scholarly journals The Bacillus subtilis yqjI Gene Encodes the NADP+-Dependent 6-P-Gluconate Dehydrogenase in the Pentose Phosphate Pathway

2004 ◽  
Vol 186 (14) ◽  
pp. 4528-4534 ◽  
Author(s):  
Nicola Zamboni ◽  
Eliane Fischer ◽  
Dietmar Laudert ◽  
Stéphane Aymerich ◽  
Hans-Peter Hohmann ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Glucose Dehydrogenase ◽  
Reducing Power ◽  
Pentose Phosphate ◽  
Gene Encoding ◽  
Metabolic Intermediates ◽  
The Third ◽  
Key Enzyme ◽  
Sequence Similarities

ABSTRACT Despite the importance of the oxidative pentose phosphate (PP) pathway as a major source of reducing power and metabolic intermediates for biosynthetic processes, almost no direct genetic or biochemical evidence is available for Bacillus subtilis. Using a combination of knockout mutations in known and putative genes of the oxidative PP pathway and 13C-labeling experiments, we demonstrated that yqjI encodes the NADP+-dependent 6-P-gluconate dehydrogenase, as was hypothesized previously from sequence similarities. Moreover, YqjI was the predominant isoenzyme during glucose and gluconate catabolism, and its role in the oxidative PP pathway could not be played by either of two homologues, GntZ and YqeC. This conclusion is in contrast to the generally held view that GntZ is the relevant isoform; hence, we propose a new designation for yqjI, gndA, the monocistronic gene encoding the principal 6-P-gluconate dehydrogenase. Although we demonstrated the NAD+-dependent 6-P-gluconate dehydrogenase activity of GntZ, gntZ mutants exhibited no detectable phenotype on glucose, and GntZ did not contribute to PP pathway fluxes during growth on glucose. Since gntZ mutants grew normally on gluconate, the functional role of GntZ remains obscure, as does the role of the third homologue, YqeC. Knockout of the glucose-6-P dehydrogenase-encoding zwf gene was primarily compensated for by increased glycolytic fluxes, but about 5% of the catabolic flux was rerouted through the gluconate bypass with glucose dehydrogenase as the key enzyme.

scholarly journals Contrasting Effects of σE on Compartmentalization of σF Activity during Sporulation of Bacillus subtilis

2004 ◽  
Vol 186 (7) ◽  
pp. 1983-1990 ◽  
Author(s):  
David W. Hilbert ◽  
Vasant K. Chary ◽  
Patrick J. Piggot
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Bacillus Subtilis ◽  
Mother Cell ◽  
Cell Fates ◽  
Gene Encoding ◽  
Primitive Form ◽  
Small Proteins ◽  
Dna Translocase

ABSTRACT Spore formation by Bacillus subtilis is a primitive form of development. In response to nutrient starvation and high cell density, B. subtilis divides asymmetrically, resulting in two cells with different sizes and cell fates. Immediately after division, the transcription factor σF becomes active in the smaller prespore, which is followed by the activation of σE in the larger mother cell. In this report, we examine the role of the mother cell-specific transcription factor σE in maintaining the compartmentalization of gene expression during development. We have studied a strain with a deletion of the spoIIIE gene, encoding a DNA translocase, that exhibits uncompartmentalized σF activity. We have determined that the deletion of spoIIIE alone does not substantially impact compartmentalization, but in the spoIIIE mutant, the expression of putative peptidoglycan hydrolases under the control of σE in the mother cell destroys the integrity of the septum. As a consequence, small proteins can cross the septum, thereby abolishing compartmentalization. In addition, we have found that in a mutant with partially impaired control of σF, the activation of σE in the mother cell is important to prevent the activation of σF in this compartment. Therefore, the activity of σE can either maintain or abolish the compartmentalization of σF, depending upon the genetic makeup of the strain. We conclude that σE activity must be carefully regulated in order to maintain compartmentalization of gene expression during development.

scholarly journals Cloning and Functional Characterization of CsUGD2 in Cucumber (Cucumis sativus L.)

2018 ◽  
Vol 47 (2) ◽  
pp. 288-293
Author(s):  
Yitong DUAN ◽  
Shuocheng ZENG
Keyword(s):  
Glucose Dehydrogenase ◽  
Functional Characterization ◽  
Protein Alignment ◽  
Cucumis Sativus L ◽  
Irreversible Oxidation ◽  
Cucumber Fruit ◽  
Key Enzyme ◽  
Cucumber Roots

UGD gene encodes UDP-glucose dehydrogenase (UGD) which is a key enzyme in the biosynthesis of cell wall, and it catalyses the irreversible oxidation of UDP-glucose (UDP-Glc) into UDP-glucuronic acid (UDP-GlcA). In cucumber, the expression level of CsUGD2 genes was higher in phloem tissues of pedicel and fruit than that in stalk. This study investigated the function of CsUGD2 in cucumber by different methods. Structure analysis indicated that CsUGD2 gene only has an exon with a length of 1,443 bp. Protein alignment suggested that UGD protein was highly conservative in different species. Phylogenetic analysis showed that CsUGD2 protein and CmoUGD2 protein form a same clade which is far away from UGDs in Arabidopsis. Real-time fluorescence quantitative analysis of CsUGD2 in different tissues of cucumber in the same period showed that CsUGD2 expressed highest in the root of cucumber. When we transformed CsUGD2 into wild type Arabidopsis, the roots of transgenic plants were shorter and the flowering time was delayed.  These results suggested that CsUGD2 may play an important role in the development of cucumber roots and only act on the development of cucumber fruit when compared with other plants fruits. However, the role of CsUGD2 in regulating the growth and development process of cucumber need to further study.

scholarly journals Genetic and Physiological Characterization of Fructose-1,6-Bisphosphate Aldolase and Glyceraldehyde-3-Phosphate Dehydrogenase in the Crabtree-Negative Yeast Kluyveromyces lactis

2022 ◽  
Vol 23 (2) ◽  
pp. 772
Author(s):  
Rosaura Rodicio ◽  
Hans-Peter Schmitz ◽  
Jürgen J. Heinisch
Keyword(s):  
Kluyveromyces Lactis ◽  
Regulation Of Gene Expression ◽  
Carbon Sources ◽  
Pentose Phosphate ◽  
Gene Encoding ◽  
Physiological Characterization ◽  
Genes Encoding ◽  
Fructose 1,6 Bisphosphate

The milk yeast Kluyveromyces lactis degrades glucose through glycolysis and the pentose phosphate pathway and follows a mainly respiratory metabolism. Here, we investigated the role of two reactions which are required for the final steps of glucose degradation from both pathways, as well as for gluconeogenesis, namely fructose-1,6-bisphosphate aldolase (FBA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In silico analyses identified one gene encoding the former (KlFBA1), and three genes encoding isoforms of the latter (KlTDH1, KlTDH2, KlGDP1). Phenotypic analyses were performed by deleting the genes from the haploid K. lactis genome. While Klfba1 deletions lacked detectable FBA activity, they still grew poorly on glucose. To investigate the in vivo importance of the GAPDH isoforms, different mutant combinations were analyzed for their growth behavior and enzymatic activity. KlTdh2 represented the major glycolytic GAPDH isoform, as its lack caused a slower growth on glucose. Cells lacking both KlTdh1 and KlTdh2 failed to grow on glucose but were still able to use ethanol as sole carbon sources, indicating that KlGdp1 is sufficient to promote gluconeogenesis. Life-cell fluorescence microscopy revealed that KlTdh2 accumulated in the nucleus upon exposure to oxidative stress, suggesting a moonlighting function of this isoform in the regulation of gene expression. Heterologous complementation of the Klfba1 deletion by the human ALDOA gene renders K. lactis a promising host for heterologous expression of human disease alleles and/or a screening system for specific drugs.

scholarly journals Metabolic Engineering of Bacillus subtilis for 2.3-BDO production by introducing an exogenous NADPH/NADP+ regeneration system

2019 ◽  
Author(s):  
Huiling Liu ◽  
Shuangying Liu ◽  
Fengyu Xie ◽  
Xian Zhang ◽  
Meijuan Xu ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Metabolic Engineering ◽  
Carbon Flux ◽  
Glucose Dehydrogenase ◽  
Transcriptional Regulator ◽  
Pentose Phosphate ◽  
Regeneration System ◽  
Nadph Regeneration ◽  
By Products ◽  
Regeneration Systems

Abstract Background: Generally, glucose is transformed into pyruvate from glycolysis before the target products acetoin and 2,3-butanediol (2,3-BDO) are formed. Pentose Phosphate Pathway (PPP) is an inefficient synthetic pathway for pyruvate production from glucose in Bacillus subtilis. Previously, it was found that engineered PPP in B. subtilis unbalanced NADH and NADPH regeneration systems and affected acetoin and 2,3 -BDO production.Results: In this study, metabolic engineering strategies were proposed to redistribute carbon flux to 2,3-BDO via reconstructing intracellular cofactors regeneration systems. Firstly, extra copies of glucose dehydrogenase (GDH)and an exogenous NADPH-dependent 2,3-BDO dehydrogenase (TDH) were introduced into the GRAS strain B. subtilis 168 to introduce an exogenous NADPH/NADP + regeneration system and broaden 2,3-BDO production pathway. It was found that overexpressing the NADPH/NADP + regeneration system effectively improved 2,3-BDO production and inhibited NADH-dependent by-products accumulation. Subsequently, the disruption of lactate dehydrogenase (encoded by ldh ) by insertion of the transcriptional regulator ALsR, essential for the expression of alsSD (encoding two key enzymes for the conversion of pyruvate to acetoin) in B. subtilis, resulted in the recombinant strain in which alsSD was overexpressed and the pathway to lactate was blocked simultaneously. On fermentation by the result engineered strain, the highest 2,3-BDO concentration increased by18.43%, while the titers of main byproducts acetoin and lactate decreased by 22.03% and 64%, respectively.Conclusion: In this study, it shows that engineering PPP and reconstructing intracellular cofactors regeneration system could be an alternative strategy in the metabolic engineering of 2,3-BDO production in B. subtilis .

scholarly journals Localization of the Bacillus subtilis murB Gene within the dcw Cluster Is Important for Growth and Sporulation

2006 ◽  
Vol 188 (5) ◽  
pp. 1721-1732 ◽  
Author(s):  
Gonçalo Real ◽  
Adriano O. Henriques
Keyword(s):  
Growth Rate ◽  
Cell Wall ◽  
Bacillus Subtilis ◽  
Ectopic Expression ◽  
Normal Growth ◽  
Wild Type ◽  
Extra Copy ◽  
Gene Encoding ◽  
Key Enzyme ◽  
High Concentrations

ABSTRACT The Bacillus subtilis murB gene, encoding UDP-N-acetylenolpyruvoylglucosamine reductase, a key enzyme in the peptidoglycan (PG) biosynthetic pathway, is embedded in the dcw (for “division and cell wall”) cluster immediately upstream of divIB. Previous attempts to inactivate murB were unsuccessful, suggesting its essentiality. Here we show that the cell morphology, growth rate, and resistance to cell wall-active antibiotics of murB conditional mutants is a function of the expression level of murB. In one mutant, in which murB was insertionally inactivated in a merodiploid bearing a second xylose-inducible PxylA-murB allele, DivIB levels were reduced and a normal growth rate was achieved only if MurB levels were threefold that of the wild-type strain. However, expression of an extra copy of divIB restored normal growth at wild-type levels of MurB. In contrast, DivIB levels were normal in a second mutant containing an in-frame deletion of murB (ΔmurB) in the presence of the PxylA-murB gene. Furthermore, this strain grew normally with wild-type levels of MurB. During sporulation, the levels of MurB were highest at the time of synthesis of the spore cortex PG. Interestingly, the ΔmurB PxylA-murB mutant did not sporulate efficiently even at high concentrations of inducer. Since high levels of inducer did not interfere with sporulation of a murB + PxylA-murB strain, it appears that ectopic expression of murB fails to support efficient sporulation. These data suggest that coordinate expression of divIB and murB is important for growth and sporulation. The genetic context of the murB gene within the dcw cluster is unique to the Bacillus group and, taken together with our data, suggests that in these species it contributes to the optimal expression of cell division and PG biosynthetic functions during both vegetative growth and spore development.

scholarly journals Blocking Chromosome Translocation during Sporulation of Bacillus subtilis Can Result in Prespore-Specific Activation of σG That Is Independent of σE and of Engulfment

2006 ◽  
Vol 188 (20) ◽  
pp. 7267-7273 ◽  
Author(s):  
Vasant K. Chary ◽  
Panagiotis Xenopoulos ◽  
Patrick J. Piggot
Keyword(s):  
Bacillus Subtilis ◽  
Rna Polymerase ◽  
Mother Cell ◽  
Chromosome Translocation ◽  
Specific Gene ◽  
Main Function ◽  
Cell Compartment ◽  
Gene Encoding ◽  
Direct Activator

ABSTRACT Formation of spores by Bacillus subtilis is characterized by cell compartment-specific gene expression directed by four RNA polymerase σ factors, which are activated in the order σF-σE-σG-σK. Of these, σG becomes active in the prespore upon completion of engulfment of the prespore by the mother cell. Transcription of the gene encoding σG, spoIIIG, is directed in the prespore by RNA polymerase containing σF but also requires the activity of σE in the mother cell. When first formed, σG is not active. Its activation requires expression of additional σE-directed genes, including the genes required for completion of engulfment. Here we report conditions in which σG becomes active in the prespore in the absence of σE activity and of completion of engulfment. The conditions are (i) having an spoIIIE mutation, so that only the origin-proximal 30% of the chromosome is translocated into the prespore, and (ii) placing spoIIIG in an origin-proximal location on the chromosome. The main function of the σE-directed regulation appears to be to coordinate σG activation with the completion of engulfment, not to control the level of σG activity. It seems plausible that the role of σE in σG activation is to reverse some inhibitory signal (or signals) in the engulfed prespore, a signal that is not present in the spoIIIE mutant background. It is not clear what the direct activator of σG in the prespore is. Competition for core RNA polymerase between σF and σG is unlikely to be of major importance.

scholarly journals Analysis of potential redundancy among Arabidopsis 6-phosphogluconolactonase isoforms in peroxisomes

2019 ◽  
Vol 71 (3) ◽  
pp. 823-836 ◽  
Author(s):  
Hannes Lansing ◽  
Lennart Doering ◽  
Kerstin Fischer ◽  
Marie-Christin Baune ◽  
Antje Von Schaewen
Keyword(s):  
Pentose Phosphate ◽  
Second Step ◽  
Higher Plant ◽  
Knock Out ◽  
Phosphogluconate Dehydrogenase ◽  
The Third ◽  
Relative Contribution ◽  
Plant Enzymes ◽  
Peroxisomal Targeting

Abstract Recent work revealed that PGD2, an Arabidopsis 6-phosphogluconate dehydrogenase (6-PGD) catalysing the third step of the oxidative pentose-phosphate pathway (OPPP) in peroxisomes, is essential during fertilization. Earlier studies on the second step, catalysed by PGL3, a dually targeted Arabidopsis 6-phosphogluconolactonase (6-PGL), reported the importance of OPPP reactions in plastids but their irrelevance in peroxisomes. Assuming redundancy of 6-PGL activity in peroxisomes, we examined the sequences of other higher plant enzymes. In tomato, there exist two 6-PGL isoforms with the strong PTS1 motif SKL. However, their analysis revealed problems regarding peroxisomal targeting: reporter–PGL detection in peroxisomes required construct modification, which was also applied to the Arabidopsis isoforms. The relative contribution of PGL3 versus PGL5 during fertilization was assessed by mutant crosses. Reduced transmission ratios were found for pgl3-1 (T-DNA-eliminated PTS1) and also for knock-out allele pgl5-2. The prominent role of PGL3 showed as compromised growth of pgl3-1 seedlings on sucrose and higher activity of mutant PGL3-1 versus PGL5 using purified recombinant proteins. Evidence for PTS1-independent uptake was found for PGL3-1 and other Arabidopsis PGL isoforms, indicating that peroxisome import may be supported by a piggybacking mechanism. Thus, multiple redundancy at the level of the second OPPP step in peroxisomes explains the occurrence of pgl3-1 mutant plants.

scholarly journals Gene conversion in the evolution of both the H-2 and Qa class I genes of the murine major histocompatibility complex.

Genetics ◽  
1990 ◽  
Vol 126 (4) ◽  
pp. 1115-1126
Author(s):  
M Kuhner ◽  
S Watts ◽  
W Klitz ◽  
G Thomson ◽  
R S Goodenow
Keyword(s):  
Major Histocompatibility Complex ◽  
Gene Conversion ◽  
Dna Sequences ◽  
Class I ◽  
Major Histocompatibility ◽  
Gene Encoding ◽  
Histocompatibility Complex ◽  
Sequence Similarities ◽  
I Gene

Abstract In order to better understand the role of gene conversion in the evolution of the class I gene family of the major histocompatibility complex (MHC), we have used a computer algorithm to detect clustered sequence similarities among 24 class I DNA sequences from the H-2, Qa, and Tla regions of the murine MHC. Thirty-four statistically significant clusters were detected; individual analysis of the clusters suggested at least 25 past gene conversion or recombination events. These clusters are comparable in size to the conversions observed in the spontaneously occurring H-2Kbm and H-2Kkm2 mutations, and are distributed throughout all exons of the class I gene. Thus, gene conversion does not appear to be restricted to the regions of the class I gene encoding their antigen-presentation function. Moreover, both the highly polymorphic H-2 loci and the relatively monomorphic Qa and Tla loci appear to have participated as donors and recipients in conversion events. If gene conversion is not limited to the highly polymorphic loci of the MHC, then another factor, presumably natural selection, must be responsible for maintaining the observed differences in level of variation.

scholarly journals Metabolic Engineering of Gluconobacter oxydans for Improved Growth Rate and Growth Yield on Glucose by Elimination of Gluconate Formation

2010 ◽  
Vol 76 (13) ◽  
pp. 4369-4376 ◽  
Author(s):  
Vera Krajewski ◽  
Petra Simić ◽  
Nigel J. Mouncey ◽  
Stephanie Bringer ◽  
Hermann Sahm ◽  
...  
Keyword(s):  
Negative Impact ◽  
Glucose Dehydrogenase ◽  
Pyruvate Decarboxylase ◽  
Growth Yield ◽  
Intermediate Formation ◽  
Gluconobacter Oxydans ◽  
Product Formation ◽  
Pentose Phosphate ◽  
Secondary Products ◽  
Gene Encoding

ABSTRACT Gluconobacter oxydans N44-1, an obligatory aerobic acetic acid bacterium, oxidizes glucose primarily in the periplasm to the end products 2-ketogluconate and 2,5-diketogluconate, with intermediate formation of gluconate. Only a minor part of the glucose (less than 10%) is metabolized in the cytoplasm after conversion to gluconate or after phosphorylation to glucose-6-phosphate via the only functional catabolic routes, the pentose phosphate pathway and the Entner-Doudoroff pathway. This unusual method of glucose metabolism results in a low growth yield. In order to improve it, we constructed mutants of strain N44-1 in which the gene encoding the membrane-bound glucose dehydrogenase was inactivated either alone or together with the gene encoding the cytoplasmic glucose dehydrogenase. The growth and product formation from glucose of the resulting strains, N44-1 mgdH::kan and N44-1 ΔmgdH sgdH::kan, were analyzed. Both mutant strains completely consumed the glucose but produced neither gluconate nor the secondary products 2-ketogluconate and 2,5-diketogluconate. Instead, carbon dioxide formation of the mutants increased by a factor of 4 (N44-1 mgdH::kan) or 5.5 (N44-1 ΔmgdH sgdH::kan), and significant amounts of acetate were produced, presumably by the activities of pyruvate decarboxylase and acetaldehyde dehydrogenase. Most importantly, the growth yields of the two mutants increased by 110% (N44-1 mgdH::kan) and 271% (N44-1 ΔmgdH sgdH::kan). In addition, the growth rates improved by 39% (N44-1 mgdH::kan) and 78% (N44-1 ΔmgdH sgdH::kan), respectively, compared to the parental strain. These results show that the conversion of glucose to gluconate and ketogluconates has a strong negative impact on the growth of G. oxydans.

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