837 research outputs found
Transformation of the Methylotrophic Actinomycete Amycolatopis methanolica with Plasmid DNA: Stimulatory Effect of a pMEA300-Encoded Gene
Amycolatopsis methanolica contains a 13.29-kb plasmid (pMEA300) present both in the free state and integrated at a unique genomic location. A pMEA300-free derivative (strain WV1) was selected, allowing further analysis of pMEA300-encoded functions. Whole cells of strain WV1 could be transformed at high frequencies (approximately 10^6 transformants per microgram of plasmid DNA) with both circular and linear plasmid DNA, provided that the pMEA300-encoded stf (stimulation of transformation frequency) gene was present. stf would encode a putative protein of 373 amino acids with Mr 40,201, resembling putative regulatory proteins involved in sporulation of Streptomyces griseus and Streptomyces coelicolor.
Metabolism of amino acid amides in Pseudomonas putida ATCC 12633
The metabolism of the natural amino acid L-valine, the unnatural amino acids D-valine, and D-, L-phenylglycine (D-, L-PG), and the unnatural amino acid amides D-, L-phenylglycine amide (D, L-PG-NH2) and L-valine amide (L-Val-NH2) was studied in Pseudomonas putida ATCC 12633. The organism possessed constitutive L-amidase activities towards L-PG-NH2 and L-Val-NH2, both following the same pattern of expression, suggesting the involvement of similarly regulated enzymes, or a common enzyme. Quite surprisingly, growth in mineral media with L-PG-NH2 resulted in variable, long lag phases of growth and strongly reduced L-amidase activities. Conversion of D-PG-NH2 into D-PG and L-PG also occurred and could be attributed to the presence of an inducible D-amidase and the racemization of the amino acid amide in combination with L-amidase activity, respectively. The further degradation of L-PG and D-PG involved constitutive L-PG aminotransferase and inducible D-PG dehydrogenase activities, respectively, both with a high degree of enantioselectivity. Amino acid racemase activity for D- and L-PG was not detected.
Regulation of Autotrophic and Heterotrophic Metabolism in Pseudomonas oxalaticus OX1:Growth on Mixtures of Acetate and Formate in Continuous Culture
Growth of Pseudomonas oxalaticus in carbon- and energy-limited continuous cultures with mixtures of acetate and formate resulted in the simultaneous utilization of both substrates at all dilution rates tested. During growth on these mixtures, acetate repressed the synthesis of ribulosebisphosphate carboxylase. The degree of this repression was dependent on the dilution rate and on the ratio of acetate and formate in the medium reservoir. At fixed acetate and formate concentrations in the infiowing medium of 30 and 100 mM, respectively, and dilution rates above 0.10 h-1, the severe repression of autotrophic enzymes resulted in a marked increase in bacterial dry weight compared to the growth yield of the organisms on the two substrates separately. Also, at these dilution rates a significant increase in isocitrate lyase activity was observed in the cells as compared to growth on acetate alone. This indicated that under these conditions more acetate was assimilated and less dissimilated since acetate was partly replaced by formate as the energy source. When formate was added to the reservoir of an acetate-limited culture (SR = 30 mM), derepression of RuBPCase synthesis was observed at formate concentrations of 50 mM and above. Below this concentration formate only served as an energy source for acetate assimilation; when its concentration was increased above 50 mM a progressively increasing contribution of carbon dioxide fixation to the total carbon assimilation was observed as the activity of RuBPCase in the cells increased. It is concluded that in Pseudomonas oxalaticus the synthesis of enzymes involved in autotrophic carbon dioxide fixation via the Calvin cycle is regulated by a repression/derepression mechanism
Screening and characterization of Lactobacillus strains producing large amounts of exopolysaccharides
A total of 182 Lactobacillus strains were screened for production of extracellular polysaccharides (EPS) by a new method: growth in liquid media with high sugar concentrations. Sixty EPS-positive strains were identified; 17 strains produced more than 100 mg/l soluble EPS. Sucrose was an excellent substrate for abundant EPS synthesis. The ability to produce glucans appears to be widespread in the genus Lactobacillus. The monosaccharide composition of EPS produced by Lactobacillus reuteri strain LB 121 varied with the growth conditions (solid compared to liquid medium) and the sugar substrates (sucrose or raffinose) supplied in the medium. Strain LB 121 produced both a glucan and a fructan on sucrose, but only a fructan on raffinose. This is the first report of fructan production by a Lactobacillus species. EPS production increased with increasing sucrose concentrations and involved extracellular sucrase-type enzymes.
Single amino acid mutations interchange the reaction specificities of cyclodextrin glycosyltransferase and the acarbose-modifying enzyme acarviosyl transferase
Acarviosyl transferase (ATase) from Actinoplanes sp. SE50/110 is a bacterial enzyme that transfers the acarviosyl moiety of the diabetic drug acarbose to sugar acceptors. The enzyme exhibits 42% sequence identity with cyclodextrin glycosyltransferases (CGTase), and both enzymes are members of the α-amylase family, a large clan of enzymes acting on starch and related compounds. ATase is virtually inactive on starch, however. In contrast, ATase is the only known enzyme to efficiently use acarbose as substrate (2 µmol min-1 mg-1); acarbose is a strong inhibitor of CGTase and of most other α-amylase family enzymes. This distinct reaction specificity makes ATase an interesting enzyme to investigate the variation in reaction specificity of α-amylase family enzymes. Here we show that a G140H mutation in ATase, introducing the typical His of the conserved sequence region I of the α-amylase family, changed ATase into an enzyme with 4-α-glucanotransferase activity (3.4 µmol min-1 mg-1). Moreover, this mutation introduced cyclodextrin-forming activity into ATase, converting 2% of starch into cyclodextrins. The opposite experiment, removing this typical His side chain in CGTase (H140A), introduced acarviosyl transferase activity in CGTase (0.25 µmol min-1 mg-1).
A plasmid from the methylotrophic actinomycete amycolatopsis-methanolica capable of site-specific integration
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