scholarly journals Enhancement of membrane vesicle production by disrupting the degP gene in Meiothermus ruber H328

AMB Express ◽  
2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yuki Asano ◽  
Manato Onishi ◽  
Kaito Nishi ◽  
Kazunori Kawasaki ◽  
Kunihiko Watanabe
Keyword(s):  
Mutant Strain ◽  
Fluorescent Dyes ◽  
Membrane Vesicle ◽  
Wild Strain ◽  
Production Capacity ◽  
Dual Function ◽  
Bacterial Cells ◽  
Tem Analysis ◽  
Meiothermus Ruber ◽  
Mutant Cells

AbstractThe phenomenon of membrane vesicle (MV) production is known to be common to all bacterial cells. Although MVs are expected to be employed in a variety of applications, improving MV productivity is essential for applications. Since the deletion of the degP gene, a periplasmic dual-function protease and chaperone, in Escherichia coli has successfully improved MV production capacity, we tried to enhance MV productivity in the thermophilic M. ruber H328 by deleting the degP gene. One gene (mrH_0331) was selected for degP gene from the H328 genome and we constructed the mutant strain ∆degP by deleting the degP gene of the H328 strain that was replaced with the htk gene showing thermophilic kanamaycin resistance by homologous recombination. The mutant strain ∆degP exhibited smooth growth but a lower level of turbidity at 60 °C although there was no difference in growth at 55 °C between the wild strain and the mutant strain. Finally, we have confirmed that incubation at 60 °C increases MV in the mutant strain ∆degP strain about fivefold by using two fluorescent dyes, DiI and FM4-64, which is followed by TEM analysis. The deletion of the degP gene presumably causes an increase in denatured proteins at 60 °C, leading to enhanced MV production. Meanwhile, the S-layer protein included in the outer membrane of the H328 strain increased in the MV fraction prepared from the mutant cells incubated at 60 °C. This indicates that this method is effective for MV production and that degP deletion enhances it in strain H328.

scholarly journals Enhancement of Membrane Vesicle Production by Disrupting the degP Gene in Meiothermus Ruber H328

2021 ◽  
Author(s):  
Yuki ASANO ◽  
Manato ONISHI ◽  
Kaito NISHI ◽  
Kazunori KAWASAKI ◽  
Kunihiko Watanabe
Keyword(s):  
Mutant Strain ◽  
Fluorescent Dyes ◽  
Membrane Vesicle ◽  
Wild Strain ◽  
Production Capacity ◽  
Dual Function ◽  
Bacterial Cells ◽  
Tem Analysis ◽  
Meiothermus Ruber ◽  
Mutant Cells

Abstract The phenomenon of membrane vesicle (MV) production is known to be common to all bacterial cells. Although MVs are expected to be employed in a variety of applications, improving MV productivity is essential for applications. Since the deletion of the degP gene, a periplasmic dual-function protease and chaperone, in Escherichia coli has successfully improved MV production capacity, we tried to enhance MV productivity in the thermophilic M. ruber H328 by deleting the degP gene. One gene (mrH_0331) was selected for degP gene from the H328 genome and we constructed the mutant strain DdegP by deleting the degP gene of the H328 strain that was replaced with the htk gene showing thermophilic kanamaycin resistance by homologous recombination. The mutant strain DdegP exhibited smooth growth but a lower level of turbidity at 60ºC although there was no difference in growth at 55ºC between the wild strain and the mutant strain. Finally, we have confirmed that incubation at 60°C increases MV in the mutant strain DdegP strain about fivefold by using two fluorescent dyes, DiI and FM4-64, which is followed by TEM analysis. The deletion of the degP gene presumably causes an increase in denatured proteins at 60°C, leading to enhanced MV production. Meanwhile, the S-layer protein included in the outer membrane of the H328 strain increased in the MV fraction prepared from the mutant cells incubated at 60°C. This indicates that this method is effective for MV production and that degP deletion enhances it in strain H328.

Selection and Characterization of ʟ-Ethionine Resistant Mutants of Trichosporon cutaneum

2005 ◽  
Vol 60 (9-10) ◽  
pp. 657-660 ◽  
Author(s):  
Nelly Georgieva ◽  
Zlatka Alexieva
Keyword(s):  
Amino Acid ◽  
Mutant Strain ◽  
Wild Strain ◽  
Amino Acid Content ◽  
Resistant Mutant ◽  
Trichosporon Cutaneum ◽  
Cell Content ◽  
In The Wild ◽  
Mutant Cells

Abstract Trichosporon cutaneum R57 and its ʟ-ethionine resistant mutant NZ94 strain were investigated. The amino acid analyses of cell content of both strains were carried out. The pool of free methionine in the mutant strain is enhanced 16.5 times. The total amount of sulphurcontaining amino acids in the mutant cells was significantly increased from 36.8 in the wild strain to 113.4 mg/g protein in the mutant strain. In the process of mutant strain cultivation there was found a high excretion of free methionine (259 μg/ml) in the medium. It was shown that the amino acid content of both wild and mutant strains would be helpful for formulating of new improved animal nutritional diets.

Protein secretion in Tetrahymena thermophila: characterization of the secretory mutant strain SB281

1985 ◽  
Vol 78 (1) ◽  
pp. 49-65 ◽  
Author(s):  
N.J. Maihle ◽  
B.H. Satir
Keyword(s):  
Plasma Membrane ◽  
Mutant Strain ◽  
Protein Secretion ◽  
Tetrahymena Thermophila ◽  
Freeze Fracture ◽  
Size Class ◽  
Secretory Product ◽  
Wild Type ◽  
Intramembrane Particle ◽  
Mutant Cells

The ciliated protozoon Tetrahymena thermophila contains membrane-bounded secretory organelles termed mucocysts, the release of which has previously been characterized ultrastructurally as a model system for the events occurring during membrane fusion and protein secretion. Recently, a series of secretory mutant strains of Tetrahymena has been isolated following mutagenesis of a parental wild-type strain designated SB210. In this study, the correlates of non-release in one unique mutant strain of this series, designated SB281, are described. SB281 appears to express a diminished (undetectable) level of the major 34000 Mr proteinaceous secretory product of Tetrahymena, as determined by Western immunoblot analysis and indirect immunofluorescence labelling. Thin-section electron-microscopic studies of these cells reveal that they possess no docked or free mature mucocysts. In addition, freeze-fracture electron microscopy demonstrates that an intramembrane particle array termed the rosette, present in the plasma membrane of wild-type cells above sites of docked mucocysts, is absent in the plasma membrane of mutant SB281 cells. A morphometric analysis of intramembrane particles in the plasma membrane of both wild-type and mutant cells indicates that both strains have a similar intramembrane particle density in both leaflets of the the plasma membrane. Although assembled rosettes are missing in the plasma membrane of mutant cells, a 15 nm intramembrane particle size class does exist in the plasma membrane of the mutant, but this size class is significantly reduced in number relative to wild-type.

Ultrasensitive fluorometric determination of iron(iii) and inositol hexaphosphate in cancerous and bacterial cells by using carbon dots with bright yellow fluorescence

The Analyst ◽  
2019 ◽  
Vol 144 (16) ◽  
pp. 5010-5021 ◽  
Author(s):  
Fangchao Cui ◽  
Jiadi Sun ◽  
Xingxing Yang ◽  
Jian Ji ◽  
Fuwei Pi ◽  
...  
Keyword(s):  
Living Cells ◽  
Dual Function ◽  
Bacterial Cells ◽  
Trace Detection ◽  
Ferric Ions ◽  
Fluorometric Determination ◽  
Inositol Hexaphosphate ◽  
Yellow Fluorescence ◽  
Bright Yellow

An ON–OFF–ON dual-function fluorescent nanoprobe is described for the trace detection of ferric ions and inositol hexaphosphate (IP6) in living cells.

scholarly journals Identification and Characterization of Genes Encoding a Putative ABC-Type Transporter Essential for Utilization of γ-Hexachlorocyclohexane in Sphingobium japonicum UT26

2007 ◽  
Vol 189 (10) ◽  
pp. 3712-3720 ◽  
Author(s):  
Ryo Endo ◽  
Yoshiyuki Ohtsubo ◽  
Masataka Tsuda ◽  
Yuji Nagata
Keyword(s):  
Abc Transporter ◽  
Sole Source ◽  
Bacterial Cells ◽  
New Genes ◽  
Hydrophobic Compounds ◽  
Dead End ◽  
Genes Encoding ◽  
Degradation Activity ◽  
Mutant Cells

ABSTRACT Sphingobium japonicum UT26 utilizes γ-hexachlorocyclohexane (γ-HCH) as its sole source of carbon and energy. In our previous studies, we cloned and characterized genes encoding enzymes for the conversion of γ-HCH to β-ketoadipate in UT26. In this study, we analyzed a mutant obtained by transposon mutagenesis and identified and characterized new genes encoding a putative ABC-type transporter essential for the utilization of γ-HCH in strain UT26. This putative ABC transporter consists of four components, permease, ATPase, periplasmic protein, and lipoprotein, encoded by linK, linL, linM, and linN, respectively. Mutation and complementation analyses indicated that all the linKLMN genes are required, probably as a set, for γ-HCH utilization in UT26. Furthermore, the mutant cells deficient in this putative ABC transporter showed (i) higher γ-HCH degradation activity and greater accumulation of the toxic dead-end product 2,5-dichlorophenol (2,5-DCP), (ii) higher sensitivity to 2,5-DCP itself, and (iii) higher permeability of hydrophobic compounds than the wild-type cells. These results strongly suggested that LinKLMN are involved in γ-HCH utilization by controlling membrane hydrophobicity. This study clearly demonstrated that a cellular factor besides catabolic enzymes and transcriptional regulators is essential for utilization of xenobiotic compounds in bacterial cells.

scholarly journals Identification and Functional Characterization of the Novel Edwardsiella tarda Effector EseJ

2015 ◽  
Vol 83 (4) ◽  
pp. 1650-1660 ◽  
Author(s):  
Hai-Xia Xie ◽  
Jin-Fang Lu ◽  
Ying Zhou ◽  
Jia Yi ◽  
Xiu-Jun Yu ◽  
...  
Keyword(s):  
Mutant Strain ◽  
Bacterial Adhesion ◽  
Sequence Similarity ◽  
Functional Characterization ◽  
Lethal Dose ◽  
Edwardsiella Tarda ◽  
Bacterial Cells ◽  
Wild Type ◽  
High Sequence Similarity ◽  
Content Type

Edwardsiella tardais a Gram-negative enteric pathogen that causes hemorrhagic septicemia in fish and gastro- and extraintestinal infections in humans. The type III secretion system (T3SS) ofE. tardahas been identified as a key virulence factor that contributes to pathogenesis in fish. However, little is known about the associated effectors translocated by this T3SS. In this study, by comparing the profile of secreted proteins of the wild-type PPD130/91 and its T3SS ATPase ΔesaNmutant, we identified a new effector by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry. This effector consists of 1,359 amino acids, sharing high sequence similarity with Orf29/30 ofE. tardastrain EIB202, and is renamed EseJ. The secretion and translocation of EseJ depend on the T3SS. A ΔeseJmutant strain adheres to epithelioma papillosum of carp (EPC) cells 3 to 5 times more extensively than the wild-type strain does. EseJ inhibits bacterial adhesion to EPC cells from within bacterial cells. Importantly, the ΔeseJmutant strain does not replicate efficiently in EPC cells and fails to replicate in J774A.1 macrophages. In infected J774A.1 macrophages, the ΔeseJmutant elicits higher production of reactive oxygen species than wild-typeE. tarda. The replication defect is consistent with the attenuation of the ΔeseJmutant in the blue gourami fish model: the 50% lethal dose (LD50) of the ΔeseJmutant is 2.34 times greater than that of the wild type, and the ΔeseJmutant is less competitive than the wild type in mixed infection. Thus, EseJ represents a novel effector that contributes to virulence by reducing bacterial adhesion to EPC cells and facilitating intracellular bacterial replication.

Variation des stérols chez Kluyveromyces lactis et chez un mutant résistant par culture en présence de doses sublétales d'amphotéricine B

1988 ◽  
Vol 34 (6) ◽  
pp. 787-792 ◽  
Author(s):  
A. Hakkou ◽  
J. Coulon ◽  
M. Mpona-Minga ◽  
R. Bonaly
Keyword(s):  
Mutant Strain ◽  
Resistant Strain ◽  
Kluyveromyces Lactis ◽  
Wild Strain ◽  
Total Sterol ◽  
Sublethal Doses

A mutant strain of Kluyveromyces lactis resistant to amphothericin B and weakly to nystatin has been isolated from subcultures of the wild strain grown in the presence of sublethal doses of amphothericin B. The mutant and the wild strain were equally sensitive to pimaricin, filipin, and candicidin. The efficacy of fungizone was very low. In comparison with the wild strain the level of sterols was two times lower in the resistant strain but the composition of these sterols was about the same in the two strains. The action of sublethal doses of amphothericin B on the composition of the sterols was the same in these two yeasts and brought a 40% decrease of the total sterol level and a modification in their distribution. This variation cannot fully explain the resistance of the yeast but it may be associated to other changes of the membranes. [Journal translation]

scholarly journals Mutant strain of Chinese hamster ovary cells with no detectable ornithine decarboxylase activity.

1985 ◽  
Vol 5 (6) ◽  
pp. 1385-1390 ◽  
Author(s):  
P Pohjanpelto ◽  
E Hölttä ◽  
O A Jänne
Keyword(s):  
Cell Line ◽  
Mutant Strain ◽  
Ornithine Decarboxylase ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster ◽  
Decarboxylase Activity ◽  
Serum Free Medium ◽  
Free Medium ◽  
Serum Free ◽  
Mutant Cells

We previously described an arginase-deficient, polyamine-dependent Chinese hamster ovary cell line which grows in serum-free medium. From this strain we isolated a new mutant strain that has no detectable catalytic ornithine decarboxylase activity. The mutant cells contain, however, immunoreactive ornithine decarboxylase-like protein roughly in the same quantity as the parent strain. The mutant and the parent cell line strains also contain similar amounts of ornithine decarboxylase-mRNA hybridizable to a specific cDNA. If polyamines are omitted from the medium, proliferation of the mutant cells is considerably retarded and ceases in 6 to 10 days. Addition of ornithine or alpha-difluoromethylornithine, a specific inhibitor of ornithine decarboxylase, has no effect on these cells. Putrescine and spermidine decreased in the mutant cells to undetectable levels during polyamine starvation, whereas spermine was reduced to 1/5th of that found in the control cultures. Polyamines appear to be indispensable for the mutant strain, but this was obvious only after the amount of polyamines, found as impurities in bovine serum albumin used in the medium, was reduced by dialysis to 10(-12) M. Because sera contain polyamines, the ability of the mutant strain to grow in serum-free medium is a great advantage in elucidation of the mechanisms of polyamine function.

scholarly journals Biophysical and proteomic analyses suggest functions of Pseudomonas syringae pv tomato DC3000 extracellular vesicles in bacterial growth during plant infection

2021 ◽  
Author(s):  
Martin Janda ◽  
Christina Ludwig ◽  
Katarzyna Rybak ◽  
Chen Meng ◽  
Egidio Stigliano ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Extracellular Vesicles ◽  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Bioinformatic Analysis ◽  
Potential Contribution ◽  
Bacterial Cells ◽  
Bacterial Survival ◽  
In Planta ◽  
Plant Infection

SummaryVesiculation is a process employed by Gram-negative bacteria to release extracellular vesicles (EVs) into the environment. Bacterial EVs contain molecular cargo from the donor bacterium and play important roles in bacterial survival and growth. Here, we describe EV production in plant-pathogenic Pseudomonas syringae pv. tomato DC3000 (Pto DC3000), the causal agent of bacterial speck disease. Cultured Pto DC3000 exhibited EV structures both on the cell surface and in the vicinity of bacterial cells, observed as outer membrane vesicle (OMV) release. We used in-solution trypsin digestion coupled to mass spectrometry to identify 369 proteins enriched in EVs recovered from cultured Pto DC3000. The predicted localization profile of EV proteins supports the production of EVs also in the form of outer-inner-membrane vesicles (OIMVs). EV production varied slightly between bacterial lifestyles and also occurred in planta. The potential contribution of EVs to Pto DC3000 plant infection was assessed using plant treatments and bioinformatic analysis of the EV-enriched proteins. While these results identify immunogenic activities of the EVs, they also point at roles for EVs in bacterial defences and nutrient acquisition by Pto DC3000.

scholarly journals Synthetic dual-function RNA reveals features necessary for target regulation

2021 ◽  
Author(s):  
Jordan J. Aoyama ◽  
Medha Raina ◽  
Gisela Storz
Keyword(s):  
Functional Organization ◽  
Regulatory Protein ◽  
Growth Conditions ◽  
Proper Function ◽  
Dual Function ◽  
Bacterial Cells ◽  
Base Pairing ◽  
Small Protein ◽  
Reading Frame ◽  
Small Proteins

Small base pairing RNAs (sRNAs) and small proteins comprise two classes of regulators that allow bacterial cells to adapt to a wide variety of growth conditions. A limited number of transcripts encoding both of these activities, regulation of mRNA expression by base pairing and synthesis of a small regulatory protein, have been identified. Given that few have been characterized, little is known about the interplay between the two regulatory functions. To investigate the competition between the two activities, we constructed synthetic dual-function RNAs, hereafter referred to as MgtSR or MgtRS, comprised of the Escherichia coli sRNA MgrR and the open reading frame encoding the small protein MgtS. MgrR is a 98 nt base pairing sRNA that negatively regulates eptB encoding phosphoethanolamine transferase. MgtS is a 31 aa small inner membrane protein that is required for the accumulation of MgtA, a magnesium (Mg 2+ ) importer. Expression of the separate genes encoding MgrR and MgtS is normally induced in response to low Mg 2+ by the PhoQP two-component system. By generating various versions of this synthetic dual-function RNA, we probed how the organization of components and the distance between the coding and base pairing sequences contribute to the proper function of both activities of a dual-function RNA. By understanding the features of natural and synthetic dual-function RNAs, future synthetic molecules can be designed to maximize their regulatory impact. IMPORTANCE Dual-function RNAs in bacteria encode a small protein and also base pair with mRNAs to act as small, regulatory RNAs. Given that only a limited number of dual-function RNAs have been characterized, further study of these regulators is needed to increase understanding of their features. This study demonstrates that a functional synthetic dual-regulator can be constructed from separate components and used to study the functional organization of dual-function RNAs, with the goal of exploiting these regulators.

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