Reconstruction of xylose utilization pathway and regulons in Firmicutes

Many Firmicutes bacteria, including solvent-producing clostridia such as Clostridium acetobutylicum, are able to utilize xylose, an abundant carbon source in nature. Nevertheless, homology searches failed to recognize all the genes for the complete xylose and xyloside utilization pathway in most of them. Moreover, the regulatory mechanisms of xylose catabolism in many Firmicutes except Bacillus spp. still remained unclear.

A comparative genomic approach was used to reconstruct the xylose and xyloside utilization pathway and analyze its regulatory mechanisms in 24 genomes of the Firmicutes. A novel xylose isomerase that is not homologous to previously characterized xylose isomerase, was identified in C. acetobutylicum and several other Clostridia species. The candidate genes for the xylulokinase, xylose transporters, and the transcriptional regulator of xylose metabolism (XylR), were unambiguously assigned in all of the analyzed species based on the analysis of conserved chromosomal gene clustering and regulons. The predicted functions of these genes in C. acetobutylicum were experimentally confirmed through a combination of genetic and biochemical techniques. XylR regulons were reconstructed by identification and comparative analysis of XylR-binding sites upstream of xylose and xyloside utilization genes. A novel XylR-binding DNA motif, which is exceptionally distinct from the DNA motif known for Bacillus XylR, was identified in three Clostridiales species and experimentally validated in C. acetobutylicum by an electrophoretic mobility shift assay.

This study provided comprehensive insights to the xylose catabolism and its regulation in diverse Firmicutes bacteria especially Clostridia species, and paved ways for improving xylose utilization capability in C. acetobutylicum by genetic engineering in the future.