LOCALIZATION AND CHARACTERIZATION OF THE CARBON-TETRACHLORIDE TRANSFORMATION ACTIVITY OF PSEUDOMONAS SP STRAIN KC

Previous research has established that Pseudomonas sp. strain KC rapid ly transforms carbon tetrachloride (CT) to carbon dioxide (45 to 55%), a nonvolatile fraction (45 to 55%), and a cell-associated fraction (s imilar to 5%) under denitrifying, iron-limited conditions. The present study provides additional characterization of the nonvolatile fractio n, demonstrates that electron transfer plays a role in the transformat ion, and establishes the importance of both extracellular and intracel lular factors. Experiments with C-14-labeled CT indicate that more tha n one nonvolatile product is produced during CT transformation by stra in KC. One of these products, accounting for about 20% of the [C-14] C T transformed, was identified as formate on the basis of its elution t ime from an ion-exchange column, its boiling point, and its conversion to (CO2)-C-14 when incubated with formate dehydrogenase. Production o f formate requires transfer of two electrons to the CT molecule. The r ole of electron transfer was also supported by experiments demonstrati ng that stationary phase cells that do not transform CT can be stimula ted to transform CT when supplemented with acetate (electron donor), n itrate (electron acceptor), or a protonophore (carbonyl cyanide m-chlo rophenylhydrazone). The location of transformation activity was also e valuated. By themselves, washed cells did not transform CT to a signif icant degree. Occasionally, CT transformation was observed by cell-fre e culture supernatant, but this activity was not reliable. Rapid and r eliable CT transformation was only obtained when washed whole cells we re reconstituted with culture supernatant, indicating that both extrac ellular and intracellular factors are normally required for CT transfo rmation. Fractionation of culture supernatant by ultrafiltration estab lished that the extracellular factor or factors are small, with an app arent molecular mass of less than 500 Da. The extracellular factor or factors were stable after lyophilization to powder and were extractabl e with acetone. Addition of micromolar levels of iron inhibited CT tra nsformation in whole cultures, but the level of iron needed to inhibit CT transformation was over 100-fold higher for washed cells reconstit uted with a 10,000-Da supernatant filtrate. Thus, the inhibitory effec ts of iron are exacerbated by a supernatant factor or factors with a m olecular mass greater than 10,000 Da.