scholarly journals The Mut+strain ofKomagataella phaffii(Pichia pastoris) expresses PAOX15 and 10 times faster than Mutsand Mut−strains: Evidence that formaldehyde or/and formate are true inducers of AOX

2019 ◽  
Author(s):  
Anamika Singh ◽  
Atul Narang
Keyword(s):  
Protein Synthesis ◽  
Recombinant Protein ◽  
Recombinant Proteins ◽  
Methylotrophic Yeast ◽  
Inducible Promoter ◽  
Wild Type ◽  
Methanol Metabolism ◽  
In The Wild ◽  
Host Strains ◽  
Better Than

AbstractThe methylotrophic yeastKomagataella phaffiiis among the most popular hosts for recombinant protein synthesis. Most recombinant proteins were expressed in the wild-type Mut+host strain from the methanol-inducible promoter PAOX1. Since methanol metabolism has undesirable consequences, two additional host strains, Muts(AOX1-) and Mut−(AOX1-AOX2-), were introduced which consume less methanol and reportedly also express recombinant protein better than Mut+. Both results follow from a simple model based on two widespread assumptions, namely methanol is transported by diffusion and the sole inducer of PAOX1. To test this model, we studied14C-methanol uptake in the Mut−strain and β-galactosidase expression in all three strains. We confirmed that methanol is transported by diffusion, but in contrast to the literature, Mut+expressed β-galactosidase 5- and 10-fold faster than Mutsand Mut−. These results imply that methanol is not the sole inducer of PAOX1— metabolites downstream of methanol also induce PAOX1. We find that formate or/and formaldehyde are probably true inducers since both induce PAOX1expression in Mut−which cannot synthesize intracellular methanol from formate or formaldehyde. Formate offers a promising substitute for methanol since it does not appear to suffer from the deficiencies that afflict methanol.

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 Comparative Proteomic Analysis of Wild-Type Physcomitrella patens and an OPDA-Deficient Physcomitrella patens Mutant with Disrupted PpAOS1 and PpAOS2 Genes after Wounding

2020 ◽  
Author(s):  
Weifeng Luo ◽  
Setsuko Komatsu ◽  
Tatsuya Abe ◽  
Hideyuki Matsuura ◽  
Kosaku Talahashi
Keyword(s):  
Protein Synthesis ◽  
Proteomic Analysis ◽  
Vascular Plants ◽  
Acid Metabolism ◽  
Physcomitrella Patens ◽  
Energy Utilization ◽  
Wild Type Plant ◽  
Wild Type ◽  
In The Wild ◽  
Energy Synthesis

Wounding is a serious environmental stress in plants. Oxylipins such as jasmonic acid play an important role in defense against wounding. Mechanisms to adapt to wounding have been investigated in vascular plants; however, those mechanisms in nonvascular plants remain elusive. To examine the response to wounding in Physcomitrella patens, a model moss, a proteomic analysis of wounded P. patens was conducted. Proteomic analysis showed that wounding increased the abundance of proteins related to protein synthesis, amino acid metabolism, protein folding, photosystem, glycolysis, and energy synthesis. 12-Oxo-phytodienoic acid (OPDA) was induced by wounding and inhibited growth. Therefore, OPDA is considered a signaling molecule in this plant. Proteomic analysis of a P. patens mutant in which the PpAOS1 and PpAOS2 genes, which are involved in OPDA biosynthesis, are disrupted showed accumulation of proteins involved in protein synthesis in response to wounding in a similar way to the wild-type plant. In contrast, the fold-changes of the proteins in the wild-type plant were significantly different from those in the aos mutant. This study suggests that PpAOS gene expression enhances photosynthesis and effective energy utilization in response to wounding in P. patens.

scholarly journals Acyadeletion mutant ofEscherichia colidevelops thermotolerance but does not exhibit a heat-shock response

1990 ◽  
Vol 55 (1) ◽  
pp. 1-6 ◽  
Author(s):  
John M. Delaney
Keyword(s):  
Protein Synthesis ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Heat Shock Response ◽  
Receptor Protein ◽  
Wild Type ◽  
E Coli ◽  
Shock Response ◽  
In The Wild ◽  
Shock Proteins

SummaryAn adenyl cyclase deletion mutant (cya) ofE. colifailed to exhibit a heat-shock response even after 30 min at 42 °C. Under these conditions, heat-shock protein synthesis was induced by 10 min in the wild-type strain. These results suggest that synthesis of heat-shock proteins inE. colirequires thecyagene. This hypothesis is supported by the finding that a presumptive cyclic AMP receptor protein (CRP) binding site exists within the promotor region of theE. coli htp Rgene. In spite of the absence of heat-shock protein synthesis, when treated at 50 °C, thecyamutant is relatively more heat resistant than wild type. Furthermore, when heat shocked at 42 °C prior to exposure at 50 °C, thecyamutant developed thermotolerance. These results suggest that heat-shock protein synthesis is not essential for development of thermotolerance inE. coli.

scholarly journals Identification of a Fourth Formate Dehydrogenase in Methylobacterium extorquens AM1 and Confirmation of the Essential Role of Formate Oxidation in Methylotrophy

2007 ◽  
Vol 189 (24) ◽  
pp. 9076-9081 ◽  
Author(s):  
Ludmila Chistoserdova ◽  
Gregory J. Crowther ◽  
Julia A. Vorholt ◽  
Elizabeth Skovran ◽  
Jean-Charles Portais ◽  
...  
Keyword(s):  
Formate Dehydrogenase ◽  
Wild Type ◽  
Methylobacterium Extorquens ◽  
Methanol Metabolism ◽  
Reading Frame ◽  
Formate Oxidation ◽  
In The Wild ◽  
Mutant Phenotypes ◽  
Small Open Reading Frame

ABSTRACT A mutant of Methylobacterium extorquens AM1 with lesions in genes for three formate dehydrogenase (FDH) enzymes was previously described by us (L. Chistoserdova, M. Laukel, J.-C. Portais, J. A. Vorholt, and M. E. Lidstrom, J. Bacteriol. 186:22-28, 2004). This mutant had lost its ability to grow on formate but still maintained the ability to grow on methanol. In this work, we further investigated the phenotype of this mutant. Nuclear magnetic resonance experiments with [13C]formate, as well as 14C-labeling experiments, demonstrated production of labeled CO2 in the mutant, pointing to the presence of an additional enzyme or a pathway for formate oxidation. The tungsten-sensitive phenotype of the mutant suggested the involvement of a molybdenum-dependent enzyme. Whole-genome array experiments were conducted to test for genes overexpressed in the triple-FDH mutant compared to the wild type, and a gene (fdh4A) was identified whose translated product carried similarity to an uncharacterized putative molybdopterin-binding oxidoreductase-like protein sharing relatively low similarity with known formate dehydrogenase alpha subunits. Mutation of this gene in the triple-FDH mutant background resulted in a methanol-negative phenotype. When the gene was deleted in the wild-type background, the mutant revealed diminished growth on methanol with accumulation of high levels of formate in the medium, pointing to an important role of FDH4 in methanol metabolism. The identity of FDH4 as a novel FDH was also confirmed by labeling experiments that revealed strongly reduced CO2 formation in growing cultures. Mutation of a small open reading frame (fdh4B) downstream of fdh4A resulted in mutant phenotypes similar to the phenotypes of fdh4A mutants, suggesting that fdh4B is also involved in formate oxidation.

scholarly journals Bacillus subtilis functional genomics: global characterization of the stringent response by proteome and transcriptome analysis

2002 ◽  
Vol 184 (9) ◽  
pp. 2500-2520 ◽  
Author(s):  
Christine Eymann ◽  
Georg Homuth ◽  
Christian Scharf ◽  
Michael Hecker
Keyword(s):  
Protein Synthesis ◽  
Bacillus Subtilis ◽  
Profile Analysis ◽  
Bacterial Species ◽  
Stringent Response ◽  
Dependent Manner ◽  
Wild Type ◽  
Stringent Control ◽  
In The Wild ◽  
Growth And Reproduction

ABSTRACT The stringent response in Bacillus subtilis was characterized by using proteome and transcriptome approaches. Comparison of protein synthesis patterns of wild-type and relA mutant cells cultivated under conditions which provoke the stringent response revealed significant differences. According to their altered synthesis patterns in response to dl-norvaline, proteins were assigned to four distinct classes: (i) negative stringent control, i.e., strongly decreased protein synthesis in the wild type but not in the relA mutant (e.g., r-proteins); (ii) positive stringent control, i.e., induction of protein synthesis in the wild type only (e.g., YvyD and LeuD); (iii) proteins that were induced independently of RelA (e.g., YjcI); and (iv) proteins downregulated independently of RelA (e.g., glycolytic enzymes). Transcriptome studies based on DNA macroarray techniques were used to complement the proteome data, resulting in comparable induction and repression patterns of almost all corresponding genes. However, a comparison of both approaches revealed that only a subset of RelA-dependent genes or proteins was detectable by proteomics, demonstrating that the transcriptome approach allows a more comprehensive global gene expression profile analysis. The present study presents the first comprehensive description of the stringent response of a bacterial species and an almost complete map of protein-encoding genes affected by (p)ppGpp. The negative stringent control concerns reactions typical of growth and reproduction (ribosome synthesis, DNA synthesis, cell wall synthesis, etc.). Negatively controlled unknown y-genes may also code for proteins with a specific function during growth and reproduction (e.g., YlaG). On the other hand, many genes are induced in a RelA-dependent manner, including genes coding for already-known and as-yet-unknown proteins. A passive model is preferred to explain this positive control relying on the redistribution of the RNA polymerase under the influence of (p)ppGpp.

scholarly journals Controlling the hypermutation: Exploitation of the MutS protein in Pseudomonas aeruginosa

2020 ◽  
Vol 2 (7A) ◽  
Author(s):  
Yue Yuan On ◽  
Martin Welch
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Type Strain ◽  
Wild Type Strain ◽  
Resistant Mutant ◽  
Inducible Promoter ◽  
Wild Type ◽  
Cdna Analysis ◽  
In The Wild ◽  
Mismatch Recognition ◽  
Muts Gene

Pseudomonas aeruginosa infections commonly develop in individuals with cystic fibrosis (CF), and its adaptation in such an unfavourable condition is always found to be related to hypermutation. In fact, most of the hypermutation is due to the defects in mutS gene which involves in the mismatch repair mechanism, causing the acceleration of mutation rate and adaptive evolution. In order to rheostatically express the MutS protein and achieve “hypomutation” (in which the rate of mutation is lower than that of wild type strain), an exogenous mutS gene with rhamnose-inducible promoter was cloned into MPAO1 mutS::Tn mutant strain. Present findings demonstrate that this system is tightly-controlled and stable, with less rifampicin-resistant mutant frequency and more fluorescence intensity from a GFP-tagged MutS expressing cells were observed when the concentration of the inducer increases. Interestingly, the results from Western blot analysis show that less MutS protein is required to suppress hypermutation in the wild type strain, as compared to our construct that behaves similar to the wild type but obviously needs more MutS expression to achieve such state. This indicates that the exogenous MutS might be lacking of other important protein to work efficiently in mismatch recognition. Therefore, based on our cDNA analysis, we found that fdxA gene next to the mutS gene is in the same operon, which could suggest that they might be functionally related in the DNA repair machinery.

scholarly journals Lipase and Protease Double-Deletion Mutant of Pseudomonas fluorescens Suitable for Extracellular Protein Production

2012 ◽  
Vol 78 (23) ◽  
pp. 8454-8462 ◽  
Author(s):  
Myunghan Son ◽  
Yuseok Moon ◽  
Mi Jin Oh ◽  
Sang Bin Han ◽  
Ki Hyun Park ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Pseudomonas Fluorescens ◽  
Abc Transporter ◽  
Recombinant Proteins ◽  
Fusion Proteins ◽  
Deletion Mutant ◽  
Protein Production ◽  
Type I ◽  
Wild Type ◽  
Content Type

ABSTRACTPseudomonas fluorescens, a widespread Gram-negative bacterium, is an ideal protein manufacturing factory (PMF) because of its safety, robust growth, and high protein production.P. fluorescenspossesses a type I secretion system (T1SS), which mediates secretion of a thermostable lipase (TliA) and a protease (PrtA) through its ATP-binding cassette (ABC) transporter. Recombinant proteins inP. fluorescensare attached to the C-terminal signal region of TliA for transport as fusion proteins to the extracellular medium. However, intrinsic TliA from theP. fluorescensgenome interferes with detection of the recombinant protein and the secreted recombinant protein is hydrolyzed, due to intrinsic PrtA, resulting in decreased efficiency of the PMF. In this research, the lipase and protease genes ofP. fluorescensSIK W1 were deleted using the targeted gene knockout method. Deletion mutantP. fluorescensΔtliAΔprtAsecreted fusion proteins without TliA or protein degradation. Using wild-typeP. fluorescensas an expression host, degradation of the recombinant protein varied depending on the type of culture media and aeration; however, degradation did not occur with theP. fluorescensΔtliAΔprtAdouble mutant irrespective of growth conditions. By homologous expression oftliAand the ABC transporter in a plasmid, TliA secreted fromP. fluorescensΔprtAandP. fluorescensΔtliAΔprtAcells was found to be intact, whereas that secreted from the wild-typeP. fluorescensandP. fluorescensΔtliAcells was found to be hydrolyzed. Our results demonstrate that theP. fluorescensΔtliAΔprtAdeletion mutant is a promising T1SS-mediated PMF that enhances production and detection of recombinant proteins in extracellular media.

scholarly journals HtrA Is Essential for Efficient Secretion of Recombinant Proteins by Lactococcus lactis

2008 ◽  
Vol 74 (23) ◽  
pp. 7442-7446 ◽  
Author(s):  
Kalpana Sriraman ◽  
Guhan Jayaraman
Keyword(s):  
Recombinant Protein ◽  
Lactococcus Lactis ◽  
Protein Secretion ◽  
Recombinant Proteins ◽  
Acid Tolerance ◽  
Wild Type ◽  
Acid Tolerance Response ◽  
Recombinant Protein Secretion ◽  
Tolerance Response ◽  
Secretion Of Recombinant Proteins

ABSTRACT HtrA is a unique protease on the extracellular surface of Lactococcus lactis. It is known to take part in the proteolysis of many secreted recombinant proteins, and the mutation of htrA can lead to the complete stabilization of recombinant proteins. In this work, we have shown that htrA mutation also leads to significant reduction of the efficiency of recombinant-protein secretion. We also show that the level of HtrA can be lowered by the suppression of the acid tolerance response (ATR) in L. lactis. Instead of using an L. lactis htrA mutant, the reduction of the HtrA level in wild-type recombinant cultures of L. lactis by ATR suppression may serve as a better strategy for the production of secreted recombinant proteins.

Molecular Analysis of Expression of the Lantibiotic Pep5 Immunity Phenotype

1999 ◽  
Vol 65 (2) ◽  
pp. 591-598 ◽  
Author(s):  
Ulrike Pag ◽  
Christoph Heidrich ◽  
Gabriele Bierbaum ◽  
Hans-Georg Sahl
Keyword(s):  
Gene Cluster ◽  
Inducible Promoter ◽  
Biosynthetic Gene Cluster ◽  
Site Directed Mutagenesis ◽  
Start Codon ◽  
Wild Type ◽  
Tightly Coupled ◽  
In The Wild ◽  
The Stability ◽  
Self Protection

ABSTRACT The lantibiotic Pep5 is produced by Staphylococcus epidermidis 5. Within its biosynthetic gene cluster, the immunity gene pepI, providing producer self-protection, is localized upstream of the structural gene pepA. Pep5 production and the immunity phenotype have been found to be tightly coupled (M. Reis, M. Eschbach-Bludau, M. I. Iglesias-Wind, T. Kupke, and H.-G. Sahl, Appl. Environ. Microbiol. 60:2876–2883, 1994). To study this phenomenon, we analyzed pepA and pepItranscription and translation and constructed a number of strains containing various fragments of the gene cluster and expressing different levels of immunity. Complementation of apepA-expressing strain with pepI intrans did not result in phenotypic immunity or production of PepI. On the other hand, neither pepA nor its product was found to be involved in immunity, since suppression of the translation of the pepA mRNA by mutation of the ATG start codon did not reduce the level of immunity. Moreover, homologous and heterologous expression of pepI from a xylose-inducible promoter resulted in significant Pep5 insensitivity. Most important for expression of the immunity phenotype was the stability ofpepI transcripts, which in the wild-type strain, is achieved by an inverted repeat with a free energy of −56.9 kJ/mol, localized downstream of pepA. We performed site-directed mutagenesis to study the functional role of PepI and constructed F13D PepI, I17R PepI, and PepI 1-65; all mutants showed reduced levels of immunity. Western blot analysis indicated that F13D PepI and PepI 1-65 were not produced correctly or were partially degraded, while I17R PepI apparently was less efficient in providing self-protection than the wild-type PepI.

scholarly journals Chloramphenicol enhances Photosystem II photodamage in intact cells of the cyanobacterium Synechocystis PCC 6803

2020 ◽  
Vol 145 (3) ◽  
pp. 227-235
Author(s):  
Sandeesha Kodru ◽  
Ateeq ur Rehman ◽  
Imre Vass
Keyword(s):  
Protein Synthesis ◽  
Photosystem Ii ◽  
Superoxide Production ◽  
Protein Synthesis Inhibitor ◽  
Wild Type ◽  
Protein Synthesis Inhibitors ◽  
Synthesis Inhibitor ◽  
Intact Cells ◽  
In The Wild ◽  
Pcc 6803

Abstract The effect of chloramphenicol, an often used protein synthesis inhibitor, in photosynthetic systems was studied on the rate of Photosystem II (PSII) photodamage in the cyanobacterium Synechocystis PCC 6803. Light-induced loss of PSII activity was compared in the presence of chloramphenicol and another protein synthesis inhibitor, lincomycin, by measuring the rate of oxygen evolution in Synechocystis 6803 cells. Our data show that the rate of PSII photodamage was significantly enhanced by chloramphenicol, at the usually applied 200 μg mL−1 concentration, relative to that obtained in the presence of lincomycin. Chloramphenicol-induced enhancement of photodamage has been observed earlier in isolated PSII membrane particles, and has been assigned to the damaging effect of chloramphenicol-mediated superoxide production (Rehman et al. 2016, Front Plant Sci 7:479). This effect points to the involvement of superoxide as damaging agent in the presence of chloramphenicol also in Synechocystis cells. The chloramphenicol-induced enhancement of photodamage was observed not only in wild-type Synechocystis 6803, which contains both Photosystem I (PSI) and PSII, but also in a PSI-less mutant which contains only PSII. Importantly, the rate of PSII photodamage was also enhanced by the absence of PSI when compared to that in the wild-type strain under all conditions studied here, i.e., without addition and in the presence of protein synthesis inhibitors. We conclude that chloramphenicol enhances photodamage mostly by its interaction with PSII, leading probably to superoxide production. The presence of PSI is also an important regulatory factor of PSII photodamage most likely via decreasing excitation pressure on PSII.

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