Evolutionary and functional relationships revealed by the dha regulon predicted by genomic context analysis

Background

The large economic interest in producing 3-hydroxypropion-aldehyde (HPA) and 1,3-propanediol is stimulating research on the dha regulon. This regulon encodes the main proteins responsible for the production of both compounds, and it has been found to be functional in only a small number of bacterial genomes, such those of the Gammaproteobacteria (Anaerovibrio, Citrobacter, Enterobacter, Ilyobacter, Lactobacillus and Klebsiella), Firmicutes (Clostridium) and Deltaproteobacteria (Pelobacter). To expand our knowledge and direct further experimental studies on the dha regulon and on related genes involved in anaerobic glycerol metabolism, an extensive genomic analysis was performed to identify the regulon in other species belonging to the Bacteria and Archaea groups.

Methods

BLAST similarity searches using the dha genes from Klebsiella pneumoniae as the seed were conducted on the National Center for Biotechnology Information database of complete prokaryotic genomes (as of May 2011). Candidate genes were thus confirmed both by sequence similarity searches (using the BLASTP program) and domain analysis. The concatenated tree was based on the alignment of the concatenated sequences of the five dha genes: these encode the three glycerol dehydratase subunits (large, medium and small, encoded by dhaB1, dhaB2 and dhaB3, respectively) and the two glycerol dehydratase-reactivation factor subunits (large and small, encoded by dhaF and dhaG, respectively). Maximum likelihood and neighbor-joining trees were generated using the MEGA program. Bootstrap support (resampled 1,000 times) was calculated, and strict consensus trees were constructed. Based on alignments of homologous sequences, conserved indels that are useful for understanding the origin of the genes of the dha regulon in the archaean Halalkalicoccus jeotgali were investigated.

Results

Comparative genomics revealed that the complete dha regulon has a restricted distribution in Bacteria, being found only in species from the Actinobacteria, Firmicutes, Fusobacteria, Gammaproteobacteria and Deltaproteobacteria. From more than 1,000 complete prokaryotic genomes analyzed, approximately 100 possess at least part of this regulon and belong to the groups mentioned above. The members of one group of Archaea (Halalkalicoccus) also carry part of this regulon. The function of this regulon has been characterized in only a few species of bacteria, but its wide distribution in these groups suggests that it may be of far greater importance than was previously recognized. Interestingly, part of this regulon, responsible for the production of HPA, is present in unique species from two groups of Bacteria and one from Archaea. In these three groups (Deltaproteobacteria, Alphaproteobacteria and Archaea), the genes dhB1, dhB2, dhB3 and dhaF and dhaG are present only in one species from each group. Moreover, these genes have similar GC content (59.3% and 60.6%), which is higher than the host genome. Interestingly, this regulon is found in a plasmid in the archaean H. jeotgali. Phylogenetic analysis reinforces the idea that these regulons have a common origin, because the genes are grouped together and so were possibly acquired by horizontal gene transfer. These genes were also analyzed for the presence of indels shared with the bacteria, but no distinctive indels were found.

Conclusions

Although in silico inferences should normally be confirmed and tested by experimentation, the present work provides a taxonomic distributional profile of the genes responsible for the anaerobic metabolism of glycerol in Bacteria and Archaea, providing new insight into the taxonomy and evolutionary history of this regulon. Curiously, some of these organisms live with only part of the regulon. In addition, this study also provides a useful framework for further investigations of the functions of these genes.