Research at the Institute of Molecular Microbiology

Denitrification - Biochemistry and regulation of nitrate and nitrite respiration; microbial emission and reduction of nitric oxide (NO) and nitrous oxide (N₂O)

Denitrification is a distinct means of bacterial energy conservation, making use of N oxides as facultative terminal electron acceptors for cellular bioenergetics under anaerobic, micro-aerophilic, and occasionally also aerobic conditions. The process reverses dinitrogen fixation and is part of the global N cycle, essential for all life forms. Discovered more than a century ago and believed to be exclusively a bacterial trait, denitrification has been found recently also in halophilic and hyperthermophilic archaea, and in the mitochondria of fungi.

We contributed to significant advances in the physiology and biochemical characterization of denitrification and pioneered the underlying molecular biology by investigating the bacterium Pseudomonas stutzeri. Around 40 genes were found to be required to encode the core structures of the denitrification apparatus. This uncovered evolutionary important relationships among denitrification enzymes and oxidases of the heme-copper family.

The activation and enzymatic transformation of N oxides is based on the metals Fe, Cu, and Mo and their redox chemistry. The metal ions are found in the denitrification enzymes in distinct organic cofactors or as protein-bound metal clusters. We have advanced the biology of the denitrification process by isolating for the first time the enzymes nitric oxide reductase and nitrous oxide reductase, characterizing their biochemistry and unraveling the genetic basis of the process. Most recently, we provided the biological material for obtaining the three-dimensional enzyme structure of the physiological state, leading to the recognition of the first known biologically active [4Cu:2S] copper-sulfur cluster.

The proteins required for the process in the gram-negative P. stutzeri are arranged in and at either side of the cytoplasmic membrane. Thus, denitrification is intimately related to cellular processes that include primary and secondary transport, protein translocation, cytochrome c biogenesis, anaerobic and N oxide-dependent gene regulation, metalloprotein assembly, and the biosynthesis of the cofactors molybdopterin and heme d₁. Nitrate and nitric oxide, in addition to being respiratory substrates, have been identified as key signal molecules for the induction of distinct N oxide-metabolizing enzymes. An important class of regulators for the environmentally induced expression of the denitrification apparatus are transcription factors of the Crp/Fnr superfamily.

A glimpse of history

Tradition in microbiology at the Karlsruhe Institute of Technology (the former University of Karlsruhe) has its roots in Walter MIGULA (1863-1938), a pioneer in bacterial systematics, who worked until 1905 at what was then the Technical Institute of the Grand Duchy of Baden. His monograph represented the authoritative system of bacteria until it was superceded by BERGEY's manual. Today, MIGULA remains known to microbiologists by his description in 1895 of the ecologically, clinically and biotechnologically important genus Pseudomonas.