Abstract
Bacteria catabolize malonate via two pathways, encoded by the mdc and mat genes. In various bacteria, transcription of these genes is controlled by the GntR family transcription factors (TFs) MatR/MdcY and/or the LysR family transcription factor MdcR. Propionate is metabolized via the methylcitrate pathway, comprising enzymes encoded by the prp and acn genes. PrpR, the Fis family sigma 54-dependent transcription factor, is known to be a transcriptional activator of the prp genes. Here, we report a detailed comparative genomic analysis of malonate and propionate metabolism and its regulation in proteobacteria. We characterize genomic loci and gene regulation and identify binding motifs for four new TFs and also new regulon members, in particular, tripartite ATP-independent periplasmic (TRAP) transporters. We describe restructuring of the genomic loci and regulatory interactions during the evolution of proteobacteria.
INTRODUCTION
Malonate can be used as a carbon source by a variety of bacteria, such as Acinetobacter calcoaceticus, Klebsiella pneumoniae, Pseudomonas fluorescens, and Pseudomonas putida (15). It is a competitive inhibitor of succinate dehydrogenase. Malonate was found in symbiotic legumes and in developing rat brains (15, 16). Malonate metabolism is essential in symbiotic nitrogen metabolism, since mutant bacteria with deleted malonate metabolic genes lose this symbiosis ability (13). In addition, malonate metabolic genes have been used for generation of the industrial strain of Streptomyces for the production of antibiotics (13).
Two groups of structural genes involved in malonate metabolism have been characterized. The first group was described for Rhizobium leguminosarum (16). It comprises three clustered malonate metabolic genes, matA, matB, and matC, and the divergently transcribed gene of the malonate regulator matR (Table 1). The MatR transcription factor (TF) belongs to the FadR subfamily of the GntR family.
Table 1.
Genes involved in malonate and propionate catabolism
| Gene | Function of producta |
|---|---|
| mdcL | Malonate transporter subunit |
| mdcM | Malonate transporter subunit |
| mdcA | Malonate/acetyl-CoA transferase, alpha subunit |
| mdcC | Acyl carrier protein, delta subunit |
| mdcD | Malonyl-CoA decarboxylase, beta subunit |
| mdcE | Protein involved in the stability of the enzyme complex or codecarboxylase, gamma subunit |
| mdcF | Malonate transporter |
| mdcG | Holo-ACP synthase |
| mdcB | Triphosphoribosyldephospho-CoA synthase, a protein involved in the formation of the prosthetic group precursor |
| mdcH | Malonyl-CoA/ACP transacylase |
| matA | Malonyl-CoA decarboxylase |
| matB | Malonyl-CoA synthetase |
| matC | Malonate transporter |
| matPQM | TRAP malonate transporter |
| matR (mdcY) | GntR family regulator of malonate metabolism |
| mdcR | LysR family regulator of malonate metabolism |
| prpB | 2-Methylisocitrate lyase |
| prpC | 2-Methylcitrate synthase |
| prpD | 2-Methylcitrate dehydratase |
| prpE | Propionyl-CoA synthetase |
| prpF | Aconitase accessory protein |
| acnD | Aconitate hydratase |
| acnB | Bifunctional aconitate hydratase 2/2-methylisocitrate dehydratase |
| acnA | Aconitate hydratase |
| prpR | Regulator of propionate metabolism |
| mutB | Metylmalonyl-CoA mutase |
| meaB | Putative auxiliary metallochaperone, involved in protection and assembly of methylmalonyl-CoA mutase (G3E family of P-loop GTPases) |
| pccA | Propionyl-CoA carboxylase, subunit alpha |
| pccB | Propionyl-CoA carboxylase, subunit beta |
| mce | Methylmalonyl-CoA epimerase |
CoA, coenzyme A; ACP, acyl carrier protein.
Another system for malonate metabolism was described for Acinetobacter calcoaceticus KCCM 4090 (14, 15). This system is encoded by the operon of structural genes mdcLMACDEGBH and the divergently transcribed regulator gene mdcY (Table 1). Like MatR, MdcY is also a FadR family TF. Some Gammaproteobacteria have a different malonate transporter gene (compared to that of A. calcoaceticus), mdcF instead of mdcLM (19). The gene organization of the malonate operon of A. calcoaceticus is similar to that of K. pneumoniae and P. putida (14, 19), but these and many other Gammaproteobacteria have another TF gene adjacent to the malonate operon, encoding the MdcR transcription factor from the LysR family. MdcR activates expression of the mdc genes and represses its own transcription (14, 19).
Propionate can also serve as a single carbon source for many bacteria. Its metabolism is strongly connected to the malonate metabolic pathway and central metabolism, e.g., the tricarboxylic acid (TCA) cycle (Fig. 1). Propionate is converted to pyruvate via the methylcitrate pathway, enzymes for which are encoded by the prpBCDE, acnB, and acnD genes (3, 4). AcnB is a bifunctional enzyme that also belongs to the TCA cycle and the glyoxylate pathway (3, 4). Propionate also participates in the citramalate cycle comprising the products of the mutB (mcm), meaB, pccBA, and mce (epi) genes (11, 17) (Table 1). PrpR, a sigma 54-dependent TF belonging to the Fis family, is known to be a transcriptional activator of the prp genes in Escherichia coli and Ralstonia eutropha HF39 (3). Moreover, it is known that a GntR family regulator gene is colocalized with the prp cluster in the Pseudomonas spp. and Vibrio cholerae, and it is likely one more propionate regulator (3).
Fig 1.
Malonate and propionate metabolism. The methylcitrate pathway is marked with red lines, the citramalate cycle with blue lines.
Here, we report a detailed comparative genomic analysis of malonate and propionate metabolism and its regulation in proteobacteria. We characterized genomic loci and gene regulation, identified binding motifs and new regulon members, and predicted novel TFs. We also describe rearrangements of genomic loci and regulatory interactions during the evolution of proteobacteria.
MATERIALS AND METHODS
Computational analysis of regulons.
The genomic sequences of the analyzed proteobacteria were obtained from GenBank (2); the genomes are listed in Table S1 in the supplemental material. Orthologs of TFs in bacterial genomes were identified by PSI-BLAST (1) searches with default parameters and confirmed by construction of phylogenetic trees for identified homologs and by colocalization with genes of the corresponding metabolic pathways. Amino acid sequence alignment was performed using the MUSCLE package (8). Phylogenetic trees were constructed with the PHYLIP package, using the Protdist program for the calculation of distances and the maximum likelihood method with default parameters for the tree construction (9). For all bacterial species that had any of the TFs described in the introduction, the comparative genomics-based reconstruction of the malonate and propionate regulons was performed. Nucleotide positional weight matrices (profiles) for all TF-binding sites (see Table S2 in the supplemental material) were constructed by the SignalX program (18), using training sets of upstream regions of genes from the analyzed bacteria. Score thresholds for identification of sites were selected so that candidate sites upstream of functionally relevant genes were accepted, while the fraction of genes preceded by candidate sites did not exceed 5% in each studied genome. Under these conditions, for long, conserved motifs, the number of candidate sites per genome did not exceed 50.
Candidate binding sites were confirmed by phylogenetic footprinting (21). In summary, we manually analyzed alignments of upstream regions of orthologous genes and identified groups of consecutive conserved positions, relying on the assumption that binding sites are more conserved than adjacent intergenic regions. A computational search for candidate TF-binding sites in upstream gene regions (−350 to +50 nucleotides [nt] relative to the start codon) was performed using the Genome Explorer package (18). The threshold scores for all types of TF-binding sites are given in Table S2 in the supplemental material. Weaker sites (with scores 10% less than the threshold) were also taken into account if their positions were similar to positions of stronger sites upstream of orthologous genes and there were no stronger competing sites in the same intergenic region. New candidate members were assigned to a regulon if they were preceded (as a single gene or as a part of an operon) by candidate TF-binding sites in at least four genomes. The requirement that a gene should be preceded by a candidate site in at least four genomes was established based on empirical evidence. A stricter requirement would lead to the loss of known regulon members, such as MatR/MdcY-regulated matC, which is present in the studied regulon in only four bacteria. A weaker criterion of three genomes yields candidate regulon members with clearly irrelevant function. In most cases, including all nontrivial predictions, the actual number of orthologs preceded by candidate sites was considerably higher (e.g., tripartite ATP-independent periplasmic [TRAP] transporters) (see Table S3 in the supplemental material). Moreover, it was also required that candidate sites were observed in sufficiently distant species, so that the site conservation could not be explained by residual sequence similarity.
Genes were considered to belong to one operon if they were transcribed in the same direction, with intergenic distances not exceeding 200 nt. Motif logos were constructed using WebLogo (7).
RESULTS AND DISCUSSION
Phylogenetic distribution of malonate metabolism genes and their regulators between bacteria. (i) MatR/MdcY.
MatR and MdcY have been experimentally identified in different bacteria (14, 16), and this seems to be the only reason for the different names. The amino acid sequences of MatR and MdcY are very similar (49% identity), these TFs are closely related according to phylogenetic analysis, and their previously predicted binding sites are also similar (14, 16, 20). Moreover, there is no evident phylogenetic separation between TFs whose genes are colocalized in genomes with either mat or mdc genes. Hence, this TF is referred to herein as MatR/MdcY.
An exhaustive BLAST search identified a number of MatR/MdcY TFs, mostly in the Alphaproteobacteria and several Betaproteobacteria and Gammaproteobacteria (see Table S3 and Fig. S1 in the supplemental material).
Phylogenetic footprinting of regions upstream of the mat and mdc genes revealed the MatR/MdcY binding motif with the consensus TTGTATACAA (14, 16, 20) (see Fig. S7A in the supplemental material). This motif coincides with the one previously predicted and confirmed by the DNase I footprint assay (14, 16).
The distribution of the mdc and mat operons in bacterial taxa is very flexible. Some bacteria have both mat and mdc genes, while others have either the mdc or mat operon. In most bacteria, the matR (mdcY) gene is colocalized with the regulated genes. Most bacteria possess only one malonate regulator of the MatR/MdcY type, although Methylobacterium spp. have two copies of this TF, one clustered with the mdc operon and the other one with tripartite ATP-independent periplasmic (TRAP) genes and, in some cases, like in Methylobacterium sp. strain 4-46, with TRAP and matAB genes.
(ii) TRAP transporters.
The TRAP transporters are characterized by the usage of an electrochemical ion gradient as the driving force for solute accumulation. The best-characterized TRAP is the high-affinity C4-dicarboxylate transport (Dct) system formed by three proteins: extracytoplasmic solute receptor subunit (DctP) and small (DctQ) and large (DctM) integral membrane proteins (10, 12, 22). Representatives of the TRAP family are present in a wide range of eubacteria and archaea. Some organisms possess a single TRAP system (E. coli), while others have several TRAP transporters (Pseudomonas aeruginosa and Bacillus halodurans). Probable substrates of the TRAP transporters are l-xylulose, gluconate, mannonate, succinate, etc. (10, 12, 22).
Many Alphaproteobacteria and some Beta- and Gammaproteobacteria have C3-dicarboxylic acid TRAP genes colocalized with genes encoding TFs or enzymes of malonate metabolism, either forming an operon with them or preceded by their own candidate binding sites. In Sinorhizobium meliloti, these TRAP components were named matPQM (6). Null mutants for each of the matPQM genes were shown to be unable to grow on the minimal medium containing malonate as the sole carbon source (6). In this study, in most cases the presence of TRAP genes in the regulated operons correlated with the absence of known malonate transporters mdcLM, mdcF, and matC. Among completely sequenced genomes, orthologs of these TRAP transporters were found only in bacteria that had malonate metabolic genes. These observations suggest that these TRAP transporters are involved in malonate transport and belong to the MatR/MdcY regulon.
(iii) MdcR.
MdcR orthologs were found in the Gammaproteobacteria and in the Burkholderiales among the Betaproteobacteria, but not in the Alphaproteobacteria. In the Gammaproteobacteria, MdcR, when present, almost always is the only malonate regulator, whereas in the Betaproteobacteria, it often accompanies other malonate regulators. A phylogenetic tree of all found MdcR TFs is shown in Fig. S2 in the supplemental material.
In most Gammaproteobacteria, MdcR controls a single operon, mdcABCDEGHLM or mdcABCDEFGH. In the Betaproteobacteria, the mdc genes have a diverse arrangement, usually as a single operon or sometimes in two operons (in Burkholderia phytofirmans PsJN and in Ralstonia eutropha JMP134), with mdcLM genes in a different locus. The mdcLM transporter genes are typical for the MdcR regulon. In several Gammaproteobacteria (e.g., Citrobacter koseri ATCC BAA-895 and Enterobacter sp. strain 638) they are replaced with mdcF, and in some Betaproteobacteria (Delftia acidovorans SPH-1 and Methylibium petroleiphilum PM1), they are replaced with matC (see Table S3 in the supplemental material).
The candidate MdcR binding motif was predicted by the phylogenetic footprinting of the mdcA upstream region. It is a 23-nt palindrome with the consensus sequence ATCATTACCCTgAgggTAATGAT (lowercase letters denote less-conserved nucleotides) (see Fig. S7B in the supplemental material). In most Gamma- and Betaproteobacteria, mdcR is not autoregulated. The exceptions are Ralstonia pickettii 12J and Psychromonas ingrahamii 37, where mdcR is located in a divergon with other mdc genes and thus shares the candidate binding site.
(iv) PrpR.
PrpR is a transcriptional activator of the prp genes in some Gammaproteobacteria (Enterobacteriales and Xanthomonadales) and Betaproteobacteria (Burkholderiales). A phylogenetic tree of all found PrpR TFs is shown in Fig. S3 in the supplemental material.
All Enterobacteriales have propionate genes organized in the divergon prpR prpBCDE. In most Xanthomonadales and Betaproteobacteria, the propionate divergon has the prpR prpBC-acnD-prpF structure. Some Betaproteobacteria have diverse propionate regulon structure and several other TFs regulating propionate metabolism (see below and Table S3 in the supplemental material).
The candidate PrpR binding motif was identified using phylogenetic footprinting of the prpB and prpR upstream regions. The predicted binding motif is a 16-nt palindrome with the consensus sequence rTTTCAwwwwTGAAAy (lowercase letters denote less-conserved nucleotides) (see Fig. S8A in the supplemental material). PrpR is a sigma 54-dependent transcription activator, and indeed, candidate sigma 54 promoters were identified upstream of all propionate gene clusters belonging to the PrpR regulon.
New regulators of the malonate and propionate metabolism. (i) MlnR* (FadR subfamily of the GntR family).
Some bacteria from several families of the Betaproteobacteria (Alcaligenaceae, Burkholderiaceae, Comamonadaceae, and Rhodocyclaceae) and Gammaproteobacteria (Ectothiorhodospiraceae and Xanthomonadaceae) have another GntR family TF adjacent to the malonate metabolism genes. This TF is related to MatR/MdcY but is not its ortholog (confirmed by PSI-BLAST and the phylogenetic tree; see Fig. S1 in the supplemental material). This protein was named MlnR* (here and below, an asterisk denotes a newly given name).
The MlnR* TF was predicted to control malonate utilization and a part of the citramalate cycle. In some Betaproteobacteria, two paralogous copies of MlnR* were found. In that case, one copy is colocalized with the matAB genes, whereas the other one is clustered on the chromosome with genes of the citramalate cycle, mutB, meaB, pccBA, and mce. In bacteria having only one copy of this TF, the mlnR* gene is clustered with both mat and the citramalate cycle genes (e.g., Azoarcus sp. strain BH72, Dechloromonas aromatica) or colocalized with either malonate metabolic genes (e.g., Bordetella spp., Ralstonia metallidurans, and Cupriavidus taiwanensis) or genes of the citramalate cycle (e.g., Delftia acidovorans, Leptothrix cholodnii, and Polaromonas naphthalenivorans). In Xanthomonadaceae, the mlnR* gene is clustered with mdc genes and matC. Alkalilimnicola ehrlichii MLHE-1 (Ectothiorhodospiraceae) has the mlnR* gene colocalized with matAB and the malonate TRAP genes matPQM, and such gene organization resembles the one in the MatR/MdcY regulon in many Alphaproteobacteria.
Phylogenetic footprinting of the mlnR* upstream regions revealed two types of candidate binding motifs. The first one, with the consensus sequence TTATTCATAATTATGAATAA (type 1; see Fig. S7C in the supplemental material), was found in the Betaproteobacteria. The second type of the predicted MlnR* binding motif was found in the Xanthomonadaceae and Alkalilimnicola ehrlichii MLHE-1. Its consensus, ATAATTACGATGTAATTAC (type 2; see Fig. S7D), partially coincides, after a shift, with the betaproteobacterial type 1 motif. The type 1 and type 2 motifs contain a common, short palindromic motif, RTAATTAY, with two repeats in the type 2 motif and only one copy in the type 1 motif (Fig. 2). The type 1 and type 2 profiles cross-recognize some sites.
Fig 2.
Common parts of the two types of MlnR* binding motifs. The common parts are marked with dashed lines; the repeated part in the type 1 and type 2 motifs is set in bold.
(ii) GntR and LysR family TFs in Burkholderia spp.
Bacteria from the genus Burkholderia have the mdc genes but lack orthologs of MatR/MdcY. MdcR orthologs among Burkholderia spp. are present only in Burkholderia multivorans ATCC 17616, Burkholderia sp. strain 383, and Burkholderia phytofirmans PsJN; moreover, MdcR binding sites upstream of the malonate metabolic genes are found only in Burkholderia phytofirmans PsJN. To predict other possible regulators of malonate metabolism in Burkholderia spp., we searched for TF genes colocalized with the mdc genes. Indeed, some Burkholderia spp. (see Table S3 in the supplemental material) have a gene encoding a different GntR family TF near the mdc genes. This TF is distantly related to MatR/MdcY and is not its ortholog (confirmed by PSI-BLAST and the phylogenetic tree; see Fig. S1 in the supplemental material). Other Burkholderia spp. contain a gene encoding a LysR family TF in the mdc locus. Orthologs of these GntR and LysR family TFs are absent in other genomes studied in this work. An attempt to find binding motifs of these regulators by phylogenetic footprinting failed because of the high degree of similarity of the entire intergenic regions of these closely related species.
(iii) PrpR* (FadR subfamily of the GntR family).
Multiple representatives of the Gammaproteobacteria (Alteromonadales, Oceanospirillales, Pseudomonadales, and Vibrionales), some Betaproteobacteria (Burkholderiales), and even Deltaproteobacteria (Geobacter metallireducens GS-15) have a GntR family, FadR subfamily TF as a regulator of propionate metabolism. This TF is hereafter referred to as PrpR*. A phylogenetic tree of all found PrpR* TFs is shown in Fig. S4 in the supplemental material.
Most Alteromonadales and Pseudomonadales have propionate metabolic genes organized in the prpR*BC-acnD-prpFD operon, while some bacteria from these taxa have shorter operons, prpR*BC-acnD-prpF or prpR*BC-acnD. Most Vibrionales have the prpR*BC-acnD-prpFE operon, while Oceanospirillales have the prpR*BCD operon. A similar operon organization, prpR*BDC, was observed in Geobacter metallireducens GS-15.
Geobacter metallireducens is the only member of the Deltaproteobacteria that has any of the regulators studied in this work. Even the congeners Geobacter sulfurreducens and Geobacter uraniireducens, though having the prp metabolic genes, lack orthologous transcription factors and thus were not considered. Among other Delta- and Epsilonproteobacteria, only Helicobacter hepaticus has a complete propionate metabolic pathway (prpEBCD genes), but it lacks any known or predicted propionate TFs.
In the Betaproteobacteria, PrpR* not only is rare but also seems to be not the main propionate regulator, even if it is present in a genome, as most propionate genes are regulated by a HutC-type TF (SdhR*; see below and Table S3 in the supplemental material). Among the Betaproteobacteria, PrpR* controls propionate metabolic genes only in Verminephrobacter eiseniae EF01-2 and Bordetella spp.
The predicted PrpR* binding motif, identified by phylogenetic footprinting, is a 12-nt palindrome with the consensus sequence ATTGTCGACAAT (see Fig. S8B in the supplemental material).
(iv) PrpQ* (XRE family).
Most Alpha- and some Betaproteobacteria have a gene encoding an XRE family TF that is likely the transcriptional regulator of propionate metabolism. This TF is hereafter referred to as PrpQ*. A phylogenetic tree of all found PrpQ* TFs is shown in Fig. S5 in the supplemental material.
The candidate PrpQ* binding motif was identified by phylogenetic footprinting of the upstream regions of the prpQ* and pccBA operons. The motif is a short (8-nt) palindrome with the consensus sequence TTTGCrAA, often present in multiple copies upstream of regulated genes (see Fig. S8C in the supplemental material). In Betaproteobacteria, the reconstructed PrpQ* regulon includes genes for a part of the methylcitrate pathway (see Table S3 in the supplemental material). In Alphaproteobacteria, PrpQ* binding sites were found upstream of genes encoding enzymes of the citramalate and/or methylcitrate pathways, such as the prp, acnD, pccBA, and mutB genes (see Table S3).
(v) SdhR* (HutC subfamily of the GntR family).
Multiple Betaproteobacteria (Burkholderiales) have a GntR family, HutC subfamily TF that, according to the colocalization, may control expression of genes encoding enzymes involved in the TCA cycle, such as succinate dehydrogenase (sdhABCD), citrate synthase (gltA), bifunctional aconitate hydratase 2/2-methylisocitrate dehydratase (acnB), malate dehydrogenase (mdh), and some other catabolic genes, in particular ygfY, functionally connected to sdhB, and tam, encoding trans-aconitate 2-methyltransferase; this enzyme catalyzes monomethyl esterification of trans-aconitate at high velocities and affinities and cis-aconitate, isocitrate, and citrate at lower velocities and affinities (5). This TF is hereafter referred to as SdhR*. A phylogenetic tree of all found SdhR* TFs is shown in Fig. S6 in the supplemental material.
Phylogenetic footprinting of sdhR*, mdh, and acnB upstream regions revealed a conserved area that is likely the SdhR* binding site. The predicted binding motif is a 26-nt palindrome with the consensus sequence TCTTATGTCTTATATAAGACATAAGA (see Fig. S8D in the supplemental material). This motif has an intrinsic symmetry, comprising two direct repeats of complementary motifs TCTTAT and ATAAGA in the distal and proximal parts of the palindrome, respectively.
In many Betaproteobacteria (see Table S3 in the supplemental material), SdhR* also controls a number of genes of the propionate utilization pathway (prpB, prpC, prpD, acnA, and prpF) and the glyoxylate shunt (malyl coenzyme A [malyl-CoA] lyase [mcl] and isocitrate lyase [aceA]).
Conclusions.
The diversity of the organization of the malonate and propionate regulons in different taxonomic groups naturally leads to the question of the evolution of these regulatory systems.
The most consistent group is the Alphaproteobacteria. The mode of malonate and propionate regulation is the same among these bacteria: the malonate metabolic genes are regulated by MatR/MdcY, and propionate utilization is under PrpQ* regulation. Both these TFs are typical for the Alphaproteobacteria and are present in only a few Beta- and Gammaproteobacteria, seemingly as a result of horizontal gene transfer (Fig. 3).
Fig 3.
Regulation in Alphaproteobacteria. Line color denotes the mode of regulation, using the following color code: yellow, MatR/MdcY, and violet, PrpQ*.
Indeed, among the Gammaproteobacteria, the horizontal transfer of matR (mdcY) occurred in the Pseudomonadales and Psychromonadaceae (confirmed by the phylogenetic tree [Fig. 4]; see also Fig. S1 in the supplemental material). Ectothiorhodospiraceae and Xanthomonadaceae have the GntR family TF named MlnR* as a malonate regulator, which is typical for the Betaproteobacteria (see below). It is likely they inherited it from their common ancestor with the Betaproteobacteria (see Fig. S1 in the supplemental material). In other Gammaproteobacteria, the malonate metabolism genes are regulated by MdcR, and this TF seems to be the original malonate regulator in this taxonomic group (Fig. 4). Propionate metabolism in the Gammaproteobacteria is mostly controlled by a GntR family TF named here PrpR*, but some Enterobacteriales and all analyzed Xanthomonadales use a Fis family TF, PrpR, as the propionate regulator. This TF is also present in the Betaproteobacteria, and according to the phylogenetic tree (see Fig. S3 in the supplemental material), either Beta- and Gammaproteobacteria inherited it from their common ancestor or the common ancestor of the Enterobacteriales and Xanthomonadales had it transferred from some ancient Betaproteobacteria. Both scenarios involve multiple losses of PrpR in a variety of lineages. The GntR family TF PrpR* is widespread among the Gammaproteobacteria (Fig. 4). This TF is also present in several Betaproteobacteria and the deltaproteobacterium Geobacter metallireducens GS-15. According to the branch localization in the phylogenetic tree, G. metallireducens got PrpR* as a result of horizontal gene transfer from a genome close to the ancestor of the Alteromonadales. It seems that some Betaproteobacteria had PrpR* transferred from the Pseudomonadales (for example, Bordetella petrii DSM 12804) (see Fig. S4 in the supplemental material), while some others inherited it from their common ancestor with the Gammaproteobacteria (e.g., Bordetella bronchiseptica RB50 and Verminephrobacter eiseniae EF01-2) (see Fig. S4). An interesting variant is seen in Bordetella avium 197N, which has two prpR* genes, one likely obtained from the Pseudomonadales and the other inherited from the common ancestor with the Gammaproteobacteria. It is plausible that in the Gammaproteobacteria we observe an intermediate stage of replacement of PrpR by PrpR* (Fig. 4).
Fig 4.
Regulation in Gammaproteobacteria. Line color denotes the mode of regulation, using the following color code: yellow, MatR/MdcY; orange, MdcR; pink, MlnR*; dark blue, PrpR; and green, PrpR*.
The most diverse regulation is seen in the Betaproteobacteria (Fig. 5 and Table 2). Overall, bacteria from this group possess at least five malonate regulators, including MatR/MdcY, which they presumably got from the Alphaproteobacteria via horizontal gene transfer (confirmed by the phylogenetic tree; see Fig. S1 in the supplemental material); MdcR, horizontally transferred (according to the phylogenetic tree; see Fig. S2 in the supplemental material) from the Gammaproteobacteria or inherited from the common ancestor of Beta- and Gammaproteobacteria; and LysR and GntR family regulators, found only in Burkholderia spp. However, most Betaproteobacteria contain a GntR family TF, MlnR*. Propionate utilization in the Betaproteobacteria is also under diverse regulation. Only few Betaproteobacteria, three closely related Comamonadaceae species, have PrpQ* as a result of the horizontal transfer from the Alphaproteobacteria (confirmed by the phylogenetic tree; see Fig. S5 in the supplemental material). Most Betaproteobacteria have SdhR*, either as the only propionate regulator or accompanied by PrpR or sometimes PrpR* (Fig. 5). It is interesting that most Betaproteobacteria that got MlnR* lack the Fis family TF PrpR, possibly due to the fact that these TFs control alternative pathways of propionate conversion (see Table S3 in the supplemental material).
Fig 5.
Regulation in Betaproteobacteria. Line color denotes the mode of regulation, using the following color code: yellow, MatR/MdcY; orange, MdcR; pink, MlnR*; light blue, SdhR*; dark blue, PrpR; green, PrpR*; violet, PrpQ*; light brown, LysR family TF (Burkholderia); and dark green, GntR family TF (Burkholderia).
Table 2.
Distribution of malonate and propionate transcription factors among various classes of proteobacteria
| Class of proteobacteria | No. of genera with TF |
||||||
|---|---|---|---|---|---|---|---|
| MatR/MdcY | MlnR* | MdcR | PrpR | PrpR* | PrpQ* | SdhR* | |
| Alpha | 12 | 0 | 0 | 0 | 0 | 17 | 0 |
| Beta | 4 | 11 | 5 | 3 | 2 | 3 | 12 |
| Gamma | 2 | 3 | 7 | 6 | 16 | 0 | 0 |
| Delta | 0 | 0 | 0 | 0 | 1 | 0 | 0 |
Overall, we have reconstructed the malonate and propionate regulons, described their flexible and diverse regulation in proteobacteria, found new TFs that control malonate and propionate metabolism, and identified their candidate binding sites by positional and sequence analyses. The comparative genomic analysis also yielded new candidate members of the malonate regulon, namely, TRAP transporters.
Supplementary Material
ACKNOWLEDGMENTS
This study was partially supported by The Russian Foundation of Basic Research (grants 09-04-92745 and 10-04-00431), the Russian Academy of Sciences (via the Molecular and Cellular Biology program), and state contracts 14.740.11.0003 and 07.514.11.4007.
Footnotes
Published ahead of print 13 April 2012
Supplemental material for this article may be found at http://jb.asm.org/.
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