Abstract
We have experimentally and computationally defined a set of genes that form a conserved metabolic module in the α-proteobacterium Caulobacter crescentus and used this module to illustrate a schema for the propagation of pathway-level annotation across bacterial genera. Applying comprehensive forward and reverse genetic methods and genome-wide transcriptional analysis, we (1) confirmed the presence of genes involved in catabolism of the abundant environmental sugar myo-inositol, (2) defined an operon encoding an ABC-family myo-inositol transmembrane transporter, and (3) identified a novel myo-inositol regulator protein and cis-acting regulatory motif that control expression of genes in this metabolic module. Despite being encoded from non-contiguous loci on the C. crescentus chromosome, these myo-inositol catabolic enzymes and transporter proteins form a tightly linked functional group in a computationally inferred network of protein associations. Primary sequence comparison was not sufficient to confidently extend annotation of all components of this novel metabolic module to related bacterial genera. Consequently, we implemented the Graemlin multiple-network alignment algorithm to generate cross-species predictions of genes involved in myo-inositol transport and catabolism in other α-proteobacteria. Although the chromosomal organization of genes in this functional module varied between species, the upstream regions of genes in this aligned network were enriched for the same palindromic cis-regulatory motif identified experimentally in C. crescentus. Transposon disruption of the operon encoding the computationally predicted ABC myo-inositol transporter of Sinorhizobium meliloti abolished growth on myo-inositol as the sole carbon source, confirming our cross-genera functional prediction. Thus, we have defined regulatory, transport, and catabolic genes and a cis-acting regulatory sequence that form a conserved module required for myo-inositol metabolism in select α-proteobacteria. Moreover, this study describes a forward validation of gene-network alignment, and illustrates a strategy for reliably transferring pathway-level annotation across bacterial species.
Highlights
Inositol, or cyclohexanehexol, is one of the most abundant carbohydrates in freshwater and terrestrial ecosystems [1]
We found 21 more examples of this same motif, which was enriched in the predicted upstream homologs of iolC, inositol 2-dehydrogenase (idhA), and the myo-inositol ATP-binding cassette (ABC) transporter operons in these species (Figure 6B and 7)
The iolA::Tn5 and Smb20712::Tn5 mutant strains did not grow on GTS with myoinositol as a sole carbon source (Table 4). These results show that S. meliloti IolA is required for growth on myo-inositol and confirm our cross-species computational prediction that the protein Smb20712 is the functional homolog of the C. crescentus periplasmic inositol-binding protein IbpA
Summary
Cyclohexanehexol, is one of the most abundant carbohydrates in freshwater and terrestrial ecosystems [1]. Several species of cyanobacteria, eubacteria and archaea are able to synthesize and derivitize inositol [2] These molecules serve functional roles as antioxidants, osmolytes, cell membrane components, and as carbon storage substrates [3,4]. The myo-inositol degradation pathway has been characterized biochemically in Klebsiella aerogenes [5,11,12,13] and Bacillus subtilis [14]. In this pathway, seven proteins convert myoinositol to CO2, acetyl CoA and dihydroxy-acetone phosphate (Figure 1). Structural and regulatory genes required for myoinositol catabolism have been identified and characterized in several gram-positive species, including B. subtilis [7,14], Clostridium perfringens [8], Corynebacterium glutamicum [9], and Lactobacillus casei [15], and in the gram-negative bacteria Rhizobium leguminosarum bv
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.