Abstract
L-rhamnose (L-Rha) is a deoxy-hexose sugar commonly found in nature. L-Rha catabolic pathways were previously characterized in various bacteria including Escherichia coli. Nevertheless, homology searches failed to recognize all the genes for the complete L-Rha utilization pathways in diverse microbial species involved in biomass decomposition. Moreover, the regulatory mechanisms of L-Rha catabolism have remained unclear in most species. A comparative genomics approach was used to reconstruct the L-Rha catabolic pathways and transcriptional regulons in the phyla Actinobacteria, Bacteroidetes, Chloroflexi, Firmicutes, Proteobacteria, and Thermotogae. The reconstructed pathways include multiple novel enzymes and transporters involved in the utilization of L-Rha and L-Rha-containing polymers. Large-scale regulon inference using bioinformatics revealed remarkable variations in transcriptional regulators for L-Rha utilization genes among bacteria. A novel bifunctional enzyme, L-rhamnulose-phosphate aldolase (RhaE) fused to L-lactaldehyde dehydrogenase (RhaW), which is not homologous to previously characterized L-Rha catabolic enzymes, was identified in diverse bacteria including Chloroflexi, Bacilli, and Alphaproteobacteria. By using in vitro biochemical assays we validated both enzymatic activities of the purified recombinant RhaEW proteins from Chloroflexus aurantiacus and Bacillus subtilis. Another novel enzyme of the L-Rha catabolism, L-lactaldehyde reductase (RhaZ), was identified in Gammaproteobacteria and experimentally validated by in vitro enzymatic assays using the recombinant protein from Salmonella typhimurium. C. aurantiacus induced transcription of the predicted L-Rha utilization genes when L-Rha was present in the growth medium and consumed L-Rha from the medium. This study provided comprehensive insights to L-Rha catabolism and its regulation in diverse Bacteria.
Highlights
L-rhamnose (L-Rha) is a deoxy-hexose sugar commonly found in plants as a part of complex pectin polysaccharides and in many bacteria as a common component of the cell wall (Buttke and Ingram, 1975; Giraud and Naismith, 2000)
For reconstruction of RhaR regulons we used an established comparative genomics approach based on identification of candidate regulator-binding sites in closely related bacterial genomes implemented in the RegPredict Web server tool (Novichkov et al, 2010)
To find the conserved DNA-binding motifs for each group of orthologous RhaR regulators, we used initial training sets of genes that are co-localized with regulator with the Caur_2209 (rhaR) orthologs, and we updated each set by the most likely RhaRregulated genes confirmed by the comparative genomics tests as well as functional considerations
Summary
L-rhamnose (L-Rha) is a deoxy-hexose sugar commonly found in plants as a part of complex pectin polysaccharides and in many bacteria as a common component of the cell wall (Buttke and Ingram, 1975; Giraud and Naismith, 2000). The resulting L-Rha and unsaturated rhamnogalacturonides can enter the cells by specific transport systems, the L-rhamnose permease RhaT in Enterobacteriaceae (Muiry et al, 1993), and the RhiT transporter in Erwinia chrysanthemi (Hugouvieux-Cotte-Pattat, 2004). In the latter species, the unsaturated galacturonyl hydrolase RhiN is used to release L-Rha and unsaturated galacturonate residues to promote their further catabolism in the cytoplasm (Hugouvieux-Cotte-Pattat, 2004; Rodionov et al, 2004).
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