The wood preservative chromated copper arsenate is a substrate for trimethylarsine biosynthesis.

The wood preservative chromated copper arsenate (CCA) is a very widely used product. As it contains both copper and arsenic, it is not dissimilar to the pigments Scheele's green and Schweinfurter's green which were found to be biologically convertible to the toxic Gosio gas (B. Gosio, Ber. 30:1024-1026, 1897) later identified by Challenger and co-workers as trimethylarsine (F. Challenger, Adv. Enzymol. 12:429-491, 1951). Thus, it was of interest to determine whether microbiological action on CCA and wood treated with CCA could result in the production of trimethylarsine. We report that the fungus Candida humicola will produce this arsine from dilute solutions of CCA and from wood soaked in CCA.

The reduction of trimethylarsine oxide by Candida humicola.

Trimethylarsine oxide, a probable intermediate in the biological transformation of arsenate, was reduced to volatile trimethylarsine by Candida humicola. A simple assay for the rate of trimethylarsine production from trimethylarsine oxide by the fungus was developed. The optimum pH for the reduction was determined as 5.1-5.2, and the optimum temperature was 40 degrees C. The rate of reduction was directly proportional to cell concentration and followed Michaelis-Menten type kinetics. There was almost no trimethylarsine produced by heated or broken cells. The reaction was inhibited by a number of electron transport inhibitors and uncouplers of oxidative phosphorylation including cyanide, azide, and 2,4-dinitrophenol. The rate of reduction was modified by arsenate, methylarsonate, dimethylarsinate, selenate, and tellurate. Preincubation of cells with trimethylarsine oxide increased the rate of reduction 69-fold; this increase in activity was blocked if the cells were incubated with the protein synthesis inhibitor cycloheximide.

The transformation of arsenicals by Candida humicola.

An analytical procedure for the separation, detection, and identification of some of the compounds produced by a cell preparation of the fungus Candida humicola from 74As-arsenate, [14C]-methylarsonate, and [14C]-dimethylarsinate has been devised and tested. It has been possible to detect five distinct 74As-containing compounds following the incubation of 74As-arsenate with a broken cell homogenate, three of which have been identified as arsenite, methylarsonate, and dimethylarsinate. With [14C]methylarsonate as substrate, [14C]dimethylarsinate and [14C]trimethylarsine oxide are produced. Products from [14C]dimethylarsinate include [14C]methylarsonate and [14C]trimethylarsine oxide as well as an unknown species. The absence of any transformation when buffer replaces the cell preparation implicates these various compounds as intermediates in a biological synthesis of trimethylarsine.

Cadmium and zinc sensitivity and tolerance in Bacillus subtilis subsp. niger and in a Pseudomonas sp.

The action of Cd2+ and Zn2+ on Bacillus subtilis subsp. niger ATCC 9372, and on a Pseudomonas sp. (possibly Pseudomonas fluorescens), isolated from cadmium-polluted soil has been determined and compared with results obtained previously with Klebsiella aerogenes. In liquid medium the lag and the mean generation time of Bacillus subtilis subsp, niger increased with increasing Cd2+ or Zn2+ concentrations whereas only the total biomass of the Pseudomonas sp. was affected. Nevertheless, the responses of both species indicated a specific action at low concentrations and a more general toxic action at high concentrations. The survival on Cd2+ - or Zn2+ - agar depended on the state of the metal ions with regard to chelating ligands and on the nutritional stage of the organisms. In admixture, the metal ions acted synergistically, particularly on the Pseudomonas sp. Resistance to both metal ions developed. It was graded to the training concentration and reciprocal cross-resistance occurred with Bacillus subtilis. subsp. niger but not with the Pseudomonas sp.

Cadmium and zinc sensitivity and tolerance in Klebsiella (Aerobacter) aerogenes.

The resistance of strains of K. aerogenes NCIB 418 'trained' to Cd2+ or Zn2+ in liquid medium is graded to the training concentration. Training to Cd2+ increased the sensitivity to Zn2+ but training to Zn2+ reduced the sensitivity to Cd2+. The trained organisms, particularly those trained to Cd2+, grow slowly in medium containing the metals and the growth rates after 20 and 200 subcultures were not significantly different. The survival of untrained organisms on Cd2+-agar decreased progressively as the Cd2+ concentration was increased, but a threshold concentration of Zn2+ was necessary before any decrease set in. Very low concentrations of Zn2+ potentiated the lethal action of Cd2+ and vice versa, whereas chelating the metal ions with citrate, aspartate or gluconate eliminated it. Nutrient-limitation, as might occur in natural environments, also had a profound effect. Mg2+-limited organisms were particularly resistant to both metals whereas glucose-, NH+4 and PO3-4-limited organisms were very sensitive to Cd2+, and glucose- and K+-limited organisms to Zn2+.

Enzymic activities of cadmium- and zinc-tolerant strains of Klebsiella (Aerobacter) aerogenes growing in glucose-limited chemostats.

The activities of phosphatases and some enzymes of glucose metabolism were determined in K. aerogenes NCIB 418 and in two strains derived from it, resistant to 50 mug Cd2+ ml-1 and 16 mug Zn2+ ml-1 respectively, during growth at D = 0.38 h-1 in medium containing beta-glycerophosphate as sole phosphorus source and supplemented with Cd2+ and Zn2+, as appropriate, for the resistant strains. The pH-activity profiles of the phosphatases differed from strain to strain but all showed maximum activity at an acid pH and this activity was very much lower in the Zn2+-resistant strain than in the control and even lower in the Cd2+-resistant strain. Resistance to either metal was associated with decreased glucose-6-phosphate dehydrogenase activity and increased phosphoglucose isomerase activity, suggesting an increased flow of carbon through the Embden-Meyerhof pathway relative to the pentose phosphate pathway, but the efficiency of the conversion of glucose into biomass was largely unaffected. Glucose phosphoenolpyruvate phosphotransferase activity was also lower in the resistant strains.