ARRANGEMENT AND REGULATION OF NITROGEN-FIXATION GENES IN KLEBSIELLA-PNEUMONIAE STUDIED BY DEREPRESSION KINETICS

Events underlying derepression of the nitrogen fixation (nif) genes in K. pneumoniae M5A1 were analyzed in vivo by comparing the effects of selective inhibitors of transcription and translation on subsequent nitrogenase activity (rate of acetylene reduction). When batch cultures were induced for derepression, an 87 min lag separated ammonium ion/O2 removal and the appearance of activity. To prevent eventual activity by adding inhibitors during this period it was found necessary to add rifampin, ammonium ion or chloramphenicol more than 50, 20 or 10 min, respectively, before activity appeared in a parallel control. When these inhibitors were added to cultures in which nitrogenase activity already appeared, further increase in activity was not stopped by rifampin or ammonium ion until 45 or 20 min, respectively, after addition. Chloramphenicol stopped further increase in nitrogenase activity almost immediately. A nif operon whose transcription/translocation consumes 40 min was indicated. When the kinetics of .beta.-galactosidase induction were analyzed under similar conditions, 7 min separated the initiation of transcription and the 1st completions of translocation. Extrapolating from this, the nif operon was found to occupy 17 average gene lengths. The nif structural genes in K. pneumoniae may be contained on 1 operon and the disparity between the kinetics of inhibition by rifampin and those by ammonium ion suggests regulation at a locus other than the operator.