scholarly journals Controlling Instability in gacS-gacARegulatory Genes during Inoculant Production of Pseudomonas fluorescens Biocontrol Strains

2000 ◽  
Vol 66 (8) ◽  
pp. 3142-3150 ◽  
Author(s):  
Brion K. Duffy ◽  
Geneviève Défago
Keyword(s):  
Pseudomonas Fluorescens ◽  
Response Regulator ◽  
Spontaneous Mutation ◽  
Scale Up ◽  
Screening Method ◽  
Carbon Sources ◽  
Transcriptional Response ◽  
Culture Conditions ◽  
Ammonium Molybdate ◽  
Spontaneous Mutants

ABSTRACT Secondary metabolism in fluorescent pseudomonads is globally regulated by gacS, which encodes a membrane-bound sensor kinase, and gacA, which encodes a transcriptional response regulator. Spontaneous mutation in either gene blocked biosynthesis of the antimicrobial compounds hydrogen cyanide, 2,4-diacetylphloroglucinol, pyoluteorin, and pyrrolnitrin by the model biocontrol strain Pseudomonas fluorescens CHA0. Spontaneous mutants also had altered abilities to utilize several carbon sources and to increase medium pH compared with the wild type, suggesting thatgacS and gacA influence primary as well as secondary bacterial metabolism. Inoculant efficacy for biocontrol was significantly reduced by contamination with regulatory mutants which accumulated during inoculum production. Spontaneous mutants accumulated in all 192 separate liquid cultures examined, typically at a frequency of 1% or higher after 12 days. During scale-up in a simulated industrial fermentation process, mutants increased exponentially and accounted for 7, 23, and 61% of the total viable cells after transfer to 20-, 100-, and 500-ml preparations, respectively. GacS−and GacA− mutants had identical phenotypes and occurred at the same frequency, indicating that the selective pressures for the two mutants were similar. We developed a simple screening method for monitoring inoculant quality based on the distinctive appearance of mutant colonies (i.e., orange color, enlarged diameter, hyperfluorescence). Mutant competitiveness was favored in a nutrient-rich medium with a high electrolyte concentration (nutrient broth containing yeast extract). We were able to control mutant accumulation and to clean up contaminated cultures by using certain mineral amendments (i.e., zinc, copper, cobalt, manganese, and ammonium molybdate) or by diluting media 1/10. Spontaneous mutants and genetic constructs had the same response to culture conditions. Zinc and medium dilution were also effective for improving the genetic stability of other P. fluorescens biocontrol strains obtained from Ghana and Italy.

scholarly journals Optimization of Cellulase Production from Bacteria Isolated from Soil

2013 ◽  
Vol 2013 ◽  
pp. 1-7 ◽  
Author(s):  
Sonia Sethi ◽  
Aparna Datta ◽  
B. Lal Gupta ◽  
Saksham Gupta
Keyword(s):  
Bacillus Subtilis ◽  
Carbon Source ◽  
Pseudomonas Fluorescens ◽  
Carbon Sources ◽  
Nitrogen Sources ◽  
Fermentation Medium ◽  
Cellulase Production ◽  
Culture Conditions ◽  
Optimum Conditions ◽  
E Coli

Cellulase-producing bacteria were isolated from soil and identified as Pseudomonas fluorescens, Bacillus subtilIs, E. coli, and Serratia marcescens. Optimization of the fermentation medium for maximum cellulase production was carried out. The culture conditions like pH, temperature, carbon sources, and nitrogen sources were optimized. The optimum conditions found for cellulase production were 40°C at pH 10 with glucose as carbon source and ammonium sulphate as nitrogen source, and coconut cake stimulates the production of cellulase. Among bacteria, Pseudomonas fluorescens is the best cellulase producer among the four followed by Bacillus subtilis, E. coli, and Serratia marscens.

scholarly journals Phyllobacterium loti sp. nov. isolated from nodules of Lotus corniculatus

2014 ◽  
Vol 64 (Pt_3) ◽  
pp. 781-786 ◽  
Author(s):  
Maximo Sánchez ◽  
Martha-Helena Ramírez-Bahena ◽  
Alvaro Peix ◽  
María J. Lorite ◽  
Juan Sanjuán ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Sequence Similarity ◽  
Carbon Sources ◽  
Lotus Corniculatus ◽  
Culture Conditions ◽  
Rrna Gene ◽  
Content Type ◽  
Link Type ◽  
The 16S Rrna Gene

Strain S658T was isolated from a Lotus corniculatus nodule in a soil sample obtained in Uruguay. Phylogenetic analysis of the 16S rRNA gene and atpD gene showed that this strain clustered within the genus Phyllobacterium . The closest related species was, in both cases, Phyllobacterium trifolii PETP02T with 99.8 % sequence similarity in the 16S rRNA gene and 96.1 % in the atpD gene. The 16S rRNA gene contains an insert at the beginning of the sequence that has no similarities with other inserts present in the same gene in described rhizobial species. Ubiquinone Q-10 was the only quinone detected. Strain S658T differed from its closest relatives through its growth in diverse culture conditions and in the assimilation of several carbon sources. It was not able to reproduce nodules in Lotus corniculatus. The results of DNA–DNA hybridization, phenotypic tests and fatty acid analyses confirmed that this strain should be classified as a representative of a novel species of the genus Phyllobacterium , for which the name Phyllobacterium loti sp. nov. is proposed. The type strain is S658T( = LMG 27289T = CECT 8230T).

scholarly journals Ice-nucleating microorganisms. Part II. Effect of carbon sources on the ice-nucleating activity of Pseudomonas fluorescens KUIN-1.

1987 ◽  
Vol 61 (10) ◽  
pp. 1285-1288
Author(s):  
Hitoshi OBATA ◽  
Kiyonori DOBASHI ◽  
Junichi TANISHITA ◽  
Tai TOKUYAMA
Keyword(s):  
Pseudomonas Fluorescens ◽  
Carbon Sources

Optimization of culture conditions and scale-up to plant scales for teicoplanin production by Actinoplanes teichomyceticus

2008 ◽  
Vol 80 (1) ◽  
Author(s):  
Hyung-Moo Jung ◽  
Sang-Yong Kim ◽  
Ponnandy Prabhu ◽  
Hee-Jung Moon ◽  
In-Won Kim ◽  
...  
Keyword(s):  
Scale Up ◽  
Culture Conditions ◽  
Actinoplanes Teichomyceticus ◽  
Optimization Of Culture Conditions

scholarly journals A screening system for carbon sources enhancing β-N-acetylglucosaminidase formation in Hypocrea atroviridis (Trichoderma atroviride)

Microbiology ◽  
2006 ◽  
Vol 152 (7) ◽  
pp. 2003-2012 ◽  
Author(s):  
Verena Seidl ◽  
Irina S. Druzhinina ◽  
Christian P. Kubicek
Keyword(s):  
Screening Method ◽  
Carbon Sources ◽  
Transcript Abundance ◽  
Phenotype Microarray ◽  
Screening System ◽  
Trichoderma Atroviride ◽  
Wild Type ◽  
Activity Measurements ◽  
In The Wild ◽  
Biolog Phenotype Microarray

To identify carbon sources that trigger β-N-acetylglucosaminidase (NAGase) formation in Hypocrea atroviridis (anamorph Trichoderma atroviride), a screening system was designed that consists of a combination of Biolog Phenotype MicroArray plates, which contain 95 different carbon sources, and specific enzyme activity measurements using a chromogenic substrate. The results revealed growth-dependent kinetics of NAGase formation and it was shown that NAGase activities were enhanced on carbon sources sharing certain structural properties, especially on α-glucans (e.g. glycogen, dextrin and maltotriose) and oligosaccharides containing galactose. Enzyme activities were assessed in the wild-type and a H. atroviridis Δnag1 strain to investigate the influence of the two NAGases, Nag1 and Nag2, on total NAGase activity. Reduction of NAGase levels in the Δnag1 strain in comparison to the wild-type was strongly carbon-source and growth-phase dependent, indicating the distinct physiological roles of the two proteins. The transcript abundance of nag1 and nag2 was increased on carbon sources with elevated NAGase activity, indicating transcriptional regulation of these genes. The screening method for the identification of carbon sources that induce enzymes or a gene of interest, as presented in this paper, can be adapted for other purposes if appropriate enzyme or reporter assays are available.

scholarly journals Putative metabolic pathway for the bioproduction of bikaverin and intermediates thereof in the wild Fusarium oxysporum LCP531 strain

AMB Express ◽  
2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Juliana Lebeau ◽  
Thomas Petit ◽  
Laurent Dufossé ◽  
Yanis Caro
Keyword(s):  
Metabolic Pathway ◽  
Carbon Sources ◽  
Nitrogen Sources ◽  
Pharmaceutical Products ◽  
Culture Conditions ◽  
Fusarium Fujikuroi ◽  
Submerged Cultures ◽  
In The Wild ◽  
Considerable Work ◽  
Pharmaceutical Uses

AbstractFungal naphthoquinones, like red bikaverin, are of interest due to their growing applications in designing pharmaceutical products. Though considerable work has been done on the elucidation of bikaverin biosynthesis pathway in Fusarium fujikuroi, very few reports are available regarding its bioproduction in F. oxysporum. We are hereby proposing a putative metabolic pathway for bikaverin bioproduction in a wild F. oxysporum strain by cross-linking the pigment profiles we obtained under two different fermentation conditions with literature. Naphthoquinone pigments were extracted with a pressurized liquid extraction method, and characterized by HPLC–DAD and UHPLC-HRMS. The results led to the conclusions that the F. oxysporum LCP531 strain was able to produce bikaverin and its various intermediates, e.g., pre-bikaverin, oxo-pre-bikaverin, dinor-bikaverin, me-oxo-pre-bikaverin, and nor-bikaverin, in submerged cultures in various proportions. To our knowledge, this is the first report of the isolation of these five bikaverin intermediates from F. oxysporum cultures, providing us with steady clues for confirming a bikaverin metabolic pathway as well as some of its regulatory patterns in the F. oxysporum LCP531 strain, based on the previously reported model in F. fujikuroi. Interestingly, norbikaverin accumulated along with bikaverin in mycelial cells when the strain grew on simple carbon and nitrogen sources and additional cofactors. Along bikaverin production, we were able to describe the excretion of the toxin beauvericin as main extrolite exclusively in liquid medium containing complex nitrogen and carbon sources, as well as the isolation of ergosterol derivate in mycelial extracts, which have potential for pharmaceutical uses. Therefore, culture conditions were also concluded to trigger some specific biosynthetic route favoring various metabolites of interest. Such observation is of great significance for selective production of pigments and/or prevention of occurrence of others (aka mycotoxins).

Induction and Transcription Studies of the Dextransucrase Gene in Leuconostoc mesenteroides NRRL B-512F

1999 ◽  
Vol 65 (12) ◽  
pp. 5504-5509 ◽  
Author(s):  
M. Quirasco ◽  
A. López-Munguía ◽  
M. Remaud-Simeon ◽  
P. Monsan ◽  
A. Farrés
Keyword(s):  
High Performance ◽  
Leuconostoc Mesenteroides ◽  
Carbon Sources ◽  
Promoter Sequence ◽  
Culture Conditions ◽  
Start Codon ◽  
Reaction Products ◽  
Product Specificity ◽  
First Time

ABSTRACT Dextransucrase production by Leuconostoc mesenteroidesNRRL B-512F in media containing carbon sources other than sucrose is reported for the first time. Dextransucrases were analyzed by gel electrophoresis and by an in situ activity assay. Their polymers and acceptor reaction products were also compared by 13C nuclear magnetic resonance and high-performance liquid chromatography techniques, respectively. From these analyses, it was found that, independently of the carbon source, L. mesenteroides NRRL B-512F produced dextransucrases of the same size and product specificity. The 5′ ends of dextransucrase mRNAs isolated from cells grown under different culture conditions were identical. Based on this evidence, we conclude that dextransucrases obtained from cells grown on the various carbon sources result from the transcription of the same gene. The control of expression occurs at this level. The low dextransucrase yields from cultures in d-glucose ord-fructose and the enhancement of dextransucrase gene transcription in the presence of sucrose suggest that an activating phenomenon may be involved in the expression mechanism. Dextransucrase mRNA has a size of approximately 4.8 kb, indicating that the gene is located in a monocistronic operon. The transcription start point was localized 34 bp upstream from the ATG start codon. The −10 and −35 sequences found, TATAAT and TTTACA, were highly homologous to the only glycosyltransferase promoter sequence reported for lactic acid bacteria.

scholarly journals New Roles for Two-Component System Response Regulators of Salmonella enterica Serovar Typhi during Host Cell Interactions

2020 ◽  
Vol 8 (5) ◽  
pp. 722
Author(s):  
Claudie Murret-Labarthe ◽  
Maud Kerhoas ◽  
Karine Dufresne ◽  
France Daigle
Keyword(s):  
Salmonella Enterica ◽  
Pathogenic Bacteria ◽  
Response Regulator ◽  
Transcriptional Response ◽  
Host Cells ◽  
System Response ◽  
Response Regulators ◽  
Salmonella Enterica Serovar Typhi ◽  
Two Component ◽  
External Stresses

In order to survive external stresses, bacteria need to adapt quickly to changes in their environment. One adaptive mechanism is to coordinate and alter their gene expression by using two-component systems (TCS). TCS are composed of a sensor kinase that activates a transcriptional response regulator by phosphorylation. TCS are involved in motility, virulence, nutrient acquisition, and envelope stress in many bacteria. The pathogenic bacteria Salmonella enterica serovar Typhi (S. Typhi) possess 30 TCSs, is specific to humans, and causes typhoid fever. Here, we have individually deleted each of the 30 response regulators. We have determined their role during interaction with host cells (epithelial cells and macrophages). Deletion of most of the systems (24 out of 30) resulted in a significant change during infection. We have identified 32 new phenotypes associated with TCS of S. Typhi. Some previously known phenotypes associated with TCSs in Salmonella were also confirmed. We have also uncovered phenotypic divergence between Salmonella serovars, as distinct phenotypes between S. Typhi and S. Typhimurium were identified for cpxR. This finding highlights the importance of specifically studying S. Typhi to understand its pathogenesis mechanisms and to develop strategies to potentially reduce typhoid infections.

scholarly journals Spontaneous Mutation at Amino Acid 544 of the Ebola Virus Glycoprotein Potentiates Virus Entry and Selection in Tissue Culture

2017 ◽  
Vol 91 (15) ◽  
Author(s):  
John B. Ruedas ◽  
Jason T. Ladner ◽  
Chelsea R. Ettinger ◽  
Suryaram Gummuluru ◽  
Gustavo Palacios ◽  
...  
Keyword(s):  
Tissue Culture ◽  
Experimental Evidence ◽  
Ebola Virus ◽  
Virus Entry ◽  
Spontaneous Mutation ◽  
Culture Conditions ◽  
Surface Glycoprotein ◽  
Virus Growth ◽  
Content Type ◽  
Zaire Ebolavirus

ABSTRACT Ebolaviruses have a surface glycoprotein (GP1,2) that is required for virus attachment and entry into cells. Mutations affecting GP1,2 functions can alter virus growth properties. We generated a recombinant vesicular stomatitis virus encoding Ebola virus Makona variant GP1,2 (rVSV-MAK-GP) and observed emergence of a T544I mutation in the Makona GP1,2 gene during tissue culture passage in certain cell lines. The T544I mutation emerged within two passages when VSV-MAK-GP was grown on Vero E6, Vero, and BS-C-1 cells but not when it was passaged on Huh7 and HepG2 cells. The mutation led to a marked increase in virus growth kinetics and conferred a robust growth advantage over wild-type rVSV-MAK-GP on Vero E6 cells. Analysis of complete viral genomes collected from patients in western Africa indicated that this mutation was not found in Ebola virus clinical samples. However, we observed the emergence of T544I during serial passage of various Ebola Makona isolates on Vero E6 cells. Three independent isolates showed emergence of T544I from undetectable levels in nonpassaged virus or virus passaged once to frequencies of greater than 60% within a single passage, consistent with it being a tissue culture adaptation. Intriguingly, T544I is not found in any Sudan, Bundibugyo, or Tai Forest ebolavirus sequences. Furthermore, T544I did not emerge when we serially passaged recombinant VSV encoding GP1,2 from these ebolaviruses. This report provides experimental evidence that the spontaneous mutation T544I is a tissue culture adaptation in certain cell lines and that it may be unique for the species Zaire ebolavirus. IMPORTANCE The Ebola virus (Zaire) species is the most lethal species of all ebolaviruses in terms of mortality rate and number of deaths. Understanding how the Ebola virus surface glycoprotein functions to facilitate entry in cells is an area of intense research. Recently, three groups independently identified a polymorphism in the Ebola glycoprotein (I544) that enhanced virus entry, but they did not agree in their conclusions regarding its impact on pathogenesis. Our findings here address the origins of this polymorphism and provide experimental evidence showing that it is the result of a spontaneous mutation (T544I) specific to tissue culture conditions, suggesting that it has no role in pathogenesis. We further show that this mutation may be unique to the species Zaire ebolavirus, as it does not occur in Sudan, Bundibugyo, and Tai Forest ebolaviruses. Understanding the mechanism behind this mutation can provide insight into functional differences that exist in culture conditions and among ebolavirus glycoproteins.

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