scholarly journals Poly(3-Hydroxybutyrate) (PHB) Polymerase PhaC1 and PHB Depolymerase PhaZa1 ofRalstonia eutrophaAre PhosphorylatedIn Vivo

2018 ◽  
Vol 84 (13) ◽  
pp. e00604-18 ◽  
Author(s):  
Janina R. Juengert ◽  
Cameron Patterson ◽  
Dieter Jendrossek
Keyword(s):  
Growth Phase ◽  
Ralstonia Eutropha ◽  
Stationary Growth Phase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Stationary Growth ◽  
Futile Cycle ◽  
Content Type ◽  
Phb Depolymerase ◽  
Phb Synthase

ABSTRACTIn this study, we screened poly(3-hydroxybutyrate) (PHB) synthase PhaC1 and PHB depolymerase PhaZa1 ofRalstonia eutrophafor the presence of phosphorylated residues during the PHB accumulation and PHB degradation phases. Thr373 of PHB synthase PhaC1 was phosphorylated during the stationary growth phase but was not modified during the exponential and PHB accumulation phases. Ser35 of PHB depolymerase PhaZa1 was identified in the phosphorylated form during both the exponential and stationary growth phases. Additional phosphosites were identified for both proteins in sample-dependent forms. Site-directed mutagenesis of the codon for Thr373 and other phosphosites of PhaC1 revealed a strong negative impact on PHB synthase activity. Modifications of Thr26 and Ser35 of PhaZa1 reduced the ability ofR. eutrophato mobilize PHB in the stationary growth phase. Our results show that phosphorylation of PhaC1 and PhaZa1 can be important for the modulation of the activities of PHB synthase and PHB depolymerase.IMPORTANCEPoly(3-hydroxybutyrate) (PHB) and related polyhydroxyalkanoates (PHAs) are important intracellular carbon and energy storage compounds in many prokaryotes. The accumulation of PHB or PHAs increases the fitness of cells during periods of starvation and under other stress conditions. The simultaneous presence of PHB synthase (PhaC1) and PHB depolymerase (PhaZa1) on synthesized PHB granules inRalstonia eutropha(alternative designation,Cupriavidus necator) was previously shown in several laboratories. These findings imply that the activities of PHB synthase and PHB depolymerase should be regulated to avoid a futile cycle of simultaneous synthesis and degradation of PHB. Here, we addressed this question by identifying the phosphorylation sites on PhaC1 and PhaZa1 and by site-directed mutagenesis of the identified residues. Furthermore, we conductedin vitroandin vivoanalyses of PHB synthase activity and PHB contents.

scholarly journals Absence of ppGpp Leads to Increased Mobilization of Intermediately Accumulated Poly(3-Hydroxybutyrate) in Ralstonia eutropha H16

2017 ◽  
Vol 83 (13) ◽  
Author(s):  
Janina R. Juengert ◽  
Marina Borisova ◽  
Christoph Mayer ◽  
Christiane Wolz ◽  
Christopher J. Brigham ◽  
...  
Keyword(s):  
Ralstonia Eutropha ◽  
Extraction Procedure ◽  
Stringent Response ◽  
Wild Type ◽  
Futile Cycle ◽  
Content Type ◽  
Phb Depolymerase ◽  
Research Activities ◽  
Phb Synthase ◽  
The Impact

ABSTRACT In this study, we constructed a set of Ralstonia eutropha H16 strains with single, double, or triple deletions of the (p)ppGpp synthase/hydrolase (spoT1), (p)ppGpp synthase (spoT2), and/or polyhydroxybutyrate (PHB) depolymerase (phaZa1 or phaZa3) gene, and we determined the impact on the levels of (p)ppGpp and on accumulated PHB. Mutants with deletions of both the spoT1 and spoT2 genes were unable to synthesize detectable amounts of (p)ppGpp and accumulated only minor amounts of PHB, due to PhaZa1-mediated depolymerization of PHB. In contrast, unusually high levels of PHB were found in strains in which the (p)ppGpp concentration was increased by the overexpression of (p)ppGpp synthase (SpoT2) and the absence of (p)ppGpp hydrolase. Determination of (p)ppGpp levels in wild-type R. eutropha under different growth conditions and induction of the stringent response by amino acid analogs showed that the concentrations of (p)ppGpp during the growth phase determine the amount of PHB remaining in later growth phases by influencing the efficiency of the PHB mobilization system in stationary growth. The data reported for a previously constructed ΔspoT2 strain (C. J. Brigham, D. R. Speth, C. Rha, and A. J. Sinskey, Appl Environ Microbiol 78:8033–8044, 2012, https://doi.org/10.1128/AEM.01693-12 ) were identified as due to an experimental error in strain construction, and our results are in contrast to the previous indication that the spoT2 gene product is essential for PHB accumulation in R. eutropha. IMPORTANCE Polyhydroxybutyrate (PHB) is an important intracellular carbon and energy storage compound in many prokaryotes and helps cells survive periods of starvation and other stress conditions. Research activities in several laboratories over the past 3 decades have shown that both PHB synthase and PHB depolymerase are constitutively expressed in most PHB-accumulating bacteria, such as Ralstonia eutropha. This implies that PHB synthase and depolymerase activities must be well regulated in order to avoid a futile cycle of simultaneous PHB synthesis and PHB degradation (mobilization). Previous reports suggested that the stringent response in Rhizobium etli and R. eutropha is involved in the regulation of PHB metabolism. However, the levels of (p)ppGpp and the influence of those levels on PHB accumulation and PHB mobilization have not yet been determined for any PHB-accumulating species. In this study, we optimized a (p)ppGpp extraction procedure and a high-performance liquid chromatography–mass spectrometry (HPLC-MS)-based detection method for the quantification of (p)ppGpp in R. eutropha. This enabled us to study the relationship between the concentrations of (p)ppGpp and the accumulated levels of PHB in the wild type and in several constructed mutant strains. We show that overproduction of the alarmone (p)ppGpp correlated with reduced growth and massive overproduction of PHB. In contrast, in the absence of (p)ppGpp, mobilization of PHB was dramatically enhanced.

scholarly journals Clostridioides difficile Phosphoproteomics Shows an Expansion of Phosphorylated Proteins in Stationary Growth Phase

mSphere ◽  
2022 ◽  
Author(s):  
Wiep Klaas Smits ◽  
Yassene Mohammed ◽  
Arnoud H. de Ru ◽  
Valentina Cordo' ◽  
Annemieke H. Friggen ◽  
...  
Keyword(s):  
Growth Phase ◽  
Posttranslational Modification ◽  
Stationary Growth Phase ◽  
Comprehensive Analysis ◽  
Limited Evidence ◽  
Stationary Growth ◽  
Content Type ◽  
Phosphorylated Proteins ◽  
Clostridioides Difficile

In this paper, we present a comprehensive analysis of protein phosphorylation in the Gram-positive enteropathogen Clostridioides difficile . To date, only limited evidence on the role of phosphorylation in the regulation of this organism has been published; the current study is expected to form the basis for research on this posttranslational modification in C. difficile .  

scholarly journals Genome-wide transcriptome analyses of the ‘Knallgas’ bacterium Ralstonia eutropha H16 with regard to polyhydroxyalkanoate metabolism

Microbiology ◽  
2010 ◽  
Vol 156 (7) ◽  
pp. 2136-2152 ◽  
Author(s):  
Katja Peplinski ◽  
Armin Ehrenreich ◽  
Christina Döring ◽  
Mechthild Bömeke ◽  
Frank Reinecke ◽  
...  
Keyword(s):  
Mutant Strain ◽  
Growth Phase ◽  
Ralstonia Eutropha ◽  
Stationary Growth Phase ◽  
Transition Phase ◽  
Wild Type ◽  
Stationary Growth ◽  
Transcription Level ◽  
Genome Wide ◽  
Transcriptome Analyses

Ralstonia eutropha H16 is probably the best-studied ‘Knallgas’ bacterium and producer of poly(3-hydroxybutyrate) (PHB). Genome-wide transcriptome analyses were employed to detect genes that are differentially transcribed during PHB biosynthesis. For this purpose, four transcriptomes from different growth phases of the wild-type H16 and of the two PHB-negative mutants PHB−4 and ΔphaC1 were compared: (i) cells from the exponential growth phase with cells that were in transition to stationary growth phase, and (ii) cells from the transition phase with cells from the stationary growth phase of R. eutropha H16, as well as (iii) cells from the transition phase of R. eutropha H16 with those from the transition phase of R. eutropha PHB−4 and (iv) cells from the transition phase of R. eutropha ΔphaC1 with those from the transition phase of R. eutropha PHB−4. Among a large number of genes exhibiting significant changes in transcription level, several genes within the functional class of lipid metabolism were detected. In strain H16, phaP3, accC2, fabZ, fabG and H16_A3307 exhibited a decreased transcription level in the stationary growth phase compared with the transition phase, whereas phaP1, H16_A3311, phaZ2 and phaZ6 were found to be induced in the stationary growth phase. Compared with PHB−4, we found that phaA, phaB1, paaH1, H16_A3307, phaP3, accC2 and fabG were induced in the wild-type, and phaP1, phaP4, phaZ2 and phaZ6 exhibited an elevated transcription level in PHB−4. In strain ΔphaC1, phaA and phaB1 were highly induced compared with PHB−4. Additionally, the results of this study suggest that mutant strain PHB−4 is defective in PHB biosynthesis and fatty acid metabolism. A significant downregulation of the two cbb operons in mutant strain PHB−4 was observed. The putative polyhydroxyalkanoate (PHA) synthase phaC2 identified in strain H16 was further investigated by several functional analyses. Mutant PHB−4 could be phenotypically complemented by expression of phaC2 from a plasmid; on the other hand, in the mutant H16ΔphaC1, no PHA production was observed. PhaC2 activity could not be detected in any experiment.

scholarly journals Conversion of Daidzein and Genistein by an Anaerobic Bacterium Newly Isolated from the Mouse Intestine

2008 ◽  
Vol 74 (15) ◽  
pp. 4847-4852 ◽  
Author(s):  
Anastasia Matthies ◽  
Thomas Clavel ◽  
Michael Gütschow ◽  
Wolfram Engst ◽  
Dirk Haller ◽  
...  
Keyword(s):  
Stationary Phase ◽  
Enzyme Induction ◽  
Growth Phase ◽  
Anaerobic Bacterium ◽  
Stationary Growth Phase ◽  
Gut Bacteria ◽  
Soy Isoflavones ◽  
Strictly Anaerobic ◽  
Stationary Growth ◽  
Mouse Intestine

ABSTRACT The metabolism of isoflavones by gut bacteria plays a key role in the availability and bioactivation of these compounds in the intestine. Daidzein and genistein are the most common dietary soy isoflavones. While daidzein conversion yielding equol has been known for some time, the corresponding formation of 5-hydroxy-equol from genistein has not been reported previously. We isolated a strictly anaerobic bacterium (Mt1B8) from the mouse intestine which converted daidzein via dihydrodaidzein to equol as well as genistein via dihydrogenistein to 5-hydroxy-equol. Strain Mt1B8 was a gram-positive, rod-shaped bacterium identified as a member of the Coriobacteriaceae. Strain Mt1B8 also transformed dihydrodaidzein and dihydrogenistein to equol and 5-hydroxy-equol, respectively. The conversion of daidzein, genistein, dihydrodaidzein, and dihydrogenistein in the stationary growth phase depended on preincubation with the corresponding isoflavonoid, indicating enzyme induction. Moreover, dihydrogenistein was transformed even more rapidly in the stationary phase when strain Mt1B8 was grown on either genistein or daidzein. Growing the cells on daidzein also enabled conversion of genistein. This suggests that the same enzymes are involved in the conversion of the two isoflavones.

scholarly journals Site-Directed Mutagenesis of the Heterotrimeric Killer Toxin Zymocin Identifies Residues Required for Early Steps in Toxin Action

2014 ◽  
Vol 80 (20) ◽  
pp. 6549-6559 ◽  
Author(s):  
Sabrina Wemhoff ◽  
Roland Klassen ◽  
Friedhelm Meinhardt
Keyword(s):  
Kluyveromyces Lactis ◽  
Killer Toxin ◽  
Cysteine Residue ◽  
Site Directed Mutagenesis ◽  
Target Cells ◽  
Directed Mutagenesis ◽  
Content Type ◽  
Protein Toxin ◽  
Assisted Delivery

ABSTRACTZymocin is aKluyveromyces lactisprotein toxin composed of αβγ subunits encoded by the cytoplasmic virus-like element k1 and functions by αβ-assisted delivery of the anticodon nuclease (ACNase) γ into target cells. The toxin binds to cells' chitin and exhibits chitinase activityin vitrothat might be important during γ import.Saccharomyces cerevisiaestrains carrying k1-derived hybrid elements deficient in either αβ (k1ORF2) or γ (k1ORF4) were generated. Loss of either gene abrogates toxicity, and unexpectedly, Orf2 secretion depends on Orf4 cosecretion. Functional zymocin assembly can be restored by nuclear expression of k1ORF2 or k1ORF4, providing an opportunity to conduct site-directed mutagenesis of holozymocin. Complementation required active site residues of α's chitinase domain and the sole cysteine residue of β (Cys250). Since βγ are reportedly disulfide linked, the requirement for the conserved γ C231 was probed. Toxicity of intracellularly expressed γ C231A indicated no major defect in ACNase activity, while complementation of k1ΔORF4 by γ C231A was lost, consistent with a role of β C250 and γ C231 in zymocin assembly. To test the capability of αβ to carry alternative cargos, the heterologous ACNase fromPichia acaciae(P. acaciaeOrf2 [PaOrf2]) was expressed, along with its immunity gene, in k1ΔORF4. While efficient secretion of PaOrf2 was detected, suppression of the k1ΔORF4-derived k1Orf2 secretion defect was not observed. Thus, the dependency of k1Orf2 on k1Orf4 cosecretion needs to be overcome prior to studying αβ's capability to deliver other cargo proteins into target cells.

scholarly journals Determination of the Catalytic Base in Family 48 Glycosyl Hydrolases

2011 ◽  
Vol 77 (17) ◽  
pp. 6274-6276 ◽  
Author(s):  
Maxim Kostylev ◽  
David B. Wilson
Keyword(s):  
Aspartic Acid ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Thermobifida Fusca ◽  
Glycosyl Hydrolases ◽  
Content Type ◽  
Modeling Data ◽  
Partial Activity

ABSTRACTThe catalytic base in family 48 glycosyl hydrolases has not been previously established experimentally. Based on structural and modeling data published to date, we used site-directed mutagenesis and azide rescue activity assays to show definitively that the catalytic base inThermobifida fuscaCel48A is aspartic acid 225. Of the tested mutants, only Cel48A with the D225E mutation retained partial activity on soluble and insoluble substrates. In azide rescue experiments, only the D225G mutation, in the smallest residue tested, showed an increase in activity with added azide.

scholarly journals The extracellular proteome of Rhizobium etli CE3 in exponential and stationary growth phase

2010 ◽  
Vol 8 (1) ◽  
pp. 51 ◽  
Author(s):  
Niurka Meneses ◽  
Guillermo Mendoza-Hernández ◽  
Sergio Encarnación
Keyword(s):  
Growth Phase ◽  
Stationary Growth Phase ◽  
Rhizobium Etli ◽  
Stationary Growth ◽  
Extracellular Proteome

scholarly journals Versatile Fungal Polyphenol Oxidase with Chlorophenol Bioremediation Potential: Characterization and Protein Engineering

2018 ◽  
Vol 84 (23) ◽  
Author(s):  
Efstratios Nikolaivits ◽  
Maria Dimarogona ◽  
Ioanna Karagiannaki ◽  
Angelina Chalima ◽  
Ayelet Fishman ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Fruits And Vegetables ◽  
Homology Model ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Gene Encoding ◽  
Content Type ◽  
Polyphenol Oxidases ◽  
Increased Activity ◽  
Work Knowledge

ABSTRACTPolyphenol oxidases (PPOs) have been mostly associated with the undesirable postharvest browning in fruits and vegetables and have implications in human melanogenesis. Nonetheless, they are considered useful biocatalysts in the food, pharmaceutical, and cosmetic industries. The aim of the present work was to characterize a novel PPO and explore its potential as a bioremediation agent. A gene encoding an extracellular tyrosinase-like enzyme was amplified from the genome ofThermothelomyces thermophilaand expressed inPichia pastoris. The recombinant enzyme (TtPPO) was purified and biochemically characterized. Its production reached 40 mg/liter, and it appeared to be a glycosylated and N-terminally processed protein.TtPPO showed broad substrate specificity, as it could oxidize 28/30 compounds tested, including polyphenols, substituted phenols, catechols, and methoxyphenols. Its optimum temperature was 65°C, with a half-life of 18.3 h at 50°C, while its optimum pH was 7.5. The homology model ofTtPPO was constructed, and site-directed mutagenesis was performed in order to increase its activity on mono- and dichlorophenols (di-CPs). The G292N/Y296V variant ofTtPPO 5.3-fold increased activity on 3,5-dichlorophenol (3,5-diCP) compared to the wild type.IMPORTANCEA novel fungal PPO was heterologously expressed and biochemically characterized. Construction of single and double mutants led to the generation of variants with altered specificity against CPs. Through this work, knowledge is gained regarding the effect of mutations on the substrate specificity of PPOs. This work also demonstrates that more potent biocatalysts for the bioremediation of harmful CPs can be developed by applying site-directed mutagenesis.

scholarly journals Proteomic Study of the Exponential-Stationary Growth Phase Transition in the Haloarchaea Natrialba magadii and Haloferax volcanii

PROTEOMICS ◽  
2018 ◽  
Vol 18 (14) ◽  
pp. 1800116 ◽  
Author(s):  
Micaela Cerletti ◽  
María Ines Giménez ◽  
Christian Tröetschel ◽  
Celeste D’ Alessandro ◽  
Ansgar Poetsch ◽  
...  
Keyword(s):  
Phase Transition ◽  
Growth Phase ◽  
Stationary Growth Phase ◽  
Haloferax Volcanii ◽  
Stationary Growth ◽  
Proteomic Study ◽  
Natrialba Magadii ◽  
Growth Phase Transition

Effect of Temperature on Growth and Alpha Toxin Production by Clostridium perfringens1

1979 ◽  
Vol 42 (11) ◽  
pp. 848-851 ◽  
Author(s):  
Y. PARK ◽  
E. M. MIKOLAJCIK
Keyword(s):  
Growth Phase ◽  
Incubation Temperature ◽  
Ground Beef ◽  
Population Level ◽  
Toxin Production ◽  
Stationary Growth Phase ◽  
Effect Of Temperature ◽  
Alpha Toxin ◽  
Stationary Growth ◽  
Incubation Temperatures

Growth and alpha toxin production by a strain of Clostridium perfringens was determined in Thioglycollate medium, beef broth with ground beef, and beef broth with ground beef and soy protein. Incubation temperatures ranged from 15 to 50 C. In Thioglycollate medium, maximum alpha toxin production occurred at 35 C and was 40 times greater than that observed at 45 C. However, generation time and maximum population were approximately the same at 35 and 45 C. At 15 C, a two log cycle reduction in viable counts occurred within 6 h. Irrespective of incubation temperature, alpha toxin levels in Thioglycollate medium declined as the incubation period was extended beyond the stationary growth phase. In the beef broth with ground beef system which was studied at 35 C only, the organism grew slower and produced less toxin than in Thioglycollate medium. The amount of alpha toxin detected was influenced to a greater extent by the incubation time and temperature, the holding time beyond the stationary growth phase, and the growth medium than by the population level of C. perfringens.

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