scholarly journals Mutagenesis development of actinoplanes sp. KCTC 9161 by N-methyl-N'-nitro-N-nitrosoguanidine (NTG) and screening for acarbose production

2018 ◽  
Vol 14 (4) ◽  
pp. 753-760
Author(s):  
Do Thi Tuyen ◽  
Nguyen The Duong ◽  
Le Thanh Hoang
Keyword(s):  
Type Ii Diabetes ◽  
Wild Type Strain ◽  
Fermentation Medium ◽  
Original Strain ◽  
Wild Type ◽  
Chromatography Method ◽  
Mutant Strains ◽  
Insulin Dependent ◽  
Mutant Lines ◽  
Layer Chromatography

Acarbose has been widely used in the therapy of type II diabetes (non-insulin dependent) because it controls blood sugar contents of patients after meals. Acarbose, a pseudo-oligosaccharide, acts as a competitive -glucosidase inhibitor. Acarbose is produced by the strains of Bacillus, Streptomyces and Actinoplanes sp. The aim of this study was to develop mutagenesis for an Actinoplanes sp. strain and screening for acarbose production. The spores of Actinoplanes sp. KCTC 9161 strain were subjected to be mutated by N-methyl-N'-nitro-N-nitrosoguanidine (NTG) for screening and finding mutant strains that were capable of production of higher acarbose (an inhibitor of α-glucosidase) higher than wild type strain. Firstly, the original NTG solution was prepared in phosphate buffer 0.05 M, pH 6.9 and the safety concentration of NTG was determined at 5 mg/ml. Then, the spores were incubated with different NTG amounts and duration. The living colonies were transferred to fermentation medium. The results obtained showed that 15 mutant strains were produced higher acarbose than wild type when used thin layer chromatography method for analysis and comparing with standard acarbose (Sigma). Three cell lines among total tested 15 mutant lines of Actinoplanes sp. KCTC 9161 produced acarbose at a higher level or indicated a higher inhibitory activity toward α-glucosidase than the original strain. Enzymatic inhibitory ativity of α-glucosidase of three mutant strains (Actinoplanes sp. KCTC- L4, L11, L14) was increased 1.3 fold higher than wild type and Actinoplanes sp. KCTC spores were very sensitive to NTG toxic, 98% spores could not survive at the treatment condition of 50 µg NTG for 30 minutes. In addition, an applicable protocol for mutating Actinoplanes sp. using NTG was suggested for further research.

scholarly journals Behavior of a Wild-Type and Two Mutant Strains of Colletotrichum gloeosporioides f. sp. aeschynomene on Northern Jointvetch in the Field

Plant Disease ◽  
1998 ◽  
Vol 82 (4) ◽  
pp. 374-379 ◽  
Author(s):  
Y. Luo ◽  
D. O. TeBeest
Keyword(s):  
Population Density ◽  
Wild Type Strain ◽  
Colletotrichum Gloeosporioides ◽  
Biological Control Agent ◽  
Field Tests ◽  
The United States ◽  
Control Agent ◽  
Sampling Time ◽  
Wild Type ◽  
Mutant Strains

The fungus Colletotrichum gloeosporioides f. sp. aeschynomene causes an anthracnose on Aeschynomene virginica and has been used as a biological control agent to control this weed in the United States. The population dynamics of a wild-type strain (3-1-3) and two mutant strains of 3-1-3 of C. gloeosporioides f. sp. aeschynomene, a benomyl-resistant strain (B21) and nitrate-nonutilizing strain (Nit A), were studied in field tests on northern jointvetch in 1994 and 1995 to determine how the strains interacted on infected plants under field conditions. Plants were co-inoculated with strains 3-1-3 and B21, strains 3-1-3 and Nit A, and strains 3-1-3, B21, and Nit A at equal and unequal initial proportions. Plants were grown and maintained under flooded conditions in small wading pools. In co-inoculation of plants with 3-1-3 and B21 from equal initial proportions, the population of 3-1-3 increased slightly until it reached a proportion of 60 to 70%, whereas the population density of B21 reached 30 to 40% at the end of growing season. From unequal initial proportions, the population density of B21 decreased from 90 to about 50%, whereas the 3-1-3 increased from 10 to 50%. The population density of 3-1-3 increased from an equal initial proportion and was significantly greater than that of Nit A on every sampling time. From unequal initial proportions, the population density of 3-1-3 increased from 10 to 90%, whereas that of Nit A declined. In co-inoculation of plants with the three strains, the population density of 3-1-3 was significantly greater than those of the mutant strains at every sampling time. The proportions of mutant strains within the total population of C. gloeosporioides f. sp. aeschynomene on plants varied according to the test conditions and the number and types of strains co-inoculated.

Sensitivity of normal and mutant strains of Escherichia coli to actinomycin-D

1972 ◽  
Vol 18 (6) ◽  
pp. 909-915 ◽  
Author(s):  
A. P. Singh ◽  
K.-J. Cheng ◽  
J. W. Costerton ◽  
E. S. Idziak ◽  
J. M. Ingram
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Wild Type Strain ◽  
Actinomycin D ◽  
Cytoplasmic Membrane ◽  
Cell Envelope ◽  
Coli Strain ◽  
Wild Type ◽  
Mutant Strains ◽  
In The Wild

The site of the cell barrier to actinomycin-D uptake was studied using a wild-type Escherichia coli strain P and its cell envelope-defective filamentous mutants, strains 6γ and 12γ, both of which 'leak' β-galactosidase and alkaline phosphatase into the medium during growth indicating both membrane and cell-wall defects. Actinomycin-D entered the cells of these two mutant strains as evidenced by the inhibition of both 14C-uracil incorporation and synthesis of the induced β-galactosidase system. Under similar conditions, no inhibition occurred in the wild-type strain and its sucrose-lysozyme prepared spheroplasts. Actinomycin-D did, however, inhibit the above-mentioned systems in the wild-type sucrose-lysozyme spheroplasts prepared in the presence of 2 mM EDTA. The experimental data indicate that although the cell wall may act as a primary barrier or sieve to actinomycin-D, the cytoplasmic membrane should be considered the final and determinative barrier to this antibiotic.

scholarly journals Cloning, Sequencing, and Characterization of the SdeAB Multidrug Efflux Pump of Serratia marcescens

2005 ◽  
Vol 49 (4) ◽  
pp. 1495-1501 ◽  
Author(s):  
Ayush Kumar ◽  
Elizabeth A. Worobec
Keyword(s):  
Serratia Marcescens ◽  
Type Strain ◽  
Wild Type Strain ◽  
Genomic Library ◽  
Efflux Pump ◽  
Sodium Dodecyl ◽  
Wild Type ◽  
Diverse Range ◽  
Mutant Strains ◽  
Encoding Genes

ABSTRACT Serratia marcescens is an important nosocomial agent known for causing various infections in immunocompromised individuals. Resistance of this organism to a broad spectrum of antibiotics makes the treatment of infections very difficult. This study was undertaken to identify multidrug resistance efflux pumps in S. marcescens. Three mutant strains of S. marcescens were isolated in vitro by the serial passaging of a wild-type strain in culture medium supplemented with ciprofloxacin, norfloxacin, or ofloxacin. Fluoroquinolone accumulation assays were performed to detect the presence of a proton gradient-dependent efflux mechanism. Two of the mutant strains were found to be effluxing norfloxacin, ciprofloxacin, and ofloxacin, while the third was found to efflux only ofloxacin. A genomic library of S. marcescens wild-type strain UOC-67 was constructed and screened for RND pump-encoding genes by using DNA probes for two putative RND pump-encoding genes. Two different loci were identified: sdeAB, encoding an MFP and an RND pump, and sdeCDE, encoding an MFP and two different RND pumps. Northern blot analysis revealed overexpression of sdeB in two mutant strains effluxing fluoroquinolones. Analysis of the sdeAB and sdeCDE loci in Escherichia coli strain AG102MB, deficient in the RND pump (AcrB), revealed that gene products of sdeAB are responsible for the efflux of a diverse range of substrates that includes ciprofloxacin, norfloxacin, ofloxacin, chloramphenicol, sodium dodecyl sulfate, ethidium bromide, and n-hexane, while those of sdeCDE did not result in any change in susceptibilities to any of these agents.

A mutation in an exbD gene reduces tagetitoxin production by Pseudomonas syringae pv. tagetis

2006 ◽  
Vol 52 (11) ◽  
pp. 1027-1035 ◽  
Author(s):  
Hyesuk Kong ◽  
Cheryl D Patterson ◽  
Robin E Mitchell ◽  
Jeffrey S Buyer ◽  
M Catherine Aime ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Wild Type Strain ◽  
Efflux Pump ◽  
Wild Type ◽  
Chain Reaction ◽  
Sequence Identity ◽  
Mutant Strains ◽  
Tonb System ◽  
Polymerase Chain ◽  
High Degree

A mutant of Pseudomonas syringae pv. tagetis EB037 with limited ability to produce tagetitoxin was isolated after transposon mutagenesis and the mutation was characterized. The mutation occurred in a gene with a high degree of sequence identity to exbD. exbD is contiguous with tonB and exbB upstream and with a gene for a TonB-dependent receptor downstream. Using reverse transcription – polymerase chain reaction with RNA from the wild-type and exbD mutant strains, we demonstrated that the mutation in exbD did not have a polar affect on the expression of downstream genes. The exbD mutant was able to grow well in conditions where iron is not freely available. Siderophore production by the exbD mutant was similar to that of the wild-type strain. We conclude that the mutation in exbD disrupts tagetitoxin production without compromising iron metabolism. The results indicate that tagetitoxin export by P. syringae pv. tagetis involves an efflux pump that requires a functional TonB system that is not essential for normal iron metabolism.Key words: Pseudomonas syringae pv. tagetis, Pseudomonas putida, tagetitoxin, exbD, exbB, tonB, TonB system, Helianthus annuus L.

Inactivation of the Crp/Fnr Family of Regulatory Genes in Listeria monocytogenes Strain F2365 Does Not Alter Its Heat Resistance at 60°C†

2006 ◽  
Vol 69 (11) ◽  
pp. 2758-2760 ◽  
Author(s):  
DARRELL O. BAYLES ◽  
GAYLEN A. UHLICH
Keyword(s):  
Listeria Monocytogenes ◽  
Heat Resistance ◽  
Thermal Tolerance ◽  
Type Strain ◽  
Transcriptional Control ◽  
Wild Type Strain ◽  
Gene Cassette ◽  
Wild Type ◽  
Mutant Strains ◽  
Virulence Regulator

A surprising facet of the Listeria monocytogenes genome is the presence of 15 genes that code for regulators in the Crp/Fnr family and include the virulence regulator PrfA. The genes under the transcriptional control of these regulators are currently undetermined, with the exception of some genes controlled by the major virulence regulator PrfA. Using 12 strains of L. monocytogenes, each with an inserted gene cassette that interrupts and renders nonfunctional a different L. monocytogenes strain F2365 Crp/Fnr regulator, we heat challenged each strain at 60°C with an immersed-coil heating apparatus, modeled the survivor data to calculate the underlying mean and mode of the heat resistance distribution for each strain, and compared the thermal tolerance of each mutant to the wild-type strain to determine if any of the Crp/Fnr mutants demonstrated altered heat tolerance. All 12 of the Crp/Fnr mutant strains tested had heat resistance characteristics similar to the wild-type strain (P > 0.05), indicating that mutations in these Crp/Fnr genes neither increased nor decreased the sensitivity of L. monocytogenes strain F2365 to mild heat.

scholarly journals Role of CpALS4790 and CpALS0660 in Candida parapsilosis Virulence: Evidence from a Murine Model of Vaginal Candidiasis

2020 ◽  
Vol 6 (2) ◽  
pp. 86
Author(s):  
Marina Zoppo ◽  
Fabrizio Fiorentini ◽  
Cosmeri Rizzato ◽  
Mariagrazia Di Luca ◽  
Antonella Lupetti ◽  
...  
Keyword(s):  
Cell Wall ◽  
Murine Model ◽  
Type Strain ◽  
Wild Type Strain ◽  
Candida Parapsilosis ◽  
Vaginal Candidiasis ◽  
Phenotypic Characterization ◽  
Wild Type ◽  
Mutant Strains

The Candida parapsilosis genome encodes for five agglutinin-like sequence (Als) cell-wall glycoproteins involved in adhesion to biotic and abiotic surfaces. The work presented here is aimed at analyzing the role of the two still uncharacterized ALS genes in C. parapsilosis, CpALS4790 and CpALS0660, by the generation and characterization of CpALS4790 and CpALS066 single mutant strains. Phenotypic characterization showed that both mutant strains behaved as the parental wild type strain regarding growth rate in liquid/solid media supplemented with cell-wall perturbing agents, and in the ability to produce pseudohyphae. Interestingly, the ability of the CpALS0660 null mutant to adhere to human buccal epithelial cells (HBECs) was not altered when compared with the wild-type strain, whereas deletion of CpALS4790 led to a significant loss of the adhesion capability. RT-qPCR analysis performed on the mutant strains in co-incubation with HBECs did not highlight significant changes in the expression levels of others ALS genes. In vivo experiments in a murine model of vaginal candidiasis indicated a significant reduction in CFUs recovered from BALB/C mice infected with each mutant strain in comparison to those infected with the wild type strain, confirming the involvement of CpAls4790 and CpAls5600 proteins in C. parapsilosis vaginal candidiasis in mice.

scholarly journals The RcsCB His-Asp Phosphorelay System Is Essential To Overcome Chlorpromazine-Induced Stress in Escherichia coli

2002 ◽  
Vol 184 (10) ◽  
pp. 2850-2853 ◽  
Author(s):  
Annie Conter ◽  
Rachel Sturny ◽  
Claude Gutierrez ◽  
Kaymeuang Cam
Keyword(s):  
Escherichia Coli ◽  
Type Strain ◽  
Wild Type Strain ◽  
Cell Envelope ◽  
Wild Type ◽  
E Coli ◽  
Induced Stress ◽  
Mutant Strains ◽  
Molecular Nature

ABSTRACT The RcsCB His-Asp phosphorelay system regulates the expression of several genes of Escherichia coli, but the molecular nature of the inducing signal is still unknown. We show here that treatment of an exponentially growing culture of E. coli with the cationic amphipathic compound chlorpromazine (CPZ) stimulates expression of a set of genes positively regulated by the RcsCB system. This induction is abolished in rcsB or rcsC mutant strains. In addition, treatment with CPZ inhibits growth. The wild-type strain is able to recover from this inhibition and resume growth after a period of adaptation. In contrast, strains deficient in the RcsCB His-Asp phosphorelay system are hypersensitive to CPZ. These results suggest that cells must express specific RcsCB-regulated genes in order to cope with the CPZ-induced stress. This is the first report of the essential role of the RcsCB system in a stress situation. These results also strengthen the notion that alterations of the cell envelope induce a signal recognized by the RcsC sensor.

scholarly journals Ohr plays a central role in bacterial responses against fatty acid hydroperoxides and peroxynitrite

2016 ◽  
Vol 114 (2) ◽  
pp. E132-E141 ◽  
Author(s):  
Thiago G. P. Alegria ◽  
Diogo A. Meireles ◽  
José R. R. Cussiol ◽  
Martín Hugo ◽  
Madia Trujillo ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Wild Type Strain ◽  
Wild Type ◽  
Glutathione Peroxidases ◽  
Mutant Strains ◽  
In The Wild ◽  
Acid Hydroperoxide ◽  
Lipoamide Dehydrogenase ◽  
Fatty Acid Hydroperoxides ◽  
Competition Assays

Organic hydroperoxide resistance (Ohr) enzymes are unique Cys-based, lipoyl-dependent peroxidases. Here, we investigated the involvement of Ohr in bacterial responses toward distinct hydroperoxides. In silico results indicated that fatty acid (but not cholesterol) hydroperoxides docked well into the active site of Ohr fromXylella fastidiosaand were efficiently reduced by the recombinant enzyme as assessed by a lipoamide-lipoamide dehydrogenase–coupled assay. Indeed, the rate constants between Ohr and several fatty acid hydroperoxides were in the 107–108M−1⋅s−1range as determined by a competition assay developed here. Reduction of peroxynitrite by Ohr was also determined to be in the order of 107M−1⋅s−1at pH 7.4 through two independent competition assays. A similar trend was observed when studying the sensitivities of a ∆ohrmutant ofPseudomonas aeruginosatoward different hydroperoxides. Fatty acid hydroperoxides, which are readily solubilized by bacterial surfactants, killed the ∆ohrstrain most efficiently. In contrast, both wild-type and mutant strains deficient for peroxiredoxins and glutathione peroxidases were equally sensitive to fatty acid hydroperoxides. Ohr also appeared to play a central role in the peroxynitrite response, because the ∆ohrmutant was more sensitive than wild type to 3-morpholinosydnonimine hydrochloride (SIN-1 , a peroxynitrite generator). In the case of H2O2insult, cells treated with 3-amino-1,2,4-triazole (a catalase inhibitor) were the most sensitive. Furthermore, fatty acid hydroperoxide and SIN-1 both induced Ohr expression in the wild-type strain. In conclusion, Ohr plays a central role in modulating the levels of fatty acid hydroperoxides and peroxynitrite, both of which are involved in host–pathogen interactions.

scholarly journals An Antibiotic Complex from Lysobacter enzymogenes Strain C3: Antimicrobial Activity and Role in Plant Disease Control

2008 ◽  
Vol 98 (6) ◽  
pp. 695-701 ◽  
Author(s):  
S. Li ◽  
C. C. Jochum ◽  
F. Yu ◽  
K. Zaleta-Rivera ◽  
L. Du ◽  
...  
Keyword(s):  
Biological Control ◽  
Fusarium Head Blight ◽  
Tall Fescue ◽  
Leaf Spot ◽  
Type Strain ◽  
Wild Type Strain ◽  
Wild Type ◽  
Head Blight ◽  
Lysobacter Enzymogenes ◽  
Mutant Strains

Lysobacter enzymogenes C3 is a bacterial biological control agent that exhibits antagonism against multiple fungal pathogens. Its antifungal activity was attributed in part to lytic enzymes. In this study, a heat-stable antifungal factor (HSAF), an antibiotic complex consisting of dihydromaltophilin and structurally related macrocyclic lactams, was found to be responsible for antagonism by C3 against fungi and oomycetes in culture. HSAF in purified form exhibited inhibitory activity against a wide range of fungal and oomycetes species in vitro, inhibiting spore germination, and disrupting hyphal polarity in sensitive fungi. When applied to tall fescue leaves as a partially-purified extract, HSAF at 25 μg/ml and higher inhibited germination of conidia of Bipolaris sorokiniana compared with the control. Although application of HSAF at 12.5 μg/ml did not reduce the incidence of conidial germination, it inhibited appressorium formation and suppressed Bipolaris leaf spot development. Two mutant strains of C3 (K19 and ΔNRPS) that were disrupted in different domains in the hybrid polyketide synthase-nonribosomal peptide synthetase gene for HSAF biosynthesis and had lost the ability to produce HSAF were compared with the wild-type strain for biological control efficacy against Bipolaris leaf spot on tall fescue and Fusarium head blight, caused by Fusarium graminearum, on wheat. Both mutant strains exhibited decreased capacity to reduce the incidence and severity of Bipolaris leaf spot compared with C3. In contrast, the mutant strains were as efficacious as the wild-type strain in reducing the severity of Fusarium head blight. Thus, HSAF appears to be a mechanism for biological control by strain C3 against some, but not all, plant pathogenic fungi.

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