Accumulation of poly(3-hydroxybutyric acid) and overproduction of exopolysaccharides in a mutant of a methylotrophic bacterium

1995 ◽  
Vol 41 (13) ◽  
pp. 55-59 ◽  
Author(s):  
Uta Breuer ◽  
Jörg-Uwe Ackermann ◽  
Wolfgang Babel
Keyword(s):  
Ammonium Phosphate ◽  
Sole Source ◽  
Chemical Mutagenesis ◽  
Methylotrophic Bacterium ◽  
Wild Type ◽  
Hydroxybutyric Acid ◽  
Serine Pathway ◽  
Methylobacterium Rhodesianum

The pink-pigmented facultatively methylotrophic bacterium Methylobacterium rhodesianum MB 126 is able to grow on methanol as the sole source of carbon and energy. Under certain conditions, e.g., limitation of ammonium, phosphate, or oxygen, carbon from methanol is channeled into poly(3-hydroxybutyric acid) (PHB) whereas other polymers or metabolites are hardly overproduced. A mutant of this strain, which we isolated after chemical mutagenesis, is impaired in its ability to synthesize PHB. Under the conditions mentioned above, the mutant still accumulated PHB, but in the absence of ammonium it simultaneously synthesized PHB and a considerable amount of an exopolysaccharide. This phenomenon was surprising insofar as the wild type did not produce exopolysaccharide in such amounts. An attempt was made to elucidate and discuss the possible reasons for these findings.Key words: methylotrophy, serine pathway bacteria, PHB, exopolysaccharides.

scholarly journals Production of the Chiral Compound (R)-3-Hydroxybutyrate by a Genetically Engineered Methylotrophic Bacterium

2010 ◽  
Vol 76 (16) ◽  
pp. 5585-5591 ◽  
Author(s):  
Tina Hölscher ◽  
Uta Breuer ◽  
Lorenz Adrian ◽  
Hauke Harms ◽  
Thomas Maskow
Keyword(s):  
Citric Acid Cycle ◽  
Sole Source ◽  
Genetically Engineered ◽  
Methylotrophic Bacterium ◽  
Cultivation Conditions ◽  
Chiral Compound ◽  
Batch Cultures ◽  
Alternative Pathways ◽  
Intracellular Degradation ◽  
Methylobacterium Rhodesianum

ABSTRACT In this study, a methylotrophic bacterium, Methylobacterium rhodesianum MB 126, was used for the production of the chiral compound (R)-3-hydroxybutyrate (R-3HB) from methanol. R-3HB is formed during intracellular degradation of the storage polymer (R)-3-polyhydroxybutyrate (PHB). Since the monomer R-3HB does not accumulate under natural conditions, M. rhodesianum was genetically modified. The gene (hbd) encoding the R-3HB-degrading enzyme, R-3HB dehydrogenase, was inactivated in M. rhodesianum. The resulting hbd mutant still exhibited low growth rates on R-3HB as the sole source of carbon and energy, indicating the presence of alternative pathways for R-3HB utilization. Therefore, transposon mutagenesis was carried out with the hbd mutant, and a double mutant unable to grow on R-3HB was obtained. This mutant was shown to be defective in lipoic acid synthase (LipA), resulting in an incomplete citric acid cycle. Using the hbd lipA mutant, we produced 3.2 to 3.5 mM R-3HB in batch and 27 mM (2,800 mg liter−1) in fed-batch cultures. This was achieved by sequences of cultivation conditions initially favoring growth, then PHB accumulation, and finally PHB degradation.

Isolation and purification of granule-associated proteins relevant for poly(3-hydroxybutyric acid) biosynthesis from methylotrophic bacteria relying on the serine pathway

1995 ◽  
Vol 41 (13) ◽  
pp. 124-130 ◽  
Author(s):  
C. G. Föllner ◽  
W. Babel ◽  
A. Steinbüchel
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Methylotrophic Bacteria ◽  
Hydroxybutyric Acid ◽  
Isolation And Purification ◽  
Small Proteins ◽  
Associated Proteins ◽  
Serine Pathway ◽  
Methylobacterium Rhodesianum ◽  
Leaky Mutants

The poly(3-hydroxybutyric acid) (PHB) granules from eight methylotrophic bacteria that use the serine pathway were isolated in a sucrose gradient (1–2 M); these bacteria included members of the genus Methylobacterium, Mycoplana rubra, and PHB-leaky mutants of Methylobacterium rhodesianum. As shown by sodium dodecyl sulfate – polyacrylamide gel electrophoresis, the granules from all investigated methylotrophic strains revealed two major bands representing small proteins. An efficient purification procedure for these two low molecular weight proteins associated with the PHB granules was developed by solubilization of the proteins with Triton X-114 and affinity chromatography on Procion Blue-H-ERD.Key words: poly(3-hydroxybutyric acid), granule-associated proteins, methylotrophic bacteria.

scholarly journals Methylobacterium populi sp. nov., a novel aerobic, pink-pigmented, facultatively methylotrophic, methane-utilizing bacterium isolated from poplar trees (Populus deltoides×nigra DN34)

2004 ◽  
Vol 54 (4) ◽  
pp. 1191-1196 ◽  
Author(s):  
Benoit Van Aken ◽  
Caroline M. Peres ◽  
Sharon Lafferty Doty ◽  
Jong Moon Yoon ◽  
Jerald L. Schnoor
Keyword(s):  
Populus Deltoides ◽  
Novel Species ◽  
Phylogenetic Analyses ◽  
Sole Source ◽  
Methylotrophic Bacterium ◽  
Carbon Source Utilization ◽  
Methylobacterium Extorquens ◽  
Poplar Trees ◽  
Methylobacterium Rhodesianum ◽  
Bacterium Strain

A pink-pigmented, aerobic, facultatively methylotrophic bacterium, strain BJ001T, was isolated from internal poplar tissues (Populus deltoides×nigra DN34) and identified as a member of the genus Methylobacterium. Phylogenetic analyses showed that strain BJ001T is related to Methylobacterium thiocyanatum, Methylobacterium extorquens, Methylobacterium zatmanii and Methylobacterium rhodesianum. However, strain BJ001T differed from these species in its carbon-source utilization pattern, particularly its use of methane as the sole source of carbon and energy, an ability that is shared with only one other member of the genus, Methylobacterium organophilum. In addition, strain BJ001T is the only member of the genus Methylobacterium to be described as an endophyte of poplar trees. On the basis of its physiological, genotypic and ecological properties, the isolate is proposed as a member of a novel species of the genus Methylobacterium, Methylobacterium populi sp. nov. (type strain, BJ001T=ATCC BAA-705T=NCIMB 13946T).

scholarly journals A novel positive selection for identifying cold-sensitive myosin II mutants in Dictyostelium.

Genetics ◽  
1995 ◽  
Vol 140 (2) ◽  
pp. 505-515 ◽  
Author(s):  
B Patterson ◽  
J A Spudich
Keyword(s):  
Positive Selection ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Time Scale ◽  
Myosin Ii ◽  
Rapid Onset ◽  
Chemical Mutagenesis ◽  
Wild Type ◽  
Cold Sensitive ◽  
Selection For

Abstract We developed a positive selection for myosin heavy chain mutants in Dictyostelium. This selection is based on the fact that brief exposure to azide causes wild-type cells to release from the substrate, whereas myosin null cells remain adherent. This procedure assays myosin function on a time scale of minutes and has therefore allowed us to select rapid-onset cold-sensitive mutants after random chemical mutagenesis of Dictyostelium cells. We developed a rapid technique for determining which mutations lie in sequences of the myosin gene that encode the head (motor) domain and localized 27 of 34 mutants to this domain. We recovered the appropriate sequences from five of the mutants and demonstrated that they retain their cold-sensitive properties when expressed from extrachromosomal plasmids.

scholarly journals Chemical Mutagenesis and Fluorescence-Based High-Throughput Screening for Enhanced Accumulation of Carotenoids in a Model Marine Diatom Phaeodactylum tricornutum

Marine Drugs ◽  
2018 ◽  
Vol 16 (8) ◽  
pp. 272 ◽  
Author(s):  
Zhiqian Yi ◽  
Yixi Su ◽  
Maonian Xu ◽  
Andreas Bergmann ◽  
Saevar Ingthorsson ◽  
...  
Keyword(s):  
High Throughput ◽  
Metabolic Pathways ◽  
Phaeodactylum Tricornutum ◽  
Mass Spectrometry Data ◽  
Marine Diatom ◽  
Chemical Mutagenesis ◽  
Carotenoid Content ◽  
Enzymatic Reactions ◽  
Wild Type ◽  
A Genome

Diatoms are a major group of unicellular algae that are rich in lipids and carotenoids. However, sustained research efforts are needed to improve the strain performance for high product yields towards commercialization. In this study, we generated a number of mutants of the model diatom Phaeodactylum tricornutum, a cosmopolitan species that has also been found in Nordic region, using the chemical mutagens ethyl methanesulfonate (EMS) and N-methyl-N′-nitro-N-nitrosoguanidine (NTG). We found that both chlorophyll a and neutral lipids had a significant correlation with carotenoid content and these correlations were better during exponential growth than in the stationary growth phase. Then, we studied P. tricornutum common metabolic pathways and analyzed correlated enzymatic reactions between fucoxanthin synthesis and pigmentation or lipid metabolism through a genome-scale metabolic model. The integration of the computational results with liquid chromatography-mass spectrometry data revealed key compounds underlying the correlative metabolic pathways. Approximately 1000 strains were screened using fluorescence-based high-throughput method and five mutants selected had 33% or higher total carotenoids than the wild type, in which four strains remained stable in the long term and the top mutant exhibited an increase of 69.3% in fucoxanthin content compared to the wild type. The platform described in this study may be applied to the screening of other high performing diatom strains for industrial applications.

Growth of rhizosphere-competent mutants of Trichoderma harzianum on carbon substrates

1988 ◽  
Vol 34 (6) ◽  
pp. 807-814 ◽  
Author(s):  
Jaleed S. Ahmad ◽  
Ralph Baker
Keyword(s):  
Trichoderma Harzianum ◽  
Carbon Sources ◽  
Sole Source ◽  
Ecological Significance ◽  
Wild Type ◽  
Wood Cellulose ◽  
Carbon Substrates ◽  
Rhizosphere Competence ◽  
Complex Carbon ◽  
Root Surfaces

When the strains of Trichoderma harzianum were grown in Czapek-Dox broth without saccharose with cotton linters, microcrystalline cellulose, wood cellulose, or xylan as a sole source of carbon, the rhizosphere-competent mutants produced significantly higher biomass than the rhizosphere-incompetent wild types. Both mutants and wild types did not readily grow on glucose, galactose, cellobiose, or xylose as sole source of carbon. The mutants, T-95 and T-12B, produced significantly higher biomass when grown on complex carbohydrates with added simple sugars. The wild types did not produce significantly greater biomass when both simple and complex sugars were the carbon sources than when either substrate was used alone. The ability of the mutants to grow more rapidly on complex carbon substrates (typical of those found on root surfaces) than their wild-type parents, and to increase biomass when simple sugars were added along with the cellulose substrate could be of ecological significance and a characteristic of rhizosphere competence.

scholarly journals A Dictyostelium mutant deficient in severin, an F-actin fragmenting protein, shows normal motility and chemotaxis.

1989 ◽  
Vol 108 (3) ◽  
pp. 985-995 ◽  
Author(s):  
E André ◽  
M Brink ◽  
G Gerisch ◽  
G Isenberg ◽  
A Noegel ◽  
...  
Keyword(s):  
Cell Function ◽  
Polyclonal Antibodies ◽  
Deae Cellulose ◽  
Cell Movement ◽  
Processing System ◽  
Chemical Mutagenesis ◽  
Deficient Mutant ◽  
Wild Type ◽  
Coding Region ◽  
Mutant Cells

A severin deficient mutant of Dictyostelium discoideum has been isolated by the use of colony immunoblotting after chemical mutagenesis. In homogenates of wild-type cells, severin is easily detected as a very active F-actin fragmenting protein. Tests for severin in the mutant, HG1132, included viscometry for the assay of F-actin fragmentation in fractions from DEAE-cellulose columns, labeling of blots with monoclonal and polyclonal antibodies, and immunofluorescent-labeling of cryosections. Severin could not be detected in the mutant using these methods. The mutation in HG1132 is recessive and has been mapped to linkage group VII. The mutant failed to produce the normal severin mRNA, but small amounts of a transcript that was approximately 100 bases larger than the wild-type mRNA were detected in the mutant throughout all stages of development. On the DNA level a new Mbo II restriction site was found in the mutant within the coding region of the severin gene. The severin deficient mutant cells grew at an approximately normal rate, aggregated and formed fruiting bodies with viable spores. By the use of an image processing system, speed of cell movement, turning rates, and precision of chemotactic orientation in a stable gradient of cyclic AMP were quantitated, and no significant differences between wild-type and mutant cells were found. Thus, under the culture conditions used, severin proved to be neither essential for growth of D. discoideum nor for any cell function that is important for aggregation or later development.

scholarly journals A Peptide Permease Mutant of Mycobacterium bovis BCG Resistant to the Toxic Peptides Glutathione andS-Nitrosoglutathione

2000 ◽  
Vol 68 (2) ◽  
pp. 429-436 ◽  
Author(s):  
Renee M. Green ◽  
Anjali Seth ◽  
Nancy D. Connell
Keyword(s):  
Mutant Strain ◽  
Mycobacterium Bovis ◽  
Mammalian Cells ◽  
Sole Source ◽  
Cosmid Library ◽  
Peptide Transporter ◽  
Wild Type ◽  
Genome Database ◽  
Mutant Strains ◽  
Oligopeptide Permease

ABSTRACT Oligopeptides play important roles in bacterial nutrition and signaling. Using sequences from the available genome database forMycobacterium tuberculosis H37Rv, the oligopeptide permease operon (oppBCDA) of Mycobacterium bovis BCG was cloned from a cosmid library. An opp mutant strain was constructed by homologous recombination with an allele ofoppD interrupted by kanamycin and streptomycin resistance markers. The deletion was complemented with a wild-type copy of theopp operon. Two approaches were taken to characterize the peptide transporter defect in this mutant strain. First, growth of wild-type and mutant strains was monitored in media containing a wide variety of peptides as sole source of carbon and/or nitrogen. Among 25 peptides ranging from two to six amino acids in length, none was capable of supporting measurable growth as the sole carbon source in either wild-type or mutant strains. The second approach exploited the resistance of permease mutants to toxic substrates. The tripeptide glutathione (γ-glutamyl-l-cyteinylglycine [GSH]) is toxic to wild-type BCG and was used successfully to characterize peptide uptake in the opp mutant. In 2 mM GSH, growth of the wild-type strain is inhibited, whereas the opp mutant is resistant to concentrations as high as 10 mM. Similar results were found with the tripeptide S-nitrosoglutathione (GSNO), thought to be a donor of NO in mammalian cells. Using incorporation of [3H]uracil to monitor the effects of GSH and GSNO on macromolecular synthesis in growing cells, it was demonstrated that theopp mutant is resistant, whereas the wild type and the mutant complemented with a wild-type copy of the operon are sensitive to both tripeptides. In uptake measurements, incorporation of [3H]GSH is reduced in the mutant compared with wild type and the complemented mutant. Finally, growth of the three strains in the tripeptides suggests that GSH is bacteriostatic, whereas GSNO is bacteriocidal.

scholarly journals Comparison of the Wild-Type Obligate Methylotrophic Bacterium Methylophilus quaylei and its Isogenic Streptomycin-Resistant Mutant via Metal Nanoparticle Generation

2019 ◽  
Vol 193 (2) ◽  
pp. 564-573 ◽  
Author(s):  
Vladimir V. Sorokin ◽  
Anna B. Pshenichnikova ◽  
Sergei V. Kalenov ◽  
Nikolay A. Suyasov ◽  
Dmitry A. Skladnev
Keyword(s):  
Resistant Mutant ◽  
Metal Nanoparticle ◽  
Methylotrophic Bacterium ◽  
Wild Type ◽  
Nanoparticle Generation

scholarly journals Generation and Molecular Characterization of New Temperature-Sensitive Plasmids Intended for Genetic Engineering of Pasteurellaceae

2005 ◽  
Vol 71 (11) ◽  
pp. 7187-7195 ◽  
Author(s):  
Robert E. Briggs ◽  
Fred M. Tatum
Keyword(s):  
Base Pair ◽  
Pasteurella Multocida ◽  
Chemical Mutagenesis ◽  
Temperature Sensitive ◽  
Origin Of Replication ◽  
Wild Type ◽  
Recognition Sequence ◽  
Nucleotide Substitutions ◽  
Serotype 1

ABSTRACT Temperature-sensitive (TS) plasmids were generated through chemical mutagenesis of a derivative of the streptomycin resistance parent plasmid pD70, isolated from Mannheimia hemolytica serotype 1. Three TS plasmids which failed to replicate at or above 42°C in M. hemolytica but which were fully functional below 31°C were selected for further analysis. Two of the TS plasmids were shown by sequencing to possess unique single-base-pair mutations. The third TS plasmid contained a unique base pair substitution and a second mutation that had been previously identified. These mutations were clustered within a 200-bp region of the presumed plasmid origin of replication. Site-directed single-nucleotide substitutions were introduced into the wild-type pD70 origin of replication to confirm that mutations identified by sequencing had conferred thermoregulated replication. Deletion analysis on the wild-type pD70 plasmid replicon revealed that approximately 720 bp are necessary for plasmid maintenance. Replication of the TS plasmids was thermoregulated in Pasteurella multocida and Haemophilus somnus as well. To consistently transform H. somnus with TS plasmid, in vitro DNA methylation with commercially available HhaI methyltransferase was necessary to protect against the organism's restriction enzyme HsoI (recognition sequence 5′-GCGC-3′) characterized herein.

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