We performed a comparative genomic reconstruction of the transcriptional regulatory network for genes involved in ILV degradation and FAD in the gamma- and betaproteobacteria (Fig. ) and FAD in the alphaproteobacteria. For ILV utilization genes, we report the identification of a novel regulator from the MerR family (LiuR) and its DNA recognition motif (ILV box). The FAD genes in the Enterobacteriales are regulated by the FadR repressor from the GntR family. Here, we report the identification of a novel transcriptional factor from the TetR family (PsrA) and its conserved motif (FAD box) that control FAD genes in other lineages of gammaproteobacteria and in betaproteobacteria. In addition to these major transcription factors, two novel TetR-like regulators were predicted to control the ILV degradation and FAD regulons in the Burkholderiales (FadP and LiuQ, respectively) using other DNA motifs. Finally, we report that the LiuR orthologs in alphaproteobacteria regulate the FAD and TCA cycle genes.
FIG. 5. Candidate regulatory elements and genes involved in ILV degradation and FAD in gamma- and betaproteobacteria. Only conserved members of the predicted ILV/FA regulons are shown. Genes are arranged by metabolic pathway. Genomes are arranged by taxonomic (more ...)
The gene content of the predicted LiuR regulons in gamma- and betaproteobacteria is considerably variable (Table ). The identified core of the LiuR regulon includes the liu and ivd genes, which are required for the conversion of CoA ethers of branched-chain carboxylic acids into CoA ethers of linear-chain carboxylic acids for their subsequent utilization through the TCA cycle (Fig. ). Although a physiological effector molecule for the LiuR regulator is unknown, we propose that one or several intermediates of the ILV degradation pathway, e.g., the CoA ethers of branched-chain carboxylic acids, might be involved in the modulation of LiuR activity.
Some of the ILV degradation genes, such as aacS, bkd, ldh, etfBA
, and etfD
, are candidate targets of LiuR regulation in only a fraction of the considered genomes. Thus, the complete leucine degradation pathway is regulated by LiuR only in Shewanella
species. Interestingly, the bkd
operon in P. aeruginosa
is regulated by the ILV-responsive transcriptional activator BkdR (24
). The LiuR regulon in the Shewanella
lineage includes genes that are involved in glutamate synthesis (gltBA
), glyoxylate shunt (aceBA
), and threonine biosynthesis (thrABC
). This apparent extension of the LiuR regulon could be explained by a metabolic connection of the LiuR-controlled metabolic pathways via acetyl-CoA, a final product of ILV degradation, which could be utilized for the amino acid biosynthesis pathways via the TCA cycle. In betaproteobacteria, the LiuR regulons are extended by a number of genes with unclear functional roles in ILV degradation (e.g., cah, aceK, paaH, gloB
, and paaI
) as well as the mdh
genes, encoding TCA cycle enzymes, and the bio
genes, which are involved in biotin biosynthesis. The latter observation is in line with the role of biotin as a cofactor of methylcrotonyl-CoA carboxylase (LiuBD).
Finally, among alphaproteobacteria, LiuR was found to control the ILV degradation genes only in R. rubrum. In contrast, orthologs of LiuR in other alphaproteobacteria were predicted to control genes involved in FAD and other pathways (Table ). The changed content of the LiuRα regulon suggests that its physiological effector might be an acyl-CoA intermediate of the FAD pathway.
As seen from previously reported transcriptome analyses (21
), most operons regulated by LiuR in S. oneidensis
were significantly induced by salt and/or alkaline stresses. The salt stress response and branched-chain amino acid metabolism seem to be linked in Shewanella
species. Firstly, leucine was shown to be an important source of branched-chain FA in the cell membrane, as the growth of Shewanella gelidimarina
on leucine as a sole carbon source resulted in a twofold increase of the branched-chain FA fraction in the membrane compared with growth on serine or alanine (28
). Secondly, the concentration of branched-chain FA in S. gelidimarina
was highly regulated by salt stress conditions and resulted in decreases in branched-chain FA content at high salinity (29
). Therefore, the LiuR-dependent derepression of ILV degradation genes in Shewanella
species reduces the pool of branched-chain acyl-CoA thioesthers (starter units for the biosynthesis of branched-chain FA) and resulted in a decrease in the branched-chain FA proportion in the membrane, thus regulating the membrane fluidity under salt stress conditions.
Unlike most members of the MerR family, LiuR seems to act solely as a repressor. Indeed, promoters activated by MerR-type transcription factors have an extended (19- to 20-bp) spacer between the −35 and −10 promoter boxes, which contains the transcription factor binding site partially overlapping the −35 element (reviewed in reference 4
). On the other hand, MerR represses (but does not activate) promoters with standard 17-bp spacers (31
). We did not observe candidate promoters with extended spacers for LiuR-regulated operons, whereas in most cases, canonical candidate promoters overlapping the LiuR binding site could be identified (data not shown).
The FAD genes in the Enterobacteriales
are regulated by the transcriptional regulatory protein FadR (8
). We performed a comparative genomic reconstruction of the FadR regulon in other taxonomic groups of gammaproteobacteria and found that despite the overall conservation of the FadR binding motif (Fig. ), the regulon composition demonstrated substantial differences (Table ). We noted that several fad
genes in the Vibrionales
and many fad
genes in Alteromonadales
species lack candidate FadR operator sites, suggesting that other factors might be involved in the regulation of these genes.
Using the comparative genomics procedure, we identified the transcriptional factor PsrA as being the master regulator of the FAD genes in five taxonomic groups of gammaproteobacteria (the Alteromonadales, Vibrionales, Xanthomonadales, Pseudomonadales, and Oceanospirillales) and several species of betaproteobacteria (Table ). The PsrA regulons in different taxonomic groups also demonstrated their significant variability. For example, the FA biosynthesis genes are regulated by PsrA in the Xanthomonadales and several betaproteobacteria, whereas the PsrA regulon in the Pseudomonadales includes a variety of cellular processes. Since FadR and PsrA cooccur in the Alteromonadales and in Vibrionales species, in some genomes, we observed an overlap between two regulons. For instance, the fadH, fadBA, and fadIJ operons in the Vibrionales and the fadIJ operon in Shewanella are coregulated by both FadR and PsrA. In the betaproteobacteria (where only two or three PsrA sites per genome were found), a novel transcriptional factor (FadP) was found to substitute the PsrA function in most of the Burkholderiales. Finally, the FAD genes in alphaproteobacteria were found to be under the candidate regulation of LiuRα. In summary, this work revealed a large diversity in the transcriptional factors controlling FAD pathways in proteobacteria.
An interesting overlap between the LiuR and PsrA regulons was observed in Shewanella species, where candidate binding motifs of both transcription factors were identified in the aceBA and etfBA regulatory regions. The former encodes the acetyl-CoA utilization genes, and thus, this observation could be explained by the fact that acetyl-CoA is a common product of both FAD and ILV degradation pathways. The latter operon (etfBA) encodes electron transfer flavoprotein, which is used as an electron acceptor by LiuR-regulated dehydrogenases of the ILV degradation pathway, as well as PsrA-regulated acyl-CoA dehydrogenase. Similarly, the etfAB and etfD genes were found under the overlapping regulation of LiuR and FadP in Polaromonas and Rhodoferax species.
We also observed a rewiring of regulatory cascades. Indeed, the cascade FadR→iclR plus IclR→aceBAK of E. coli and Salmonella spp. corresponds to a streamlined interaction, FadR→aceBAK in Yersinia spp., whereas the aceBA genes in Shewanella spp. are controlled by the novel regulator PsrA. Three feed-forward loops, LiuR→tyrR, TyrR→(liu, ivd, and bkd), and LiuR→(liu, ivd, and bkd), are present in 8 of 12 Shewanella spp., whereas in the remaining Shewanella spp., the tyrR gene is not regulated by LiuR.
The reconstructed regulatory network suggests that the LiuR and PsrA regulons are the most widespread regulators of the ILV degradation and FAD genes in gamma- and betaproteobacteria (Fig. ), respectively. In contrast, the LiuQ and FadP regulons have the narrowest phylogenetic distribution, being identified only in the order Burkholderiales. The FadR regulon was identified in four taxonomic orders of gammaproteobacteria, namely, the Enterobacteriales, Pasteurellales, Vibrionales, and Alteromonadales, where it acts either with PsrA (e.g., in Vibrionales) or alone (e.g., in the Enterobacteriales) to control the FAD genes. Based on these observations, we suggest that the most parsimonious evolutionary scenario for the ILV and FA regulons is as follows. LiuQ and PsrA were likely present in the common ancestor of gamma-, and betaproteobacteria, and they have been partially or fully substituted by LiuQ and FadP in the Burkholderiales and by FadR in some groups of gammaproteobacteria.
The results of this comparative genomics study demonstrate significant variability in the design and composition of the regulatory networks for the control of genes from central metabolic pathways. A similar extreme flexibility of transcriptional regulatory networks across various taxonomic groups of bacteria was reported in previous studies (2
). The well-characterized FadR regulon that served as a prototype regulon for FAD not only underwent many changes by itself but may not even be the ancestral one. Although the overall picture for the core of the FA and ILV degradation regulons seems to be rather consistent (Fig. ), many additional members of these regulons whose current functional annotations do not allow us to attribute them to the ILV/FA catabolic pathways were identified (Tables to ).
The reconstructed regulatory network needs to be integrated with functionally related networks, and few remaining gaps, such as the regulation of ILV degradation in the Xanthomonadales and alphaproteobacteria, need to be filled. Of course, although we are convinced that most computationally identified regulatory interactions reported here are real, each particular prediction requires experimental validation.