Role of prokaryotic type I and III pantothenate kinases in the coenzyme A biosynthetic pathway of Bacillus subtilis

2014 ◽  
Vol 60 (5) ◽  
pp. 297-305 ◽  
Author(s):  
Yuta Ogata ◽  
Hiroki Katoh ◽  
Munehiko Asayama ◽  
Shigeru Chohnan
Keyword(s):  
Bacillus Subtilis ◽  
Recombinant Proteins ◽  
Biosynthetic Pathway ◽  
Kinase Activity ◽  
Wild Type Strain ◽  
Coenzyme A ◽  
Monovalent Cation ◽  
Type I ◽  
Wild Type

Pantothenate kinases (CoaAs) catalyze the phosphorylation of pantothenate in the first step of the coenzyme A (CoA) biosynthetic pathway. These bacterial enzymes have been categorized into 3 types, the prokaryotic type I, II, and III CoaAs. Bacteria typically carry a single CoaA gene on their genome, but Bacillus subtilis possesses 2 proteins homologous to type I and III CoaAs, known as BsCoaA and BsCoaX, respectively. Both recombinant proteins exhibited the expected kinase activity and the characteristic properties of type I and III CoaAs, i.e., regulation by CoASH and acyl-CoAs in BsCoaA and the requirement of a monovalent cation in BsCoaX. Both gene disruptants appeared to grow in a manner similar to the wild-type strain. With the BsCoaX disruptant, the BsCoaA had the ability to completely fill the intracellular CoA pool, whereas the BsCoaA disruptant did not. These findings clearly indicate that these 2 CoaAs are employed together in the CoA biosynthetic pathway in B. subtilis and that the contribution of the type I CoaA (BsCoaA) to the formation of the intracellular CoA pool is larger than that of the type III CoaA (BsCoaX).

scholarly journals Biochemical and Mutational Analyses of AcuA, the Acetyltransferase Enzyme That Controls the Activity of the Acetyl Coenzyme A Synthetase (AcsA) in Bacillus subtilis

2008 ◽  
Vol 190 (14) ◽  
pp. 5132-5136 ◽  
Author(s):  
Jeffrey G. Gardner ◽  
Jorge C. Escalante-Semerena
Keyword(s):  
Bacillus Subtilis ◽  
Coenzyme A ◽  
Effective Means ◽  
Wild Type ◽  
Modification System ◽  
Vitro Assay ◽  
Acetyl Coenzyme A ◽  
Acetyl Coenzyme

ABSTRACT The acuABC genes of Bacillus subtilis comprise a putative posttranslational modification system. The AcuA protein is a member of the Gcn5-related N-acetyltransferase (GNAT) superfamily, the AcuC protein is a class I histone deacetylase, and the role of the AcuB protein is not known. AcuA controls the activity of acetyl coenzyme A synthetase (AcsA; EC 6.2.1.1) in this bacterium by acetylating residue Lys549. Here we report the kinetic analysis of wild-type and variant AcuA proteins. We contrived a genetic scheme for the identification of AcuA residues critical for activity. Changes at residues H177 and G187 completely inactivated AcuA and led to its rapid turnover. Changes at residues R42 and T169 were less severe. In vitro assay conditions were optimized, and an effective means of inactivating the enzyme was found. The basic kinetic parameters of wild-type and variant AcuA proteins were obtained and compared to those of eukaryotic GNATs. Insights into how the isolated mutations may exert their deleterious effect were investigated by using the crystal structure of an AcuA homolog.

scholarly journals Role of Arg-Gingipain A in Virulence of Porphyromonas gingivalis

1998 ◽  
Vol 66 (3) ◽  
pp. 1159-1166 ◽  
Author(s):  
Masayuki Tokuda ◽  
Thonthi Karunakaran ◽  
Margaret Duncan ◽  
Nobushiro Hamada ◽  
Howard Kuramitsu
Keyword(s):  
Porphyromonas Gingivalis ◽  
Wild Type Strain ◽  
Type I Collagen ◽  
Northern Blotting ◽  
Type I ◽  
Wild Type ◽  
Self Aggregation ◽  
Wild Type Parent ◽  
Rat Tail

ABSTRACT In order to access the role of the Porphyromonas gingivalis Arg-gingipain proteases in the virulence of this organism, a mutant defective in the rgpA gene was constructed in strain 381. This mutant, MT10, displayed only 40% of the Arg-specific cysteine protease activity of the wild-type strain. In addition, MT10, as well as the recently characterized protease mutant G-102, which is defective in the rgpB gene, displayed reduced self-aggregation, hemagglutination, and the ability to bind to immobilized type I collagen compared to levels of the wild-type parent. However, unlike mutant G-102, the rgpA mutant displayed increased binding to epithelial cells relative to that of the parental organism. Mutant MT10 also did not express detectable levels of the FimA protein as assessed by both Western and Northern blotting or fimbriae visible by electron microscopy of the cells. Furthermore, the ability of MT10 to degrade rat tail collagen fibers when it was cultured at 37°C was markedly attenuated compared to that of strain 381. These results suggest that Arg-gingipain A may play a significant role in the pathogenicity of P. gingivalis by altering the colonization and toxic properties of the organism.

scholarly journals A Polysaccharide Deacetylase Gene (pdaA) Is Required for Germination and for Production of Muramic δ-Lactam Residues in the Spore Cortex of Bacillus subtilis

2002 ◽  
Vol 184 (21) ◽  
pp. 6007-6015 ◽  
Author(s):  
Tatsuya Fukushima ◽  
Hiroki Yamamoto ◽  
Abdelmadjid Atrih ◽  
Simon J. Foster ◽  
Junichi Sekiguchi
Keyword(s):  
Bacillus Subtilis ◽  
Phase Contrast ◽  
Biosynthetic Pathway ◽  
Wild Type Strain ◽  
Dipicolinic Acid ◽  
Northern Hybridization ◽  
Predicted Amino Acid Sequence ◽  
Wild Type ◽  
Contrast Microscopy ◽  
Acid Release

ABSTRACT The predicted amino acid sequence of Bacillus subtilis yfjS (renamed pdaA) exhibits high similarity to those of several polysaccharide deacetylases. β-Galactosidase fusion experiments and results of Northern hybridization with sporulation sigma mutants indicated that the pdaA gene is transcribed by EσG RNA polymerase. pdaA-deficient spores were bright by phase-contrast microscopy, and the spores were induced to germination on the addition of l-alanine. Germination-associated spore darkening, a slow and partial decrease in absorbance, and slightly lower dipicolinic acid release compared with that by the wild-type strain were observed. In particular, the release of hexosamine-containing materials was lacking in the pdaA mutant. Muropeptide analysis indicated that the pdaA-deficient spores completely lacked muramic δ-lactam. A pdaA-gfp fusion protein constructed in strain 168 and pdaA-deficient strains indicated that the protein is localized in B. subtilis spores. The biosynthetic pathway of muramic δ-lactam is discussed.

scholarly journals Selective Removal of Aberrant Extender Units by a Type II Thioesterase for Efficient FR-008/Candicidin Biosynthesis in Streptomyces sp. Strain FR-008

2008 ◽  
Vol 74 (23) ◽  
pp. 7235-7242 ◽  
Author(s):  
Yongjun Zhou ◽  
Qingqing Meng ◽  
Delin You ◽  
Jialiang Li ◽  
Shi Chen ◽  
...  
Keyword(s):  
Biosynthetic Pathway ◽  
Biochemical Analysis ◽  
Wild Type Strain ◽  
Type I ◽  
Type Ii ◽  
Wild Type ◽  
Acyl Carrier Proteins ◽  
Peptide Synthetase ◽  
In The Wild

ABSTRACT Gene fscTE, encoding a putative type II thioesterase (TEII), was associated with the FR-008/candicidin gene cluster. Deletion of fscTE reduced approximately 90% of the FR-008/candicidin production, while the production level was well restored when fscTE was added back to the mutant in trans. FscTE was unable to compensate for the release of the maturely elongated polyketide as site-directed inactivation of the type I thioesterase (TEI) totally abolished FR-008/candicidin production. Direct biochemical analysis of FscTE in parallel with its homologue TylO from the tylosin biosynthetic pathway demonstrated their remarkable preferences for acyl-thioesters (i.e., propionyl-S-N-acetylcysteamine [SNAC] over methylmalonyl-SNAC and acetyl-SNAC over malonyl-SNAC) and thus concluded that TEII could maintain effective polyketide biosynthesis by selectively removing the nonelongatable residues bound to acyl carrier proteins. Overexpression of FscTE under the strong constitutive ermE*p promoter in the wild-type strain did not suppress FR-008/candicidin formation, which confirmed its substrate specificity in vivo. Furthermore, successful complementation of the fscTE mutant was obtained with fscTE and tylO, whereas no complementation was detected with nonribosomal peptide synthetase (NRPS) TEII tycF and srfAD, reflecting substrate specificities of TEIIs distinctive from those of either polyketide synthases or NRPSs.

scholarly journals NMR and LC-MS-Based Metabolomics to Study Osmotic Stress in Lignan-Deficient Flax

Molecules ◽  
2021 ◽  
Vol 26 (3) ◽  
pp. 767
Author(s):  
Kamar Hamade ◽  
Ophélie Fliniaux ◽  
Jean-Xavier Fontaine ◽  
Roland Molinié ◽  
Elvis Otogo Nnang ◽  
...  
Keyword(s):  
Stress Response ◽  
Osmotic Stress ◽  
Biosynthetic Pathway ◽  
Potential Role ◽  
Plant Secondary Metabolites ◽  
Wild Type ◽  
Osmotic Stress Response ◽  
Transgenic Flax ◽  
Insight Into

Lignans, phenolic plant secondary metabolites, are derived from the phenylpropanoid biosynthetic pathway. Although, being investigated for their health benefits in terms of antioxidant, antitumor, anti-inflammatory and antiviral properties, the role of these molecules in plants remains incompletely elucidated; a potential role in stress response mechanisms has been, however, proposed. In this study, a non-targeted metabolomic analysis of the roots, stems, and leaves of wild-type and PLR1-RNAi transgenic flax, devoid of (+) secoisolariciresinol diglucoside ((+) SDG)—the main flaxseed lignan, was performed using 1H-NMR and LC-MS, in order to obtain further insight into the involvement of lignan in the response of plant to osmotic stress. Results showed that wild-type and lignan-deficient flax plants have different metabolic responses after being exposed to osmotic stress conditions, but they both showed the capacity to induce an adaptive response to osmotic stress. These findings suggest the indirect involvement of lignans in osmotic stress response.

scholarly journals Enhanced production of Clavulanic acid by improving glycerol utilization using reporter-guided mutagenesis of an industrial Streptomyces clavuligerus strain

2021 ◽  
Author(s):  
Chang-Hun Shin ◽  
Hang Soo Cho ◽  
Hyung-Jin Won ◽  
Ho Jeong Kwon ◽  
Chan-Wha Kim ◽  
...  
Keyword(s):  
Clavulanic Acid ◽  
Wild Type Strain ◽  
Random Mutagenesis ◽  
Streptomyces Clavuligerus ◽  
Wild Type ◽  
Mutant Selection ◽  
Enhanced Production ◽  
Industrial Strains ◽  
Glycerol Utilization

Abstract Clavulanic acid (CA) produced by Streptomyces clavuligerus is a clinically important β-lactamase inhibitor. It is known that glycerol utilization can significantly improve cell growth and CA production of S. clavuligerus. We found that the industrial CA-producing S. clavuligerus strain OR generated by random mutagenesis consumes less glycerol than the wild-type strain; we then developed a mutant strain in which the glycerol utilization operon is overexpressed, as compared to the parent OR strain, through iterative random mutagenesis and reporter-guided selection. The CA production of the resulting S. clavuligerus ORUN strain was increased by approximately 31.3 per cent (5.21 ± 0.26 g/L) in a flask culture and 17.4 per cent (6.11 ± 0.36 g/L) in a fermenter culture, as compared to that of the starting OR strain. These results confirmed the important role of glycerol utilization in CA production and demonstrated that reporter-guided mutant selection is an efficient method for further improvement of randomly mutagenized industrial strains.

scholarly journals The ketogenic diet increases Neuregulin 1 expression via elevating histone acetylation and its anti-seizure effect requires ErbB4 kinase activity

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Jin Wang ◽  
Jie Huang ◽  
Shan Yao ◽  
Jia-Hui Wu ◽  
Hui-Bin Li ◽  
...  
Keyword(s):  
Histone Acetylation ◽  
Ketogenic Diet ◽  
Chromatin Immunoprecipitation ◽  
Kinase Activity ◽  
Control Diet ◽  
Synaptic Activity ◽  
Therapeutic Interventions ◽  
Type I ◽  
Neuregulin 1

Abstract Background The ketogenic diet (KD)has been considered an effective treatment for epilepsy, whereas its underlying mechanisms remain obscure. We have previously reported that the KD feeding increased Neuregulin 1 (NRG1) expression in the hippocampus; disruption of NRG1 signaling by genetically deleting its receptor-ErbB4 abolished KD’s effects on inhibitory synaptic activity and seizures. However, it is still unclear about the mechanisms underlying the effect of KD on NRG1 expression and whether the effects of KD require ErbB4 kinase activity. Methods The effects of the KD on NRG1 expression were assessed via western blotting and real-time PCR. Acetylation level at the Nrg1 promoter locus was examined using the chromatin immunoprecipitation technique. Kainic acid (KA)-induced acute seizure model was utilized to examine the effects of KD and histone deacetylase inhibitor-TSA on seizures. Synaptic activities in the hippocampus were recorded with the technique of electrophysiology. The obligatory role of ErbB4 kinase activity in KD’s effects on seizures and inhibitory synaptic activity was evaluated by using ErbB kinase antagonist and transgenic mouse-T796G. Results We report that KD specifically increases Type I NRG1 expression in the hippocampus. Using the chromatin immunoprecipitation technique, we observe increased acetylated-histone occupancy at the Nrg1 promoter locus of KD-fed mice. Treatment of TSA dramatically elevates NRG1 expression and diminishes the difference between the effects of the control diet (CD) and KD. These data indicate that KD increases NRG1 expression via up-regulating histone acetylation. Moreover, both pharmacological and genetic inhibitions of ErbB4 kinase activity significantly block the KD’s effects on inhibitory synaptic activity and seizure, suggesting an essential role of ErbB4 kinase activity. Conclusion These results strengthen our understanding of the role of NRG1/ErbB4 signaling in KD and shed light on novel therapeutic interventions for epilepsy.

scholarly journals Role of SpoVA Proteins in Release of Dipicolinic Acid during Germination of Bacillus subtilis Spores Triggered by Dodecylamine or Lysozyme

2006 ◽  
Vol 189 (5) ◽  
pp. 1565-1572 ◽  
Author(s):  
Venkata Ramana Vepachedu ◽  
Peter Setlow
Keyword(s):  
Bacillus Subtilis ◽  
Small Molecules ◽  
Spore Germination ◽  
Dipicolinic Acid ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Ph Optimum ◽  
Inner Membrane ◽  
Temperature Sensitive Mutants

ABSTRACT The release of dipicolinic acid (DPA) during the germination of Bacillus subtilis spores by the cationic surfactant dodecylamine exhibited a pH optimum of ∼9 and a temperature optimum of 60°C. DPA release during dodecylamine germination of B. subtilis spores with fourfold-elevated levels of the SpoVA proteins that have been suggested to be involved in the release of DPA during nutrient germination was about fourfold faster than DPA release during dodecylamine germination of wild-type spores and was inhibited by HgCl2. Spores carrying temperature-sensitive mutants in the spoVA operon were also temperature sensitive in DPA release during dodecylamine germination as well as in lysozyme germination of decoated spores. In addition to DPA, dodecylamine triggered the release of amounts of Ca2+ almost equivalent to those of DPA, and at least one other abundant spore small molecule, glutamic acid, was released in parallel with Ca2+ and DPA. These data indicate that (i) dodecylamine triggers spore germination by opening a channel in the inner membrane for Ca2+-DPA and other small molecules, (ii) this channel is composed at least in part of proteins, and (iii) SpoVA proteins are involved in the release of Ca2+-DPA and other small molecules during spore germination, perhaps by being a part of a channel in the spore's inner membrane.

scholarly journals Induction of Dendritic Cell Production of Type I and Type III Interferons by Wild-Type and Vaccine Strains of Measles Virus: Role of Defective Interfering RNAs

2013 ◽  
Vol 87 (14) ◽  
pp. 7816-7827 ◽  
Author(s):  
R. Shivakoti ◽  
M. Siwek ◽  
D. Hauer ◽  
K. L. W. Schultz ◽  
D. E. Griffin
Keyword(s):  
Dendritic Cell ◽  
Measles Virus ◽  
Type I ◽  
Wild Type ◽  
Cell Production ◽  
Type Iii

Retrovirus-induced interference with collagen I gene expression in Mov13 fibroblasts is maintained in the absence of DNA methylation

1991 ◽  
Vol 11 (1) ◽  
pp. 47-54
Author(s):  
H Chan ◽  
S Hartung ◽  
M Breindl
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Chromatin Structure ◽  
Transcriptional Activity ◽  
Xenopus Laevis Oocytes ◽  
Regulatory Sequences ◽  
Type I ◽  
Wild Type ◽  
Col1a1 Gene

We have studied the role of DNA methylation in repression of the murine alpha 1 type I collagen (COL1A1) gene in Mov13 fibroblasts. In Mov13 mice, a retroviral provirus has inserted into the first intron of the COL1A1 gene and blocks its expression at the level of transcriptional initiation. We found that regulatory sequences in the COL1A1 promoter region that are involved in the tissue-specific regulation of the gene are unmethylated in collagen-expressing wild-type fibroblasts and methylated in Mov13 fibroblasts, confirming and extending earlier observations. To directly assess the role of DNA methylation in the repression of COL1A1 gene transcription, we treated Mov13 fibroblasts with the demethylating agent 5-azacytidine. This treatment resulted in a demethylation of the COL1A1 regulatory sequences but failed to activate transcription of the COL1A1 gene. Moreover, the 5-azacytidine treatment induced a transcription-competent chromatin structure in the retroviral sequences but not in the COL1A1 promoter. In DNA transfection and microinjection experiments, we found that the provirus interfered with transcriptional activity of the COL1A1 promoter in Mov13 fibroblasts but not in Xenopus laevis oocytes. In contrast, the wild-type COL1A1 promoter was transcriptionally active in Mov13 fibroblasts. These experiments showed that the COL1A1 promoter is potentially transcriptionally active in the presence of proviral sequences and that Mov13 fibroblasts contain the trans-acting factors required for efficient COL1A1 gene expression. Our results indicate that the provirus insertion in Mov13 can inactivate COL1A1 gene expression at several levels. It prevents the developmentally regulated establishment of a transcription-competent methylation pattern and chromatin structure of the COL1A1 domain and, in the absence of DNA methylation, appears to suppress the COL1A1 promoter in a cell-specific manner, presumably by assuming a dominant chromatin structure that may be incompatible with transcriptional activity of flanking cellular sequences.

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