H16. A fourth β-ketothiolase or a combination of several of the other β-ketothiolases contributed to a maximum of only 30% (wt/wt of CDW) of the remaining (co)polymer.

scholarly journals Exopolyphosphatases PPX1 and PPX2 from Corynebacterium glutamicum

2009 ◽  
Vol 75 (10) ◽  
pp. 3161-3170 ◽  
Author(s):  
Steffen N. Lindner ◽  
Sandra Knebel ◽  
Hendrik Wesseling ◽  
Siegfried M. Schoberth ◽  
Volker F. Wendisch
Keyword(s):  
Corynebacterium Glutamicum ◽  
Type Strain ◽  
Wild Type Strain ◽  
Deletion Mutants ◽  
Gene Products ◽  
Wild Type ◽  
Inorganic Polyphosphate ◽  
Deletion Mutations ◽  
In The Wild ◽  
Growth Experiments

ABSTRACT Corynebacterium glutamicum accumulates up to 300 mM of inorganic polyphosphate (PolyP) in the cytosol or in granules. The gene products of cg0488 (ppx1) and cg1115 (ppx2) were shown to be active as exopolyphosphatases (PPX), as overexpression of either gene resulted in higher exopolyphosphatase activities in crude extracts and deletion of either gene with lower activities than those of the wild-type strain. PPX1 and PPX2 from C. glutamicum share only 25% identical amino acids and belong to different protein groups, which are distinct from enterobacterial, archaeal, and yeast exopolyphosphatases. In comparison to that in the wild type, more intracellular PolyP accumulated in the Δppx1 and Δppx2 deletion mutations but less when either ppx1 or ppx2 was overexpressed. When C. glutamicum was shifted from phosphate-rich to phosphate-limiting conditions, a growth advantage of the deletion mutants and a growth disadvantage of the overexpression strains compared to the wild type were observed. Growth experiments, exopolyphosphatase activities, and intracellular PolyP concentrations revealed PPX2 as being a major exopolyphosphatase from C. glutamicum. PPX2His was purified to homogeneity and shown to be active as a monomer. The enzyme required Mg2+ or Mn2+ cations but was inhibited by millimolar concentrations of Mg2+, Mn2+, and Ca2+. PPX2 from C. glutamicum was active with short-chain polyphosphates, even accepting pyrophosphate, and was inhibited by nucleoside triphosphates.

scholarly journals Arabidopsis thaliana cbp80, c2h2, and flk Knockout Mutants Accumulate Increased Amounts of Circular RNAs

Cells ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 1937
Author(s):  
Anna Philips ◽  
Katarzyna Nowis ◽  
Michal Stelmaszczuk ◽  
Jan Podkowiński ◽  
Luiza Handschuh ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Circular Rnas ◽  
Wild Type ◽  
Proper Order ◽  
Knockout Mutants ◽  
In The Wild ◽  
And Function

Circular RNAs (circRNAs) are the products of the non-canonical splicing of pre-mRNAs. In contrast to humans and animals, our knowledge of the biogenesis and function of circRNAs in plants is very scarce. To identify proteins involved in plant circRNA generation, we characterized the transcriptomes of 18 Arabidopsis thaliana knockout mutants for genes related to splicing. The vast majority (>90%) of circRNAs were formed in more than one variant; only a small fraction of circRNAs was mutant-specific. Five times more circRNA types were identified in cbp80 and three times more in c2h2 mutants than in the wild-type. We also discovered that in cbp80, c2h2 and flk mutants, the accumulation of circRNAs was significantly increased. The increased accumulation of circular transcripts was not accompanied by corresponding changes in the accumulation of linear transcripts. Our results indicate that one of the roles of CBP80, C2H2 and FLK in splicing is to ensure the proper order of the exons. In the absence of one of the above-mentioned factors, the process might be altered, leading to the production of circular transcripts. This suggests that the transition toward circRNA production can be triggered by factors sequestering these proteins. Consequently, the expression of linear transcripts might be regulated through circRNA production.

scholarly journals A sterol C-14 reductase encoded by FgERG24B is responsible for the intrinsic resistance of Fusarium graminearum to amine fungicides

Microbiology ◽  
2011 ◽  
Vol 157 (6) ◽  
pp. 1665-1675 ◽  
Author(s):  
Xin Liu ◽  
Jing Fu ◽  
Yingzi Yun ◽  
Yanni Yin ◽  
Zhonghua Ma
Keyword(s):  
Fusarium Graminearum ◽  
Type Strain ◽  
Wild Type Strain ◽  
Ergosterol Biosynthesis ◽  
Deletion Mutants ◽  
Wild Type ◽  
Head Blight ◽  
Intrinsic Resistance ◽  
In The Wild ◽  
Increased Sensitivity

Fusarium graminearum, the causal agent of wheat head blight, shows intrinsic resistance to amine fungicides. It is commonly accepted that the amines target sterol C-14 reductase and sterol Δ8–Δ7 isomerase of ergosterol biosynthesis, encoded by the genes ERG24 and ERG2, respectively. Analysis of the genome sequence of F. graminearum revealed that the fungus contains two paralogous FgERG24 genes (FgERG24A and FgERG24B), which are homologous to the ERG24 of Saccharomyces cerevisiae. In this study, we disrupted FgERG24A and FgERG24B in F. graminearum. Compared to the wild-type strain HN9-1, FgERG24A and FgERG24B deletion mutants did not show recognizable phenotypic changes in mycelial growth on potato dextrose agar or in virulence on wheat heads. HPLC analysis showed that the amount of ergosterol in FgERG24A or FgERG24B deletion mutants was not significantly different from that in the wild-type strain. These results indicate that neither of the two genes is essential for growth, pathogenicity or ergosterol biosynthesis in F. graminearum. FgERG24B deletion mutants exhibited significantly increased sensitivity to amine fungicides, including tridemorph, fenpropidin and spiroxamine, but not to non-amine fungicides. In contrast, FgERG24A deletion mutants did not show changed sensitivity to any amine tested. The resistance of the FgERG24B deletion mutant to amines was restored by genetic complementation of the mutant with wild-type FgERG24B. These results indicate that FgERG24B controls the intrinsic resistance of F. graminearum to amines. The finding of this study provides new insights into amine resistance in filamentous fungi.

Influence of double-stranded RNAs on growth, sporulation, pathogenicity, and survival of Chalara elegans

1995 ◽  
Vol 73 (7) ◽  
pp. 1001-1009 ◽  
Author(s):  
Zamir K. Punja
Keyword(s):  
Plant Pathogen ◽  
Dry Weight ◽  
Complete Loss ◽  
Thielaviopsis Basicola ◽  
Wild Type ◽  
C Elegans ◽  
Chalara Elegans ◽  
In The Wild ◽  
Rna Fragments ◽  
Type Strains

Three strains of Chalara elegans from diverse geographical areas that contained multiple (4 or 5) double-stranded RNA fragments were compared with spontaneously derived cultures from these strains that were either partially cured or completely free of dsRNA. In the wild-type strains, presence of the dsRNAs was found to significantly enhance phialospore production and pigmentation of colonies, whereas radial growth and mycelial dry weight accumulation were reduced. The rate and overall percentage of phialospore germination on 1% Noble water agar were also significantly reduced by the presence of the dsRNAs. In two partially cured strains (only one 2.8-kb fragment remaining), pathogenicity to various plant tissues was significantly enhanced when compared with the wild-type strains containing multiple dsRNA. However, survival in field soil was enhanced in one strain and reduced in the other. In the completely cured strain, the loss of multiple dsRNA fragments was associated with enhanced growth, reduced phialospore production, and a complete loss of pathogenicity and capability for survival in soil. These results indicate that the effects of dsRNAs in C. elegans vary with the strain. In general, the presence of multiple dsRNAs in this fungus enhanced sporulation, altered colony morphology, and reduced growth and pathogenicity. However, since the complete loss of dsRNA was found to eliminate pathogenicity and reduce survival, it suggests that some dsRNA fragments in C. elegans may confer an advantage to this soil-borne facultative plant pathogen. Key words: black root rot, soil-borne plant pathogen, Thielaviopsis basicola.

scholarly journals Importance of Different tfd Genes for Degradation of Chloroaromatics by Ralstonia eutropha JMP134

2002 ◽  
Vol 184 (15) ◽  
pp. 4054-4064 ◽  
Author(s):  
Iris Plumeier ◽  
Danilo Pérez-Pantoja ◽  
Sabina Heim ◽  
Bernardo González ◽  
Dietmar H. Pieper
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Ralstonia Eutropha ◽  
Major Product ◽  
Kinetic Properties ◽  
Gene Module ◽  
Wild Type ◽  
In The Wild ◽  
Gene Modules ◽  
Chloromuconate Cycloisomerase

ABSTRACT The tfdC I D I E I F I, and tfdD II C II E II F II gene modules of plasmid pJP4 of Ralstonia eutropha JMP134 encode complete sets of functional enzymes for the transformation of chlorocatechols into 3-oxoadipate, which are all expressed during growth on 2,4-dichlorophenoxyacetate (2,4-D). However, activity of tfd I-encoded enzymes was usually higher than that of tfd II-encoded enzymes, both in the wild-type strain grown on 2,4-D and in 3-chlorobenzoate-grown derivatives harboring only one tfd gene module. The tfdD II-encoded chloromuconate cycloisomerase exhibited special kinetic properties, with high activity against 3-chloromuconate and poor activity against 2-chloromuconate and unsubstituted muconate, thus explaining the different phenotypic behaviors of R. eutropha strains containing different tfd gene modules. The enzyme catalyzes the formation of an equilibrium between 2-chloromuconate and 5-chloro- and 2-chloromuconolactone and very inefficiently catalyzes dehalogenation to form trans-dienelactone as the major product, thus differing from all (chloro)muconate cycloisomerases described thus far.

scholarly journals Effects of Homologous Phosphoenolpyruvate-Carbohydrate Phosphotransferase System Proteins on Carbohydrate Uptake and Poly(3-Hydroxybutyrate) Accumulation in Ralstonia eutropha H16

2011 ◽  
Vol 77 (11) ◽  
pp. 3582-3590 ◽  
Author(s):  
Chlud Kaddor ◽  
Alexander Steinbüchel
Keyword(s):  
Ralstonia Eutropha ◽  
Insertion Site ◽  
Carbon Sources ◽  
Sugar Transport ◽  
Dry Weight ◽  
Wild Type ◽  
Phosphotransferase System ◽  
Homologous Proteins ◽  
Content Type ◽  
Negative Effect

ABSTRACTSeven gene loci encoding putative proteins of the phosphoenolpyruvate-carbohydrate phosphotransferase system (PEP-PTS) were identified in the genome ofRalstonia eutrophaH16 byin silicoanalysis. Except theN-acetylglucosamine-specific PEP-PTS, an additional complete PEP-PTS is lacking in strain H16. Based on these findings, we generated single and multiple deletion mutants defective mainly in the PEP-PTS genes to investigate their influence on carbon source utilization, growth behavior, and poly(3-hydroxybutyrate) (PHB) accumulation. As supposed, the H16 ΔfrcACBand H16 ΔnagFECmutants exhibited no growth when cultivated on fructose andN-acetylglucosamine, respectively. Furthermore, a transposon mutant with aptsM-ptsHinsertion site did not grow on both carbon sources. The observed phenotype was not complemented, suggesting that it results from an interaction of genes or a polar effect caused by the Tn5::mobinsertion.ptsM,ptsH, andptsIsingle, double, and triple mutants stored much less PHB than the wild type (about 10 to 39% [wt/wt] of cell dry weight) and caused reduced PHB production in mutants lacking the H16_A2203, H16_A0384,frcACB, ornagFECgenes. In contrast, mutant H16 ΔH16_A0384 accumulated 11.5% (wt/wt) more PHB than the wild type when grown on gluconate and suppressed partially the negative effect of theptsMHIdeletion on PHB synthesis. Based on our experimental data, we discussed whether the PEP-PTS homologous proteins inR. eutrophaH16 are exclusively involved in the complex sugar transport system or whether they are also involved in cellular regulatory functions of carbon and PHB metabolism.

The nitrate-tolerant symbiosis of Glycine max (L.) Merr. nts382 and Bradyrhizobium japonicum is also tolerant of ammonium

2000 ◽  
Vol 77 (10) ◽  
pp. 1432-1438
Author(s):  
J Kevin Vessey ◽  
Bert Luit
Keyword(s):  
Glycine Max ◽  
Bradyrhizobium Japonicum ◽  
Continuous Flow ◽  
Dry Weight ◽  
Hydroponic Culture ◽  
Wild Type ◽  
Whole Plant ◽  
In The Wild ◽  
Glycine Max L ◽  
Wild Type Parent

Previously, we have shown that the Glycine max (L.) Merr. -Bradyrhizobium japonicum symbiosis is very sensitive to inhibition by NH4+. The current study addresses whether the supernodulating soybean mutant, nts382, which is known to be tolerant of NO3-, is also tolerant of NH4+. The nts382 mutant and its wild-type parent, Bragg, were grown in continuous-flow hydroponic culture in the presence of 0, 0.25, 0.5, or 1.0 mM 15N-enriched NH4+. Plants were harvested at 14, 21, and 28 days after inoculation. Both cultivars had the highest dry weight (DW) at each harvest date when grown on 0.25 mM NH4+. At 0.25 mM NH4+, whole plant DW increased by 5.3- and 3.2-fold in Bragg and nts382, respectively, compared with the 0.0 mM NH4+ control by the end of the experiment. As expected, whole-plant nodulation (nodules per plant), DW-specific nodulation (nodules per gram root dry weight), and nodule DW were severely inhibited in Bragg at all levels of NH4+. However, in nts382, whole-plant nodulation was not affected by NH4+ treatment, and nodule DW increased by as much as fivefold. Whereas DW-specific nodulation decreased by 94% in Bragg, this parameter decreased by only 52% in the nts382 mutant. Likewise, while the nitrogen derived from the atmosphere decreased by approximately 40% in NH4+-supplied Bragg, it increased 2.8-fold at 0.25 and 0.5 mM NH4+ in nts382. This study demonstrates that both nodulation and N2 fixation in nts382 are more tolerant of NH4+ than in the wild-type Bragg.

scholarly journals A Role of Calcium Signaling Genes in Heterokaryon Incompatibility in Neurospora crassa

2015 ◽  
Vol 3 (4) ◽  
pp. 668-679
Author(s):  
Ravi Gedela ◽  
Ranjan Tamuli
Keyword(s):  
Neurospora Crassa ◽  
Mating Type ◽  
Gene Deletion ◽  
Deletion Mutants ◽  
Gene Products ◽  
Wild Type ◽  
Heterokaryon Incompatibility ◽  
Knockout Mutants ◽  
Wild Type Control

We have studied the Ca2+-signaling knockout mutants for their role in mating-type-associated heterokaryon incompatibility in Neurospora crassa.  The found results showed on heterokaryons homokaryosis for DNCU05225, DNCU06366, DNCU06650, DNCU07075, and ∆NCU07966 Ca2+-signaling knockout mutants (Neurospora crassa unit number, NCU) displayed heterokaryon het compatibility; however heterokaryons heterokaryosis for DNCU05225, DNCU063665, DNCU06650, DNCU07075, and ∆NCU07966 mutants displayed het incompatibility like the wild-type control.  In addition to that Two Ca2+-signaling knockout mutants DNCU02283, and DNCU09655 were tested for mating-type-associated heterokaryon incompatibility; these results showed, heterokaryons homokaryosis and heterokaryons heterokaryosis for DNCU02283, DNCU09655 mutants displayed het incompatibility.  Cell death and hyphal compartmentation due to mating type associated incompatibility were confirmed by uptake of vital dye Evan’s blue.  Thus, these results of NCU05225, NCU06366, NCU06650, NCU07075, and NCU07966 Ca2+-signaling gene products could play a role in mating-type-associated heterokaryon incompatibility in N. crassa.  In this article, we are reporting initially screened Ca2+-signaling gene deletion mutants of these five acts as recessive suppressors of mating type associated vegetative incompatibility in N. crassa.Int J Appl Sci Biotechnol, Vol 3(4): 668-679

scholarly journals Accumulation of the PhaP Phasin of Ralstonia eutropha Is Dependent on Production of Polyhydroxybutyrate in Cells

2001 ◽  
Vol 183 (14) ◽  
pp. 4217-4226 ◽  
Author(s):  
Gregory M. York ◽  
Björn H. Junker ◽  
JoAnne Stubbe ◽  
Anthony J. Sinskey
Keyword(s):  
Regulatory Mechanism ◽  
Ralstonia Eutropha ◽  
Dry Weight ◽  
Gene Replacement ◽  
Pha Synthase ◽  
Wild Type ◽  
Phb Production ◽  
Associated Proteins ◽  
Mixed Populations ◽  
In Cells

ABSTRACT Polyhydroxyalkanoates (PHAs) are polyoxoesters that are produced by diverse bacteria and that accumulate as intracellular granules. Phasins are granule-associated proteins that accumulate to high levels in strains that are producing PHAs. The accumulation of phasins has been proposed to be dependent on PHA production, a model which is now rigorously tested for the phasin PhaP of Ralstonia eutropha. R. eutropha phaC PHA synthase and phaP phasin gene replacement strains were constructed. The strains were engineered to express heterologous and/or mutant PHA synthase alleles and aphaP-gfp translational fusion in place of the wild-type alleles of phaC and phaP. The strains were analyzed with respect to production of polyhydroxybutyrate (PHB), accumulation of PhaP, and expression of thephaP-gfp fusion. The results suggest that accumulation of PhaP is strictly dependent on the genetic capacity of strains to produce PHB, that PhaP accumulation is regulated at the level of both PhaP synthesis and PhaP degradation, and that, within mixed populations of cells, PhaP accumulation within cells of a given strain is not influenced by PHB production in cells of other strains. Interestingly, either the synthesis of PHB or the presence of relatively large amounts of PHB in cells (>50% of cell dry weight) is sufficient to enable PhaP synthesis. The results suggest that R. eutropha has evolved a regulatory mechanism that can detect the synthesis and presence of PHB in cells and that PhaP expression can be used as a marker for the production of PHB in individual cells.

Freeze-substituted fungal cells of Nectria haematococca: A comparison of the macroconidial walls of the adhesion-competent wild type with an adhesion-reduced mutant

1990 ◽  
Vol 48 (3) ◽  
pp. 688-689
Author(s):  
Thecan Caesar-Ton That ◽  
Lynn Epstein
Keyword(s):  
Freeze Substitution ◽  
Uranyl Acetate ◽  
Wild Type ◽  
Nectria Haematococca ◽  
Fruit Extract ◽  
Dialysis Tubing ◽  
Tube Emergence ◽  
Contrast Microscopy ◽  
In The Wild ◽  
Freeze Damage

Nectria haematococca mating population I (anamorph, Fusarium solani) macroconidia attach to its host (squash) and non-host surfaces prior to germ tube emergence. The macroconidia become adhesive after a brief period of protein synthesis. Recently, Hickman et al. (1989) isolated N. haematococca adhesion-reduced mutants. Using freeze substitution, we compared the development of the macroconidial wall in the wild type in comparison to one of the mutants, LEI.Macroconidia were harvested at 1C, washed by centrifugation, resuspended in a dilute zucchini fruit extract and incubated from 0 - 5 h. During the incubation period, wild type macroconidia attached to uncoated dialysis tubing. Mutant macroconidia did not attach and were collected on poly-L-lysine coated dialysis tubing just prior to freezing. Conidia on the tubing were frozen in liquid propane at 191 - 193C, substituted in acetone with 2% OsO4 and 0.05% uranyl acetate, washed with acetone, and flat-embedded in Epon-Araldite. Using phase contrast microscopy at 1000X, cells without freeze damage were selected, remounted, sectioned and post-stained sequentially with 1% Ba(MnO4)2 2% uranyl acetate and Reynold’s lead citrate. At least 30 cells/treatment were examined.

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