N-2 fixation in marine heterotrophic bacteria: Dynamics of environmental and molecular regulation

Molecular and immunological techniques were used to examine N-2 fixation in a ubiquitous heterotrophic marine bacterium, the facultative anaerobic Vibrio natriegens. When batch cultures were shifted from aerobic N-replete to anaerobic N-deplete conditions, transcriptional and posttranslational regulation of N-2 fixation was observed, Levels of nifHDK mRNA encoding the nitrogenase enzyme mere highest at 140 min postshift and undetectable between 6 and 9 h later, Immunologically determined levels of nitrogenase enzyme (Fe protein) were highest between 6 and 15 h postshift, and nitrogenase activity peaked between 6 and 9 h postshift, declining by a factor of 2 after 12-15 h, Unlike their regulation in cyanobacteria, Fe protein and nitrogenase activity were present when nifHDK mRNA was absent in V. natriegens, indicating that nitrogenase is stored and stable under anaerobic conditions, Both nifHDK mRNA and Fe protein disappeared within 40 min after cultures were shifted from N2-fixing conditions (anaerobic, N-deplete) to non-N-2-fixing conditions (aerobic, N-enriched) but reappeared when shifted to conditions favoring N-2 fixation, Thus, unlike other N-2-fixing heterotrophic bacteria, nitrogenase must be resynthesized after aerobic exposure in V. natriegens. Immunological detection based on immunoblot (Western) analysis and immunogold labeling correlated positively with nitrogenase activity; no localization of nitrogenase was observed, Because V. natriegens continues to fix N-2 for many hours after anaerobic induction, this species may play an important role in providing ''new'' nitrogen in marine ecosystems.