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 methylamine and formaldehyde metabolism in Arthrobacter P1. Formaldehyde is the inducing signal for the synthesis of the RuMP cycle enzyme hexulose phosphate synthase

The inducing potential of formaldehyde on the synthesis of hexulose phosphate synthase, a key enzyme of the RuMP cycle in Arthrobacter P1, was investigated in resting cell suspensions. Induction of this enzyme only occurred at formaldehyde concentrations of 0.5 mM and below. No evidence was obtained for the involvement of an inactivation-activation mechanism that controlled the activity of existing hexulose phosphate synthase molecules. Addition of formaldehyde at a rate of 1 mmol · l-1 · h-1 to cells of Arthrobacter P1 growing in batch culture on the "heterotrophic" substrates glucose or acetate resulted in the immediate and very rapid synthesis of hexulose phosphate synthase. These results, obtained under carbon excess conditions, clearly show that induction by formaldehyde is the overriding control mechanism in the regulation of the synthesis of this enzyme in Arthrobacter P1. The regulation of the synthesis of this key enzyme of the RuMP cycle of formaldehyde fixation in Arthrobacter P1 (induction by formaldehyde) is therefore completely different from that generally observed for the enzymes of the RuBP cycle of CO2 fixation in facultatively autotrophic bacteria (repression/derepression mechanism). Addition of methylamine at a rate of 1 mmol · l-1 · h-1 to batch cultures growing on "heterotrophic" substrates resulted in accumulation of the C1 substrate, but not in repression of the synthesis of amine oxidase. The slow start of methylamine utilization in cells growing on glucose suggests that methylamine either is a relatively weak inducer or, because in the absence of the methylamine transport system in non-induced cells, a sufficiently high intracellular level of methylamine is only slowly built up

Regulation of methylamine and formaldehyde metabolism in Arthrobacter P1. Effect of pulse-wise addition of "heterotrophic" substrates to C1 substrate-limited continuous cultures

The regulation of methylamine and formaldehyde metabolism in Arthrobacter P1 was investigated in carbon-limited continuous cultures. To avoid toxic effects of higher formaldehyde concentrations, formaldehyde-limited cultures were established in smooth substrate transitions from choline-limitation. Evidence was obtained that the synthesis of enzymes involved in the conversion of methylamine into formaldehyde and in formaldehyde fixation is induced sequentially in this organism. Compared to growth with methylamine the molar growth yield on formaldehyde was approximately 30% higher. This difference is mainly due to the expenditure of energy for the uptake of methylamine from the medium. The addition of a pulse of a "heterotrophic" substrate, glucose or acetate, to C1 substrate-limited continuous cultures resulted in relief of carbon limitation and transient synthesis of increasing amounts of cell material. Concomitantly, a significant decrease in the specific activities of hexulose phosphate synthase was observed. However, the total activity of hexulose phosphate synthase in these cultures remained clearly in excess of that required to fix the formaldehyde that became available in time. The observed strong decrease in the specific activities of this RuMP cycle enzyme strongly suggests that its synthesis is controlled via catabolite repression exerted by the metabolism of "heterotrophic" substrates

Regulation of methanol oxidation and carbon dioxide fixation in Xanthobacter strain 25a grown in continuous culture

The regulation of C1-metabolism in Xanthobacter strain 25a was studied during growth of the organism on acetate, formate and methanol in chemostat cultures. No activity of methanol dehydrogenase (MDH), formate dehydrogenase (FDS) or ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisC/O) could be detected in cells grown on acetate alone over a range of dilution rates tested. Addition of methanol or formate to the feed resulted in the immediate induction of MDH and FDH and complete utilization (D = 0.10 h-1) of acetate and the C1-substrates. The activities of these enzymes rapidly dropped at the higher growth rates, which suggests that their synthesis is further controlled via repression by "heterotrophic" substrates such as acetate. Synthesis of RuBisC/O already occurred at low methanol concentrations in the feed, resulting in additive growth yields on acetate/methanol mixtures. The energy generated in the oxidation of formate initially allowed an increased assimilation of acetate (and a decreased dissimilation), resulting in enhanced growth yields on the mixture. RuBisC/O activity could only be detected at the higher formate/acetate ratios in the feed. The data suggest that synthesis of RuBisC/O and CO2 fixation via the Calvin cycle in Xanthobacter strain 25a is controlled via a (de)repression mechanism, as is the case in other facultatively autotrophic bacteria. Autotrophic CO2 fixation only occurs under conditions with a diminished supply of "heterotrophic" carbon sources and a sufficiently high availability of suitable energy sources. The latter point is further supported by the clearly more pronounced derepressing effect exerted by methanol compared to formate.

Regulation of methylamine and formaldehyde metabolism in Arthrobacter P1. Formaldehyde is the inducing signal for the synthesis of the RuMP cycle enzyme hexulose phosphate synthase

The inducing potential of formaldehyde on the synthesis of hexulose phosphate synthase, a key enzyme of the RuMP cycle in Arthrobacter P1, was investigated in resting cell suspensions. Induction of this enzyme only occurred at formaldehyde concentrations of 0.5 mM and below. No evidence was obtained for the involvement of an inactivation-activation mechanism that controlled the activity of existing hexulose phosphate synthase molecules. Addition of formaldehyde at a rate of 1 mmol · l-1 · h-1 to cells of Arthrobacter P1 growing in batch culture on the "heterotrophic" substrates glucose or acetate resulted in the immediate and very rapid synthesis of hexulose phosphate synthase. These results, obtained under carbon excess conditions, clearly show that induction by formaldehyde is the overriding control mechanism in the regulation of the synthesis of this enzyme in Arthrobacter P1. The regulation of the synthesis of this key enzyme of the RuMP cycle of formaldehyde fixation in Arthrobacter P1 (induction by formaldehyde) is therefore completely different from that generally observed for the enzymes of the RuBP cycle of CO2 fixation in facultatively autotrophic bacteria (repression/derepression mechanism). Addition of methylamine at a rate of 1 mmol · l-1 · h-1 to batch cultures growing on "heterotrophic" substrates resulted in accumulation of the C1 substrate, but not in repression of the synthesis of amine oxidase. The slow start of methylamine utilization in cells growing on glucose suggests that methylamine either is a relatively weak inducer or, because in the absence of the methylamine transport system in non-induced cells, a sufficiently high intracellular level of methylamine is only slowly built up.

Emendation of Xanthobacter flavus as a Motile Species

Xanthobacter flavus 301T (T = type strain) and other strains, including H4-14, both of which were previously described as nonmotile, were reproducibly motile and peritrichously flagellated during the log phase when they were cultured in medium lacking tricarboxylic acid cycle intermediates. Therefore, the species description is emended to include motility and flagellation. Similarly, Xanthobacter autotrophicus was found to be flagellated and motile, but this finding was not consistently reproducible and was mainly found with the mutant strain Fe-s.

Characterization of Xanthobacter strains H4-14 and 25a and enzyme profiles after growth under autotrophic and heterotrophic conditions

All Xanthobacter strains studied are versatile autotrophic bacteria, able to grow on methanol and other substrates. Strain 25a, a yellow-pigmented, pleomorphic, Gram-negative bacterium, capable of autotrophic growth on methanol, formate, thiosulfate, and molecular hydrogen, was isolated from an enrichment culture inoculated with soil from a subtropical greenhouse. Subsequent studies showed that the organism also grows on a wide range of multicarbon substrates. Ammonia, nitrate and molecular nitrogen were used as nitrogen sources. The taxonomic relationship of strains H4-14 and 25a with previously described Xanthobacter strains was studied by numerical classification. Strain H4-14 was identified as a X. flavus strain, but the precise position of strain 25a remained uncertain. It probably belongs to a new species of the genus Xanthobacter. The levels of various enzymes involved in autotrophic and heterotrophic metabolism were determined following growth of strains H4-14 and 25a in batch and continuous cultures. The mechanisms involved in controlling ribulose-1,5-bisphosphate carboxylase/oxygenase synthesis in Xanthobacter strains appear to be comparable to those observed for other autotrophic bacteria, namely repression by organic compounds and derepression by autotrophic energy sources, such as methanol and hydrogen.

Clinical validation study of dried blood spot for determining everolimus concentration in patients with cancer

Contains fulltext : 189818.pdf (publisher's version ) (Open Access

Use of incretin agents and risk of pancreatic cancer: A population-based cohort study

Marine protected areas (MPA) are rapidly being established to minimize the impact of anthropogenic disturbances, yet, while climate change is acknowledged as a growing threat, very limited research exists about how to directly incorporate climate-related disturbances into MPA design. Using the conservation planning software Marxan and the Indo-west Pacific as a study region, an illustrative approach is developed here that incorporates climate change projections into the process of identifying priority areas for marine conservation. Conservation targets were set at 10% and 30% of areas that continually held sea-surface temperatures less than 1 °C above maximum non-extreme historic temperatures (derived from satellite imagery from 1984-2009). This approach allowed for continuity in conservation objectives across both space and time by identifying the geographic extent of thermal stress in the region and illustrating how conditions would change in future years. Achievement of targets was found to be flexible, but some areas were more important than others for achieving these targets. Interannual trend analyses were carried out for three climate models under two climate change scenarios to examine spatial and temporal patterns of thermal stress. Spatial patterns of thermal stress varied throughout the region. Results of the conservation approach were compared to the trend results to see whether the trends might be a simpler approach for accounting for climate change impacts in conservation planning (i.e., one feature could be used instead of more than 1000). The interannual analyses had a low overlap with the Marxan results, and hence are not a suitable substitute for the approach shown here. This study showed that inclusion of climate-related disturbances in marine conservation planning is feasible and should become common practice, together with targets for biodiversity. © 2012 Elsevier Ltd