Genetic and computational identification of a conserved bacterial metabolic module

Abstract

We have experimentally and computationally defined a set of genes that form a conserved metabolic module in the alpha-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 alpha-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 alpha-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.

Figure 1. Biochemical pathway of myo-inositol degradation in C. crescentus.

Functional assignments of the catabolic genes are based on biochemical work in B. subtilis . All of the genes for this catabolic pathway are present in the C. crescentus myo-inositol catabolic locus diagrammed in Figure 2, except the gene iolJ. Protein CC3250, annotated as a fructose bisphosphate aldolase, has a high similarity to IolJ of B. subtilis (BLAST score<e⁻⁴⁰). This gene is upregulated 1.9-fold in myo-inositol relative to glucose. We propose it is the most likely candidate to carry out the enzymatic function of IolJ in C. crescentus.

Figure 2. Genomic organization of the C. crescentus myo-inositol module and regulation of gene expression by myo-inositol and glucose.

(A) Diagram of the C. crescentus chromosome with microarray expression data overlayed. Colored lines represent genes that are significantly up- (yellow) or down-regulated (blue) during growth on myo-inositol relative to glucose as the sole carbon source. The complete set of genes that are significantly up- or down-regulated in myo-inositol versus glucose can be found in Table S1. (B) Chromosomal organization of the myo-inositol transport operon, and (C) the myo-inositol degradation operon. Right-facing arrows indicate genes are on the plus strand of the chromosome; left-facing arrows indicate genes are on the minus strand. The color of the genes corresponds to their degree of regulation in myo-inositol relative to glucose (see color scale in the center of panel A). Black triangles represent locations of Himar transposon insertions identified in a forward screen for C. crescentus strains that cannot utilize myo-inositol as the sole carbon source. Vertical black lines indicate the location of the cis-acting regulatory motif GGAA-N6-TTCC (see Figure 4).

Figure 3. Regulation of genes in the myo-inositol module by the transcriptional regulator iolR and a conserved promoter sequence.

(A) β-galactosidase assays of the idhA, ibpA and iolC promoters in the wild-type and ΔiolR backgrounds. (B) β-galactosidase assays of motif 1 (m1) and motif 2 (m2) mutated versions of the iolC promoter assayed in a wild-type background. The scale on the Y axis is the same for A and B. All β-galactosidase assays were conducted in PYE complex medium. (C) Schematic of the iolC promoter showing the location of the two conserved motifs; the wild-type and mutated versions of motif 1 are shown above the motif location map; wild-type and mutated versions of motif 2 are shown below.

Figure 4. Identification of a conserved motif in the promoters of genes regulated by myo-inositol.

(A) The upstream regions of the genes and operons that were shown to be required for growth on myo-inositol were searched for common sequence motifs using MEME . This search identified a conserved palindromic motif (MEME e value = 4.4 e⁻⁰⁴). (B) A weblogo cartoon showing the relative nucleotide frequency at each of the 15 positions in the promoter motif, where frequency is proportional to the height of the letter.

Figure 5. Computational prediction of the myo-inositol module in C. crescentus.

A probabilistic network of protein associations constructed from coexpression, coinheritance, colocation, and coevolution data from C. crescentus predicts that genes at two disjoint chromosomal loci (CC0859–CC0861 and CC1296–CC1302) are functionally associated (i.e. these genes are predicted to be part of the same KEGG pathway or GO process [46]). Genes are shown as nodes and associations as edges, with edge widths denoting the strength of association (confidence interval for narrow lines is 30–60%; confidence for thick lines is >60%).

Figure 6. Cross-species module prediction in five other α-proteobacteria.

(A) A multiple network alignment constructed using the Graemlin algorithm shows extensive conservation of the myo-inositol module across four of five related α-proteobacterial species (Sinorhizobium meliloti, Mesorhizobium loti, Agrobacterium tumefaciens, and Brucella melitensis) at both the protein level and the level of inter-protein association. Here, nodes represent groups of sequence-homologous proteins; individual blocks within a node correspond to specific proteins and are color coded by species (see color key). Edges between nodes are likewise color coded by species, with edge widths representing association strength (confidence interval for narrow lines is 30–60%; confidence for thick lines is >60%). (B) Network alignment boosts the signal for promoter motif finding (MEME e value = 4.3 e⁻⁷⁵). The same palindromic motif is found in the upstream regions of iolC, idhA, and in the promoter region of the ibpA-iatA-iatP operon in all species except B. japonicum, in which the myo-inositol module is less conserved. A weblogo cartoon is shown below the aligned sequences, where the relative nucleotide frequency at each of the 15 positions in the promoter motif is proportional to the height of the letter.

Figure 7. Genomic organization of the conserved myo-inositol module in five α-proteobacteria.

Of the six species analyzed, the myo-inositol metabolic module is most compact in C. crescentus (yellow), where is distributed between only two chromosomal loci. A. tumefaciens (red) and S. meliloti (orange) exhibit homologous chromosomal organization of the module, distributed at four chromosomal sites. B. melitensis (green) and M. loti (blue) have predicted module components at three sites, although the organization is different: in M. loti, idhA is transcribed as part of the transporter operon, while idhA in B. melitensis is adjacent to iolR but transcribed off the opposite strand. Genes are shown as colored boxes on a horizontal line. Boxes above the line are genes on the plus strand; boxes below the horizontal line are on the minus strand. Vertical black arrows indicate the location of the cis-acting regulatory motif GGAA-N6-TTCC (see Figure 4 and Figure 6B). Cases where we have identified more than one of these motifs in a particular promoter are only marked with a single arrow.