Pyocyanine media

Investigations by Burton et al. (1947, 1948) were concerned with finding a medium optimal for pyocyanine production by P. aeruginosa ATCC 9027 grown in stationary culture. Other workers, in investigating the biosynthesis of pyocyanine from C-labeled compounds, have subsequently used media similar (at least quahtatively) to that recommended by Burton et aL (1947, 1948) and modified by Hellinger (1951)- The composition of such media, and the yields of pyocyanine obtained, are outlined in Table 1. 

Supplementation of medium U with methionine or other possible methyl donors increased pyocyanine production by some strains (Bj, B, B, BJ (Kurachi, 1959a and d). Kurachi (1959a) postulated that methionine acts as a methyl donor in the bios3Uithesis of pyocyanine. Further evidence for this postulate has been obtained (Sheikh and MacDonald, I964) and will be discussed later. 

Frank and deMoss (1959) incubated a mixture of washed cells of P. aeruginosa, salts, and alanine- C for 24 hours, and then isolated and purified the pyocyanine produced. With uniformly-labeled L-alanine C as the substrate, the ratio of the specific activity of the pyocyanine synthesized to the specific activity of the alanine added was close to the value expected if the carbon atoms of the L-alanine were the sole source of carbon for the synthesis of pyocyanine. In another experiment, C-labeled cells were produced by growing P. aeruginosa on a medium containing uniformly-labeled L-alanine- C as the sole source of carbon. Such cells, when incubated with unlabeled alanine and salts, produced pyocyanine which contained little radioactivity. One can conclude from the above results that the pyocyanine synthesized was derived from alanine and not from preformed intermediates in the cells. The authors showed also that when DL-alanine-1- C and DL-alanine-2- C were tested as precursors in the system described, the former compound contributed relatively less radioactive carbon for the biosynthesis of pyocyanine than did the latter. Interpretation of these results is difficult because the distribution of radioactivity in the pyocyanine produced was not known, and because results obtained with DL-alanine may not be directly comparable with those obtained with L-alanine. 

Some experiments were done to compare quinic and shikimic acid as carbon sources for pyocyanine biosynthesis in a medium also containing uniformly-labeled glyceroP C and L-alanine- C (MacDonald, 1963). The best incorporation (74%) of the carbon atoms of quinic acid into the carbon atoms of pyocyanine was obtained when quinic acid was added at a concentration of 1 % at the time of inoculation with a small inoculum. If quinic acid was added when pigment formation had just started, or if quinic acid was added at a concentration of 0.25 %, or if a large inoculum of washed cells was used, the incorporation of quinic acid into pyocyanine was less (21— 46 %). The best incorporation (29 or 31 %) of the... 

When serine-3- C (0.5%) was added to the medium, the specific activity of the N-methyl group of pyocyanine was 20% of that of the serine added and 75 % of that of the whole pyocyanine molecule. Sheikh and MacDonald (1964) concluded that the methyl group of methionine was a more immediate precursor of the N-methyl group of pyocyanine than was carbon-3 of serine. They also implied that in the biosynthesis of pyocyanine, an intact methyl group transfer from methionine was more likely than oxidation through a one-carbon pool, but they did not give any further experimental evidence to prove this point. 

FIGURE 3. Light-induced atebrin binding and its uncoupling effect at different temperatures in broken chloroplasts. The medium contained 50 mM NaCl, 50 mM KCl, 3 mM MgCl2, 1 mM ADP, 3 mM KH2PO4, 2 mM Tricine buffer, pH 8.0, 20 ]1M pyocyanine and 30 yg Chl/ml. 

Envelope-free chloroplasts were prepared and essayed as previously (de Kouchkovsky et al., 1982) for the ApH (9-aminoacridine method) and ATP synthesis (ferricyanide photoreduction). AH was related to the electro-chromic absorbance variation at 520 nm measured on the same sample than the other phenomena, just shifting the analytic wavelength and using appropriate filters. The assay medium was sorbitol 0.2 M + Tricine 0.01 M + Hepes 0.01 M + KCl 0.01 M + MgCl2 6 mM + K2HPO4 2 mM + ADP 0.5 mM + 9-aminoacridine 4 yM. DimethyIquinone (0.5 mM) or pyocyanin (50 yM) were added when indicated. Chlorophyll concentration was 15 yM. Aerobic conditions. 

Once washed, type C chloroplasts (Hall, 1972) were prepared by conventional procedures from young pea leaves (Pisum sativum var Feltham first). Chloro-plast ATPase was measured in a thermostatted glass vessel fitted with a pH-sensitive electrode. The medium contained 50 mM KCl, 2 mM tricine-NaOH, pH 8.0, 5 mM MgCl2, 0.1 mM EDTA, 5 M pyocyanine and 1 mM KP. and 10-20 g chlorc phyll/ml. For DTT activations the medium also contained 4 mM DTT and was illuminated for 4 minutes, while for trypsin activation, illumination was with 1 Kig/ml trypsin for 30 seconds, followed by the addition of an excess of trypsin inhibitor. Light was provided by a 150 W projector lamp. After the indicated periods of darkness (see each figure), MgATP was add d to a final concentration of 2 mM and the resulting ATPase activity monitored by fall in pH. Basal activity was that without prior illumination. 

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