METABOLIC PATHWAYS IN PARACOCCUS-DENITRIFICANS AND CLOSELY RELATED BACTERIA IN RELATION TO THE PHYLOGENY OF PROKARYOTES

Denitrification and methylotrophy in Paracoccus denitrificans are discussed. The properties of the enzymes of denitrification: the nitrate-nitrite antiporter, nitrate reductase, nitrite reductase, nitric oxide reductase and nitrous oxide reductase are described. The genes for none of these proteins have yet been cloned and sequenced from P. denitrificans. A number of sequences are available for enzymes from Escherichia coli, Pseudomonas stutzeri and Pseudomonas aeruginosa. It is concluded that pathway specific c-type cytochromes are involved in denitrification. At least 40 genes are involved in denitrification. In methanol oxidation at least 20 genes are involved. In this case too pathway specific c-type cytochromes are involved. The sequence homology between the quinoproteins methanol dehydrogenase, alcoholdehydrogenase and glucose dehydrogenase is discussed. This superfamily of proteins is believed to be derived from a common ancestor. The moxFJGI operon determines the structural components of methanol dehydrogenase and the associated c-type cytochrome. Upstream of this operon 3 regulatory proteins were found. The moxY protein shows the general features of a sensor protein and the moxX protein those of a regulatory protein. Thus a two component regulatory system is involved in both denitrification and methylotrophy. The phylogeny of prokaryotes based on 16S rRNA sequence is discussed. It is remarkable that the 16S rRNA of Thiosphaera pantotropha is identical to that of P. denitrificans. Still these bacteria show a number of differences. T. pantotropha is able to denitrify under aerobic circumstances and it shows heterotrophic nitrification. Nitrification and heterotrophic nitrification are found in species belonging to the beta-and gamma-subdivisions of purple non-sulfur bacteria. Thus the occurrence of heterotrophic nitrification in T. pantotropha, which belongs to the alpha-subdivision of purple non-sulfur bacteria is a remarkable property. Furthermore T. pantotropha contains two nitrate reductases of which the periplasmic one is supposed to be involved in aerobic denitrification. The nitrite reductase is of the Cu-type and not of the cytochrome cd1 type as in P. denitrificans. Also the cytochrome b of the Qbc complex of T. pantotropha is highly similar to its counterpart in P. denitrificans. It is hypothesized that the differences between these two organisms which both contain large megaplasmids is due to a combination of loss of genetic information and plasmid-coded properties. The distribution of a number of complex metabolic systems in eubacteria and in a number of species belonging to the alpha-group of purple non sulphur bacteria is reviewed. Two possibilities to explain this haphazard distribution are considered: 1. Lateral gene transfer between distantly related micro organisms occurs frequently. 2. The eubacterial ancestors must have possessed already these properties. The distribution of these properties is due to sporadic loss during evolutionary divergence. With respect to the occurrence and frequency of lateral gene transfer two opposing views exist. According to molecular biologists lateral gene transfer occurs frequently and is very easy. Bacteria are supposed to form one large gene pool. On the other hand population geneticists have provided evidence that strong systems operate that establish reproductive isolation between diverged species and even between closely related cell lines. Data on amino acid sequences of nitrogenase proteins, cytochromes c, cytochrome oxidases, beta-subunits of ATP synthase and tryptophan biosynthetic enzymes of various micro organisms were reviewed. In all these cases phylogenetic trees could be constructed based on the amino acid sequence data. In all cases this phylogenetic tree was similar to the one based on 16S rRNA homology. Only in one case evidence for the occurrence of lateral gene transfer was obtained. Therefore it is concluded that lateral gene transfer played a minor role in the distribution of complex metabolic systems among prokaryotes. It must be stressed that this does not exclude the possibility that lateral gene transfer occurred frequently in the initial stage of bacterial evolution. It is hypothesized that the appearance of nitrogen fixation, denitrification and cytochrome oxidase formation were early events in the evolution of micro organisms. Both systems are supposed to have evolved only once. Subsequently the capacity to fix nitrogen or to denitrify must have been lost many times, just as photosynthetic capacity is supposed to have been lost many times. During evolution many systems have been lost leading to a haphazard distribution of metabolic characters among bacteria. As an example it is suggested that organisms with a respiratory chain similar to that of Escherichia coli arose by loss of the capacity to form the Qbc complex and c-type cytochromes. The remaining systems could be controlled much better however than in the ancestral organisms.