Arch. Microbiol. 114, 51-54 (1977) Archives of Micrnbiology 9 by Springer-Verlag 1977 The Effect of Inorganic Phosphate on Cyanogenesis by Pseudomonas aeruginosa R. MEGANATHAN and P. A. CASTRIC Department of Biological Sciences, Duquesne University, Pittsburgh, PA 15219, U.S.A. Abstract. The biosynthesis of hydrogen cyanide (HCN) by a strain of Pseudomonas aeruginosa is found to be significantly influenced by inorganic phosphate. Opti- mum HCN production occurs when the phosphate concentration is between 1 and 10 raM. Above and below this concentration the amount of HCN pro- duced decreases sharply and at 0.1 and 100raM phosphate low HCN production occurs. If a culture growing at 0.1 mM phosphate and producing low HCN is shifted to 10 mM phosphate, HCN biosyn- thesis resumes. Experiments with chloramphenicol indicate that de novo-protein synthesis is required for the process. Key words." Hydrogen cyanide biosynthesis - Pseudo- monas aeruginosa - Phosphate effect on HCN - Secondary metabolism. Hydrogen cyanide (HCN) biosynthesis has been demonstrated in many microorganisms. It is wide- spread among fungi (Hutchinson, 1973) but is confined to a few species in the genera Chromobacterium and Pseudomonas among bacteria (Patty, 1921; Lorck, 1948; Michaels and Corpe, 1965; Wissing, 1968; Brysk et al., 1969). Several investigators have con- sidered this process to be due to cell autolysis (Michaels and Corpe, 1965; Wissing, 1968). Recently it was demonstrated that the process of HCN bio- synthesis has all the characteristics of secondary me- tabolism (Castric, 1975). These characteristics (Wein- berg, 1971) are: (1) synthesis during or subsequent to late log growth, (2) restricted taxonomic distribution among bacteria, and, (3) requirements for production, such as metal ion, temperature, oxygen and pH which are narrower than those required for cell growth. Another characteristic of secondary metabolism, inhibition by phosphate, is also shared by the bio- synthesis of HCN in P. aeruginosa (Castric, 1975). The role of this essential compound in both the stimulation and the inhibition of cyanogenesis in this organism is examined in this paper. A preliminary report of these findings has appeared (Meganathan and Castric, 1976). MATERIALS AND METHODS Organism. The bacterium used throughout the study was Pseudo- monas aeruginosa, strain 9-D2, which had been isolated from a septic human burn by S. D. Kominos, Mercy Hospital, Pitts- burgh, PA 15219. Stocks were prepared by growing the organ- ism to approximately 1 x 109 viable cells/ml in 20 mM L-glutamate plus salts (see below). 1.0 ml aliquots were placed into sterile tubes containing 0.05 ml dimethyl sulfoxide. The tubes were then stored at - 82~C. Growth Medium. The complete synthetic medium consisted of: 2 mM MgSO4; 5 mM K2HPO~; 5 mM NaH2PO4; 0.02 mM ferric citrate; 50 mM tris-(hydroxymethyl)-aminomethane; 20 mM L-glu- tamate; 12.5 mM glycine; 5.0 mM D,L-methionine. This medium minus glutamate, glyeine, and methionine is referred to as the salts solution. The medium was adjusted to a pH of 7.5 at 24~C with concentrated HC1 and sterilized by autoclaving. Preparation of Cells. Frozen cell stock, 1 ml, was inoculated into 100 mI of medium in a 250 ml Erlenmeyer flask and grown to late log phase at 35~ with shaking at a speed of 200 RPM. The cells were centrifuged at 12000 x g for 15 rain and resuspended in 10 ml of salts. A portion of these cells, giving a final concentration of i x 109 viable cells/ml, was inoculated into 5 ml of medium in the incubation vessel. Apparatus for Cell Growthand Cyanide Collection. The basic appara- tus used for cell growth and cyanide trapping consisted of a 2 x 9 cm flat-bottomed test tube, containing a small magnetic stirring bar, and two 0.2 cm inner diameter glass tubes fitted into a no. 9 rubber stopper. This portion of the incubation vessel was then inserted into a specimen jar which acts as a water jacket. The mouth of the culture vessel contained a no. 2 stopper fitted with two 0.2 cm inner diameter glass tubes to direct air flow. The entire apparatus was placed on a magnetic stirring motor allowing continuous and constant stirring of the culture. Water at 35~C circulated through the specimen jar maintained a constant temperature. Culture aeration and HCN collection were carried out by passing filter sterilized air at 25 ml per min, as measured by a flowmeter,