Multiple levels of transcriptional regulation control glycolate metabolism in Paracoccus denitrificans

ABSTRACT The hydroxyacid glycolate is a highly abundant carbon source in the environment. Glycolate is produced by unicellular photosynthetic organisms and excreted at petagram scales to the environment, where it serves as growth substrate for heterotrophic bacteria. In microbial metabolism, glycolate is first oxidized to glyoxylate by the enzyme glycolate oxidase. The recently described β-hydroxyaspartate cycle (BHAC) subsequently mediates the carbon-neutral assimilation of glyoxylate into central metabolism in ubiquitous Alpha- and Gammaproteobacteria. Although the reaction sequence of the BHAC was elucidated in Paracoccus denitrificans, little is known about the regulation of glycolate and glyoxylate assimilation in this relevant alphaproteobacterial model organism. Here, we show that regulation of glycolate metabolism in P. denitrificans is surprisingly complex, involving two regulators, the IclR-type transcription factor BhcR that acts as an activator for the BHAC gene cluster, and the GntR-type transcriptional regulator GlcR, a previously unidentified repressor that controls the production of glycolate oxidase. Furthermore, an additional layer of regulation is exerted at the global level, which involves the transcriptional regulator CceR that controls the switch between glycolysis and gluconeogenesis in P. denitrificans. Together, these regulators control glycolate metabolism in P. denitrificans, allowing the organism to assimilate glycolate together with other carbon substrates in a simultaneous fashion, rather than sequentially. Our results show that the metabolic network of Alphaproteobacteria shows a high degree of flexibility to react to the availability of multiple substrates in the environment. IMPORTANCE Algae perform ca. 50% of the photosynthetic carbon dioxide fixation on our planet. In the process, they release the two-carbon molecule glycolate. Due to the abundance of algae, massive amounts of glycolate are released. Therefore, this molecule is available as a source of carbon for bacteria in the environment. Here, we describe the regulation of glycolate metabolism in the model organism Paracoccus denitrificans. This bacterium uses the recently characterized β-hydroxyaspartate cycle to assimilate glycolate in a carbon- and energy-efficient manner. We found that glycolate assimilation is dynamically controlled by three different transcriptional regulators: GlcR, BhcR, and CceR. This allows P. denitrificans to assimilate glycolate together with other carbon substrates in a simultaneous fashion. Overall, this flexible and multi-layered regulation of glycolate metabolism in P. denitrificans represents a resource-efficient strategy to make optimal use of this globally abundant molecule under fluctuating environmental conditions.

T he two-carbon compound glycolate is the simplest α-hydroxyacid.Photosynthetic organisms that rely on carbon dioxide fixation via the Calvin-Benson-Bassham (CBB) cycle produce this molecule as part of their photorespiration process (1).Subsequently, glycolate can either be recycled into cellular metabolism using an inefficient and energetically costly metabolic pathway (2) or excreted (3).The latter route predominates in unicellular photosynthetic organisms, such as eukaryotic microalgae and Cyanobacte ria.Due to the abundance of these ubiquitous phototrophs in marine and freshwater habitats, an annual flux of one petagram (10 9 tons) of glycolate has been estimated (4).Hence, glycolate is a readily available source of carbon for heterotrophic environmental microorganisms.
In microbial metabolism, glycolate is first oxidized to glyoxylate via the enzyme glycolate oxidase (5)(6)(7).In addition, glyoxylate can be generated as breakdown product of ubiquitous purine bases and allantoin (8), as well as ethylenediaminetetraacetate (EDTA) and nitrilotriacetate (NTA) (9,10).There are two known metabolic routes for subsequent net assimilation of glyoxylate.The well-studied glycerate pathway is used by Escherichia coli and other bacteria to convert two molecules of glyoxylate into one molecule of 2-phosphoglycerate, releasing one molecule of carbon dioxide in the process (7,11).The β-hydroxyaspartate cycle (BHAC) (12,13), on the other hand, was recently shown to convert two molecules of glyoxylate into oxaloacetate via four enzymatic steps without the release of CO 2 (14).In contrast to the glycerate pathway, the BHAC has a much higher energy and carbon efficiency, and has already been successfully applied in metabolic engineering efforts in bacteria (15) and plants (16).
Notably, the BHAC is the dominant glycolate assimilation route in the environmentally relevant groups of Alpha-and Gammaproteobacteria, and was recently shown to play an important role in global glycolate conversions, in particular in marine environments (14).In a field study, enzymes of the BHAC were shown to be upregulated during a bloom of marine algae, following increased glycolate concentrations.Metagenomic data further supported the global prevalence of the BHAC.However, despite the ecological relevance of the BHAC, the question on how glycolate and glyoxylate metabolism are regulated at the molecular and cellular level in Alpha-and Gammaproteobacteria remained unanswered.
In respect to glycolate metabolism, it is known that production of the four enzymes of the BHAC is strongly induced in P. denitrificans during growth on glycolate, compared to growth on succinate.Furthermore, it was reported that bhcR, a gene coding for a putative transcriptional regulator, is positioned adjacent to the enzyme-encoding genes of the BHAC in the genome of P. denitrificans.BhcR was found to bind to the promoter region of the bhc gene cluster, while, in turn, this interaction was negatively affected by glyoxylate (14).
In this work, we show that BhcR functions as an activator of the bhc gene cluster and is required for both growth on glyoxylate and glycolate in P. denitrificans.In addition, we identify and characterize GlcR, a previously unknown transcriptional repressor of the GntR family that regulates glycolate oxidase in P. denitrificans.By extending our investigation to the global level, we found that the transcription factor CceR controls the metabolic switch between glycolysis and gluconeogenesis.Furthermore, we show that P. denitrificans co-assimilates glycolate and other carbon substrates simultaneously, not sequentially.Collectively, our work demonstrates multiple levels of transcriptional regulation in glycolate metabolism and highlights the surprising flexibility of the central metabolic network of Alphaproteobacteria in response to carbon substrate availability.

Glyoxylate assimilation via the BHAC is regulated by BhcR
We first focused on understanding the regulation of the BHAC, which mediates the second step of glycolate metabolism in P. denitrificans.To investigate the role of the transcription factor BhcR in regulating this pathway, we characterized the protein bioinformatically and experimentally.Amino acid sequence analysis showed that BhcR contains an IclR-type helix-turn-helix domain and an IclR-type effector-binding domain (see Uniprot: https://www.uniprot.org/uniprotkb/A1B8Z4),indicating that the protein belongs to the IclR-type family of transcriptional regulators.In a phylogenetic tree of 1,083 sequences from 29 subfamilies within the IclR-type family, BhcR formed a close sister group to a clade of IclR and AllR homologs (Fig. S1).
IclR, the namesake representative of the family, regulates expression of the glyoxylate shunt operon (aceBAK) in E. coli and other bacteria.The protein forms a tetramer that acts as transcriptional repressor.IclR is allosterically regulated by glyoxylate and pyruvate, which control the oligomerization state of IclR.Pyruvate stabilizes tetramer formation, whereas glyoxylate favors dimer formation and releases IclR from the DNA (33).AllR acts as transcriptional repressor of the allantoin and glyoxylate utilization operons in E. coli.It binds to the gcl promoter and the allS-allA intergenic region.Similarly to IclR, DNA binding of AllR is decreased by increasing concentrations of glyoxylate (34).
In IclR, glyoxylate and pyruvate occupy the same binding site.With the exception of one residue, this ligand-binding site is conserved in AllR (Table 1; Fig. S2), whereas in BhcR, the putative binding site shows some marked differences.Amino acids that bind to the oxygen atoms of glyoxylate or pyruvate are conserved between IclR and BhcR (except for the presence of isoleucine in place of alanine at position 161).In contrast, a hydrophobic patch of residues that interacts with the methyl group of pyruvate in IclR is apparently lacking in BhcR (Table 1).
To study the DNA-binding properties of BhcR in detail, we purified the regulator from P. denitrificans and performed binding experiments with the putative promoter region of the bhc gene cluster (P bhc ) (Fig. 1a; Fig. S3).This region contains putative −35 and −10 boxes upstream of the bhcR and bhcA start codons (Fig. 1b).In electrophoretic mobi lity shift assays (EMSAs), the interaction of BhcR with P bhc was negatively affected by increasing concentrations of glyoxylate, as previously described (14).We subsequently tested molecules that are structurally similar to glyoxylate as potential ligands of BhcR.We found that the DNA-binding interaction was positively affected by the presence of pyruvate or oxalate (Fig. 1a; Fig. S3), suggesting that these two molecules stabilize the tetrameric DNA-binding form of BhcR, analogous to the reported interaction of pyruvate with IclR (33).Interestingly, P. denitrificans was not capable of growth on oxalate as sole source of carbon and energy (Fig. S4), indicating that the observed in vitro interaction of BhcR with this compound might not be relevant in vivo.
Next, we generated a P. denitrificans ΔbhcR deletion strain and tested its growth on different carbon sources.In this strain, bhcR was replaced by a kanamycin resistance cassette in the same transcriptional direction.We also created a control strain, in which we inserted the kanamycin cassette in the opposite transcriptional direction to exclude polar effects.Both deletion strains were unable to grow on glycolate or glyoxylate (Fig. 2a and b), whereas growth rates on acetate or succinate were unchanged.Growth rates on pyruvate or glucose were very slightly, however significantly, decreased (Fig. 2c).Complementation of a ΔbhcR strain by expressing bhcR from a plasmid under the control of a medium-strength constitutive promoter recovered the ability to grow on glycolate or glyoxylate (Fig. S5).The inability of the bhcR deletion strains to grow on glycolate or glyoxylate, similar to a bhcABCD deletion strain (14), suggests that BhcR acts as an activator that is required for transcription of the bhc gene cluster.We sought to further investigate this hypothesis by generating P bhc promoter-based reporter strains with mCherry as reporter.We tested mCherry production in the ΔbhcR, ΔbhcABCD, and wild-type (WT) strain (Fig. 2d).When grown on succinate, only low fluorescence levels were observed in all three strains, indicating a basal expression of the bhc gene cluster.Supplementation of succinate medium with increasing concentrations of glyoxylate caused a gradual increase in fluorescence in the WT and ΔbhcABCD back grounds, suggesting an increase in P bhc promoter activity.Notably, in these experiments, promoter activity was positively correlated with the assumed intracellular concentration of glyoxylate.The ΔbhcABCD strain that cannot further convert glyoxylate (presumably cluster (P bhc ) was incubated with increasing amounts of purified BhcR protein (0x/400x/2,000x/4,000x/10,000x/20,000x/30,000x/40,000x molar excess) and subsequently separated by electrophoresis to visualize DNA bound to BhcR and free DNA; a 255-bp DNA fragment derived from the coding region of bhcA was used as a negative control.BhcR specifically forms a complex with the P bhc DNA fragment.Bottom, the P bhc -BhcR complex (40,000x molar excess BhcR) was incubated with increasing concentrations (0.1 mM; 0.5 mM; 5 mM) of glyoxylate, pyruvate, or oxalate, and subsequently separated by electrophoresis to assess the effect of these metabolites on complex formation.Increasing concentrations of glyoxylate decrease the binding of BhcR to the P bhc DNA fragment, whereas the opposite effect is observed for increasing concentrations of pyruvate or oxalate.(b) DNA binding of BhcR in the P bhc promoter region.Potential −35 and −10 regions upstream of the bhcR and bhcA genes were identified using BPROM (35).A potential palindromic binding site for BhcR was identified upstream of the −35 region of bhcA.resulting in higher intracellular glyoxylate levels) exhibited significantly higher expres sion from the P bhc promoter compared to the WT strain, in which glyoxylate is continu ously converted via the BHAC.In contrast to these two strains, expression from the P bhc promoter remained basal in the ΔbhcR background even in the presence of glyoxylate, supporting the role of BhcR as activator of the bhc gene cluster in vivo.
How can the in vivo function of BhcR as activator of P bhc be reconciled with the in vitro data that showed decreased DNA binding in the presence of glyoxylate?The intergenic region between the divergently transcribed genes bhcR and bhcA contains two predicted promoters (Fig. 1b).The most plausible hypothesis is that BhcR has a dual function: it might repress its own expression in the absence of glyoxylate by interacting with a binding site upstream of bhcR, but it might activate the expression of the bhc gene cluster in the presence of glyoxylate by interacting with a binding site upstream of bhcA.In the presence of glyoxylate, BhcR would dissociate from the putative binding site upstream of bhcR, resulting in increased BhcR production.Subsequently, increased titers of BhcR would bind to the binding site upstream of bhcA, so that the BhcABCD enzymes are produced.This assumed dual function would explain the decreased (but not abolished) in vitro DNA binding of BhcR in the presence of glyoxylate, especially if the affinity to the binding site upstream of bhcA would be rather low.Notably, such a dual role as activator and repressor was previously described for other IclR family regulators (36,37) and for the transcriptional regulator RamB, a member of the ScfR family, in P. denitrificans (32).

Pden_4400 encodes for GlcR, a novel repressor of the glycolate oxidase gene cluster
Next, we studied the regulation of glycolate oxidation in P. denitrificans.We hypothe sized that glycolate is converted into glyoxylate by the three-subunit enzyme glycolate oxidase (GlcDEF), encoded by the genes Pden_4397-99, and verified the role of this gene cluster by generating a Pden_4397-99 deletion strain, which was unable to grow on glycolate as sole carbon source (Fig. S7).The gene Pden_4400, adjacent to this gene cluster, is annotated as a transcriptional regulator of the GntR family.This resembles the situation in E. coli, where the GntR-family regulator GlcC serves as transcriptional activator of glcDEF (5,38).GlcC is part of the FadR subfamily of the GntR transcrip tion factor family (39).In a phylogenetic tree containing sequences of GlcC homologs, Pden_4400 homologs, and sequences from other clades within the FadR subfamily (283 sequences in total; Fig. S8), Pden_4400 and its close homologs form a well-defined clade that clusters together with the GlcC clade, as well as the PdhR (regulator of pyruvate dehydrogenase [40]) and LldR (regulator of lactate dehydrogenase [41,42]) clades.These four subfamilies share ca.35 conserved amino acid residues (Fig. S9), and the amino acid identity between E. coli GlcC and Pden_4400 is 32%.This suggests that Pden_4400 might fulfill a similar role as GlcC, but is not simply an alphaproteobacterial homolog of this transcriptional activator.We therefore designate Pden_4400 as glcR.
Homologs of glcR can be found adjacent to glcDEF in many Paracoccus strains, but also in other Rhodobacterales (e.g., Methylarcula, Puniceibacterium, Rhodobacter) as well as in some Rhizobiales (e.g., Afipia, Chenggangzhangella) (Table S1), suggesting that control of glycolate oxidase production via GlcR is conserved across different alphapro teobacterial clades.To study the interactions of GlcR with DNA and small molecule ligands in detail, we purified the transcriptional regulator and investigated its DNA-binding capabilities.In EMSAs, we could demonstrate specific binding of the protein to a DNA fragment containing the putative promoter region of the glc gene cluster (P glc ).DNA binding was decreased in the presence of glycolate, whereas glyoxylate did not alter the DNA binding of GlcR (Fig. 3a and b).We subsequently purified GlcR fused to an N-terminal maltose-binding protein (MGlcR) to increase its solubility for fluorescence polarization experiments.These experiments confirmed previous results with the non-tagged protein and allowed us to determine a K D for MGlcR of 225 ± 5 nM at 10 nM DNA.Titration of the P glc -MGlcR complex with increasing concentrations of glycolate demonstrated a notable decrease in binding, whereas the same effect was not observed for glyoxylate (Fig. 3c  and d).
Subsequently, we generated two P. denitrificans deletion strains of glcR and tested their growth on different carbon sources.As for bhcR, the glcR gene was replaced with a kanamycin resistance cassette in either the same or the opposite direction of transcrip tion to exclude any polar effects.Interestingly, the growth rate of the deletion strains on was incubated with increasing amounts of purified GlcR protein (0x/20x/100x/200x/500x/1,000x/1,500x/2,000x molar excess) and subsequently separated by electrophoresis to visualize DNA bound to GlcR and free DNA; a 156-bp DNA fragment derived from the coding region of glcD was used as a negative control.GlcR specifically forms a complex with the P glc DNA fragment.(b) The P glc -GlcR complex (1,000x molar excess of GlcR) was incubated with increasing concentrations of glycolate or glyoxylate (0.5 mM, 1 mM, 2 mM) and subsequently separated by electrophoresis to assess the effect of these metabolites on DNA:GlcR complex formation.Increasing concentrations of glycolate decrease the binding of GlcR to P glc , whereas increasing concentrations of glyoxylate did not result in altered DNA binding of GlcR.(c) Fluorescence polarization experiments with increasing concentrations of MGlcR and the P glc region (blue) or the tetO sequence as negative control (black).Three independent experiments were conducted for each combination, and a K D of 225 ± 5 nM was determined for MGlcR with 10 nM P glc .(d) Fluorescence polarization experiments with increasing concentrations of an effector (glycolate or glyoxylate; 0, 0.1, 1, 10, 100 mM) and 750 nM MGlcR and 10 nM P glc .These results confirm that glycolate causes decreased binding of GlcR to P glc .glycolate was not significantly different from the WT (Fig. 4a and c).However, the growth rates on glyoxylate, but also on succinate and acetate, were slightly decreased compared to the WT (Fig. 4b and c).Taken together, these data strongly suggest that GlcR-unlike GlcC -does not act as activator, but as repressor.In the glcR deletion strain, GlcDEF is constitutively produced, which explains the WT-like behavior of the deletion strain on glycolate, and the slightly decreased growth rate of the deletion strain on glyoxylate, succinate, and acetate due to increased protein production burden.
We independently confirmed the role of GlcR in P. denitrificans using P glc promoterbased reporter strains.We tested under which conditions mCherry was produced from a P glc fusion in the ΔglcR, ΔglcDEF, and WT background (Fig. 4d).When growing on succinate, fluorescence only increased in the ΔglcR background, but not in the other two strains.This increase is consistent with the finding that GlcR acts as repressor in vitro.When growing on succinate and different concentrations of glycolate, fluorescence also increased in the ΔglcDEF background, but only slightly in the WT.This might be explained by relatively low intracellular glycolate levels in the WT, as glycolate is further metabolized.In contrast, glycolate is expected to accumulate in the ΔglcDEF strain, which is incapable of converting glycolate further to glyoxylate due to the lack of glycolate oxidase, resulting in increased expression from P glc .Finally, with glycolate as sole carbon source, fluorescence also increased in the WT background (whereas the ΔglcDEF strain was unable to grow under these conditions).

Growth of P. denitrificans on two carbon substrates does not result in diauxie
Having characterized the regulatory circuits of glycolate oxidase and the BHAC at the molecular level, we aimed at studying glycolate and glyoxylate metabolism under more complex growth conditions at the cellular level.To that end, we grew P. denitrificans on glycolate (or glyoxylate) together with either glucose, a glycolytic carbon substrate, or pyruvate, a gluconeogenic carbon substrate, to determine the effect of substrate co-feeding on growth.
We first grew P. denitrificans either on a single carbon substrate or on two carbon substrates, mixed in three different ratios.Growth on glycolate (µ = 0.51 h −1 ) was faster than growth on glyoxylate (0.28 h −1 ), whereas the growth rates on pyruvate (µ = 0.45 h −1 ) and glucose (µ = 0.38 h −1 ) were between these two values.When growing on a mix of glycolate and glucose (Fig. 5a), the growth rate of P. denitrificans was not different from the growth rate on glycolate alone, whereas the growth rate of the bacterium was very similar to the growth rate on glucose alone when growing on a mix of glyoxylate and glucose (Fig. 5b).The same pattern was also observed when glucose was replaced with pyruvate (Fig. 5c and d).Notably, we did not observe any diauxic growth behavior (i.e., a first growth phase, an intermediate lag phase, and a second growth phase) on any of the tested carbon substrate mixtures.
Collectively, these data suggested that P. denitrificans does not assimilate the two carbon substrates sequentially, but rather in a co-utilizing manner.We therefore set out to investigate the regulation of central carbon metabolism and the uptake hierarchy of carbon substrates in P. denitrificans in more detail, with a special focus on glycolate and glyoxylate.

CceR regulates glycolysis and gluconeogenesis in P. denitrificans
To this end, we investigated the role of the transcription factor CceR (central carbon and energy metabolism regulator) in glycolate and glyoxylate metabolism of P. denitrificans.This protein was previously described as key regulator of carbon and energy metabolism in the Alphaproteobacterium Rhodobacter sphaeroides.CceR was also identified in P. denitrificans, where it was predicted to share largely the same regulon as in R. sphaeroides (43).Specifically, we aimed to determine whether CceR controls glycolate/glyoxylate assimilation pathways, uptake of these substrates into the cell, or both.We, therefore, generated two P. denitrificans ΔcceR strains, in which the gene was replaced with a kanamycin resistance cassette in either the same or the opposite direction of transcrip tion.Subsequently, we determined the growth rates of the ΔcceR and WT strains on 21 different carbon sources, including glycolate and glyoxylate (Fig. 6).Notably, ΔcceR strains had reduced growth rates on all gluconeogenic carbon sources, but not on the five glycolytic carbon sources.This partially contrasts the situation in R. sphaeroides, where the growth rates of ΔcceR were not significantly decreased on the gluconeo genic carbon sources acetate, tartrate, aspartate, and isoleucine (43), indicating few, but distinct, differences in the regulation of central carbon metabolism between both bacteria.We then analyzed the proteome of P. denitrificans WT and ΔcceR during growth on glyoxylate to identify the CceR regulon and its potential effects on C2 metabolism (Fig. 7).Notably, several key enzymes of gluconeogenesis were downregulated in the ΔcceR strain, including malic enzyme (MaeB) and PEP carboxykinase (PckA), as well as fructose 1,6-bisphosphate aldolase (Fba).In contrast, several glycolytic enzymes were upregulated in the ΔcceR strain, despite growing on a gluconeogenic carbon substrate.These included a gluconate transporter (GlnT) as well as gluconate kinase (GlnK) and glucokinase (Glk), glucose 6-phosphate isomerase (Pgi), phosphofructokinase (Pfk), and pyruvate kinase (Pyk), as well as three enzymes of the Embden-Meyerhof-Parnas pathway and four enzymes of the Entner-Doudoroff pathway (Zwf, Pgl, Edd, Eda), the main glycolytic route in Paracoccus versutus (44), a close relative of P. denitrificans.
Based on these results, we hypothesize that the decreased growth rate of the ΔcceR strain on gluconeogenic carbon substrates might be due to futile cycling, where glucose is first produced, but then catabolized again by glycolytic enzymes that are constitutively produced in this mutant.In contrast, growth of the ΔcceR strain on glycolytic carbon sources is not negatively affected, as high activity of the glycolytic pathways is required for efficient catabolism under these conditions.Furthermore, our proteomics data supported the conclusion that CceR acts as a repressor of glycolytic pathways and as an activator of gluconeogenic enzymes in P. denitrificans, analogous to the role of this regulator in R. sphaeroides (43).Although the CceR regulon of P. denitrificans is not fully identical to its counterpart in R. sphaeroides, there are still large overlaps (Table S2).Notably, key enzymes in energy metabolism (ATP synthase and NADH dehydrogenase) and the tricarboxylic acid (TCA) cycle (succinate dehydrogenase, 2-oxoglutarate dehydrogenase, fumarase) are part of the CceR regulon in R. sphaeroides, but not in P. denitrificans, suggesting that energy conservation and oxidation of acetyl-CoA to CO 2 are under the control of different regulatory mechanisms in the latter.
Finally, we investigated the substrate uptake hierarchy and substrate consumption rates of the WT and ΔcceR strains during growth on glycolytic and gluconeogenic carbon substrates.To this end, these strains were grown on glycolate, glucose, or mixtures thereof, and substrate uptake rates were quantified via liquid chromatography-mass spectrometry (LC-MS) measurements and luminescence-based assays, respectively.
On glycolate, the ΔcceR strain showed a slightly decreased growth rate, as observed before.When growing on glycolate and glucose, uptake of glycolate started and finished earlier than uptake of glucose (Fig. 8a and b).However, uptake of the two different carbon substrates largely overlapped.Once glycolate was fully consumed, we observed a slightly slower growth phase during which the remaining glucose was used up.This simultaneous uptake of glycolate and glucose was observed for both the WT and ΔcceR strains, and was independent of initial substrate concentrations.On glycolate and glucose as simultaneous growth substrates, the ΔcceR strain was growing similar to the WT strain, which could be explained by the fact that the constitutive activity of glycolytic enzymes was not futile anymore under these conditions.Yet, in all cases, the substrate consumption rates of both glycolate and/or glucose during exponential phase were not significantly changed compared to the WT (Fig. 8c and d).Overall, this data suggested that CceR controls the glycolysis-gluconeogenesis switch only at the level of the respective assimilation pathways, but not via changes in substrate uptake rate or hierarchy.

DISCUSSION
Glycolate and its downstream metabolite glyoxylate are abundant in the environment and are thus readily available carbon sources for heterotrophic microorganisms.Our work aimed at deciphering the regulation of glycolate and glyoxylate assimilation in P. denitrificans, an Alphaproteobacterium that relies on glycolate oxidase and the BHAC to funnel these C2 compounds into central carbon metabolism.We determined that BhcR, an IclR-type regulatory protein, controls the BHAC.BhcR is closely related to other glyoxylate-binding regulators and acts as an activator of the bhc gene cluster.Furthermore, we discovered that GlcR, a previously unknown member of the GntR family of transcriptional regulators, acts as a repressor to control the production of glycolate oxidase.We subsequently extended our investigation toward the regulation of central carbon metabolism in P. denitrificans and determined that different carbon substrates are assimilated largely simultaneously, and that the global regulator CceR controls the switch between glycolysis and gluconeogenesis.Taken together, this demonstrates that assimilation of glycolate is highly coordinated with central carbon metabolism in P. denitrificans to achieve optimal growth under fluctuating environmental conditions.When glycolate enters the cell, it de-represses the glcDEF gene cluster by interacting with GlcR.Glycolate oxidase converts glycolate into glyoxylate, which subsequently activates expression of bhcABCD via BhcR.The BHAC converts two glyoxylate molecules into oxaloacetate, which enables the biosynthesis of all biomass precursor molecules via the TCA cycle and gluconeogenesis.The global regulator CceR ensures that production of glycolytic enzymes is repressed during gluconeogenic growth.When a glycolytic carbon substrate, such as glucose, is present as well, it is assimilated concomitantly with glycolate.
Our work elucidates the multi-layered regulatory mechanisms that control the assimilation of glycolate and glyoxylate by P. denitrificans for the first time.This repre sents a significant advance in our understanding of the BHAC-mediated metabolism of these compounds, a process which, due to the omnipresence of glycolate-releasing phototrophs, is likely to impact the global carbon cycle (14).
The assimilation of multiple carbon substrates by bacteria has been studied since the seminal work of Monod in the 1940s (45,46).Bacteria can consume two nutrients either simultaneously or sequentially.Sequential consumption results in a growth curve with two consecutive exponential phases, referred to as diauxie.Both diauxie and simultane ous utilization of two carbon sources are common in microorganisms.The regulatory mechanism responsible for diauxie, known as catabolite repression, allows bacteria to selectively express enzymes for the preferred carbon source even when another one is present (47).The observed simultaneous assimilation of a glycolytic and a gluconeogenic carbon source by P. denitrificans can be rationalized based on the conserved topology of central carbon metabolism.When both types of carbon source are present, some precursor molecules for biomass (e.g., glucose 6-phosphate and ribose 5-phosphate) can be synthesized more efficiently from the glycolytic substrate, whereas other biomass precursors (e.g., oxaloacetate and 2-oxoglutarate) can be synthesized more efficiently from the gluconeogenic substrate.Therefore, it is advantageous for the bacterium to make use of both carbon sources simultaneously (48).Notably, a general growth-rate composition formula that was validated for the growth of E. coli on co-utilized glycolytic and gluconeogenic carbon substrates (49) does not seem to be valid for P. denitrificans (Table S3).This might be due to the fact that this formula only takes the regulatory effect of the cAMP-Crp system (50) on catabolic pathways into account.However, the cAMP-Crp system that controls the hierarchical use of different carbon sources is not present in P. denitrificans.Therefore, a specific growth-rate composition formula would have to be developed and validated separately for P. denitrificans and presumably for other Alphaproteobacteria, taking into account the differences in the global regulatory systems that result from the different lifestyles and ecological niches of these versatile microorganisms.
Future work should focus on further elucidating the regulatory nodes that control carbon metabolism in P. denitrificans and related Alphaproteobacteria, while placing these findings in an ecological context.It is as of yet unknown how these environmen tally abundant microorganisms prioritize the assimilation of the multitude of carbon substrates that are available in their natural habitats.Additional work on pure cultures of alphaproteobacterial model organisms should therefore be combined with experiments that investigate assimilation of multiple substrates by natural or synthetic microbial communities (including P. denitrificans).From an application perspective, it will be important to translate the newly gained knowledge about the transcription factors BhcR and GlcR into the development of robust biosensors for glyoxylate and glyco late, respectively.Established methods for the engineering of sensor modules with a reliable output and applicability for high-throughput screening methods are available (51).A biosensor for the rapid quantification of glycolate would be relevant not only to screen the flux from the CBB cycle into photorespiratory pathways under different conditions, but also to monitor the glycolate output of the crotonyl-CoA/ethylmalonyl-CoA/hydroxybutyryl-CoA (CETCH) cycle, a promising synthetic pathway for CO 2 fixation (52,53).
In summary, our results provide new insights into the regulation of carbon metabo lism in P. denitrificans and pave the way toward a systems-level understanding of the organism in the future, especially in concert with genome-scale metabolic models that are now available for this bacterium (54,55).

Chemicals and reagents
Unless otherwise stated, all chemicals and reagents were acquired from Sigma-Aldrich (St. Louis, MO, USA) and were of the highest purity available.Sodium glycolate was acquired from Alfa Aesar (Haverhill, MA, USA).

Strains, media, and cultivation conditions
All strains used in this study are listed in Table S4.E. coli DH5α (for genetic work), ST18 (56) (for plasmid conjugation to P. denitrificans), and BL21 AI (for protein production) were grown at 37°C in lysogeny broth (LB) (57), unless stated otherwise.
P. denitrificans DSM 413 (58) and its derivatives were grown at 30°C in LB or in mineral salt medium with TE3-Zn trace elements (59), supplemented with various carbon sources.To monitor growth, the optical density at 600 nm (OD 600 ) of culture samples was determined on a photospectrometer (Merck Chemicals GmbH, Darmstadt, Germany) or in Infinite 200 Pro plate reader systems (Tecan, Männedorf, Switzerland).

Vector construction
All plasmids used in this study are listed in Table S5.
To create a plasmid for heterologous overexpression of glcR in E. coli, this gene (Pden_4400) was cloned into the expression vector pET16b (Merck Chemicals).To this end, the respective gene was amplified from genomic DNA of P. denitrificans DSM 413 using the primers provided in Table S6.The resulting PCR product was digested with suitable restriction endonucleases (Thermo Fisher Scientific) as given in Table S6 and ligated into the expression vector pET16b that had been digested with the same enzymes to create a vector for heterologous production of GlcR.The gene encoding for BhcR (Pden_3922) had been cloned into pET16b previously (14).To heterologously produce MBP-GlcR, the glcR gene was codon-optimized using Geneious Prime (Biomat ters, Inc., Boston, MA, USA) and ordered from Twist Bioscience (South San Francisco, CA, USA), including terminal BsmBI endonuclease sites.This fragment was inserted into an expression vector (pMBP-sfgfp_dropout) encoding for an N-terminal maltose-binding protein gene (malE) by Golden Gate assembly with the BsmBI isoschizomer Esp3I.
To create constructs for gene deletion in P. denitrificans, the upstream and down stream flanking regions of the bhcR/glcR/cceR/glcDEF genes from P. denitrificans DSM 413 were cloned into the gene deletion vector pREDSIX (60).To this end, the flanking regions were amplified from genomic DNA of P. denitrificans DSM 413 with the primers given in Table S6.The resulting PCR products were used to perform Gibson assembly with the vector pREDSIX, which had been digested with MfeI.Subsequently, the resulting vector was digested with NdeI, and a kanamycin resistance cassette, which had been cut out of the vector pRGD-Kan (60) with NdeI, was ligated into the cut site to generate the final vectors for gene deletion.In each case, vectors with forward orientation and reverse orientation of the kanamycin resistance cassette were generated.
To create the promoter probe vector pTE714, the mCherry gene was amplified with the primers mCherry_fw and mCherry_rv using the vector pTE100-mCherry (61) as template.The PCR product was digested with NdeI and EcoRI and subsequently ligated into the backbone of pTE100 (61) (digested with AseI and MfeI), yielding the pTE714 plasmid.
To create reporter plasmids for P. denitrificans, the intergenic regions between bhcR/bhcA (Pden_3922/Pden_3921) and glcR/glcD (Pden_4400/4399), respectively, were cloned into the promoter probe vector pTE714.The respective regions were amplified from genomic DNA of P. denitrificans DSM413 with the primers provided in Table S6.The resulting PCR products were digested with suitable restriction endonucleases (Thermo Fisher Scientific, Waltham, MA, USA) as given in Table S6 and ligated into likewise digested pTE714.
To create the complementation vector pTE104-bhcR, the bhcR gene was amplified with the primers bhcR_104_fw and bhcR_104_rv using the vector pET16b-BhcR ( 14) as template.The PCR product was digested with XbaI and KpnI and subsequently ligated into the backbone of pTE104 (61) (digested with KpnI and SpeI), yielding the pTE104-bhcR plasmid.
Successful cloning of all desired constructs was verified by Sanger sequencing (Microsynth, Göttingen, Germany).

Production and purification of recombinant proteins
For heterologous overproduction of BhcR and GlcR, the corresponding plasmid encoding the respective protein was first transformed into chemically competent E. coli BL21 AI cells.The cells were then grown on LB agar plates containing 100 µg mL −1 ampicillin at 37°C overnight.A starter culture in selective LB medium was inoculated from a single colony on the next day and left to grow overnight at 37°C in a shaking incubator.The starter culture was used on the next day to inoculate an expression culture in selective TB medium in a 1:100 dilution.The expression culture was grown at 37°C in a shaking incubator to an OD 600 of 0.5 to 0.7, induced with 0.5 mM IPTG and 0.2% L-arabinose and subsequently grown overnight at 18°C in a shaking incubator.Cells were harvested at 6,000 × g for 15 min at 4°C, and cell pellets were stored at −20°C until purification.Cell pellets were resuspended in twice their volume of buffer A (BhcR: 100 mM KCl, 20 mM HEPES-KOH pH 7.5, 10 mM MgCl 2 , 4 mM β-mercaptoethanol, 5% glycerol and one tablet of SIGMAFAST protease inhibitor cocktail, EDTA-free per L; GlcR: 500 mM NaCl, 20 mM Tris pH 8.0, 15 mM imidazole, 1 mM β-mercaptoethanol, 5% glycerol and one tablet of SIGMAFAST protease inhibitor cocktail, EDTA-free per L).The cell suspension was treated with a Sonopuls GM200 sonicator (BANDELIN electronic GmbH & Co. KG, Berlin, Germany) at an amplitude of 50% to lyse the cells and subsequently centrifuged at 50,000 × g and 4°C for 1 h.The filtered supernatant (0.45 µm filter; Sarstedt, Nümbrecht, Germany) was loaded onto Protino Ni-NTA Agarose (Macherey-Nagel, Düren, Germany) in a gravity column, which had previously been equilibrated with five column volumes of buffer A. The column was washed with 20 column volumes of buffer A and five column volumes of 85% buffer A and 15% buffer B and the His-tagged protein was eluted with buffer B (buffer A with 500 mM imidazole).The eluate was desalted using PD-10 desalting columns (GE Healthcare, Chicago, IL, USA) and buffer C (BhcR: 100 mM KCl, 20 mM HEPES-KOH pH 7.5, 10 mM MgCl 2 , 5% glycerol and 1 mM DTT; GlcR: 100 mM NaCl, 20 mM Tris pH 8.0, 1 mM DTT, 5% glycerol).This was followed by purification on a size exclusion column (Superdex 200 pg, HiLoad 16/600; GE Healthcare, Chicago, IL, USA) connected to an ÄKTA Pure system (GE Healthcare, Chicago, IL, USA) using buffer C. 2 mL concentrated protein solution was injected, and flow was kept constant at 1 mL min −1 .Elution fractions containing pure protein were determined via SDS-PAGE analysis (62) on 12.5% gels.Purified proteins in buffer C were subsequently used for downstream experiments.
For heterologous overproduction of MBP-GlcR, the corresponding plasmid encoding for the respective protein was first transformed into chemically competent E. coli BL21 AI cells.The cells were then grown on LB agar plates containing 34 µg mL −1 chloram phenicol at 37°C overnight.A starter culture in selective LB medium was inoculated from a single colony on the next day and left to grow overnight at 37°C in a shaking incubator.The starter culture was used on the next day to inoculate an expression culture in selective TB medium with a starting OD 600 of 0.05.The expression culture was grown at 37°C in a shaking incubator to an OD 600 of 1.0, induced with 0.5 mM IPTG and 0.025% L-arabinose and subsequently grown overnight at 20°C in a shaking incubator.Cells were harvested at 4,000 × g for 20 min at 4°C, and cell pellets were stored at −70°C until purification.Cell pellets were resuspended in twice their volume of buffer A (50 mM HEPES pH 7.5, 500 mM KCl) with 5 mM MgCl 2 and DNase I (Roche, Basel, Switzerland).The cell suspension was treated with a Sonopuls GM200 sonicator (BANDELIN electronic GmbH & Co. KG, Berlin, Germany) at an amplitude of 50% in order to lyse the cells and subsequently centrifuged at 100,000 × g and 4°C for 45 min.The filtered supernatant (0.45-µm filter; Sarstedt, Nümbrecht, Germany) was loaded onto a Ni-NTA column (HisTrap HP 1 mL, Cytiva, Marlborough, MA, USA) using the fast protein liquid chromatography (FPLC) system (Äkta Start, Cytiva).The system had previously been equilibrated with buffer A + 25 mM imidazole.The column was washed with buffer A and 75 mM imidazole, and MBP-GlcR was eluted with buffer A + 500 mM imidazole.The eluate was desalted using a HiTrap desalting column (Sephadex G-25 resin, Cytiva) and protein elution buffer (25 mM Tris-HCl pH 7.4, 100 mM NaCl).

Genetic modification of P. denitrificans
Transfer of replicative plasmids into P. denitrificans was performed via conjugation using E. coli ST18 as donor strain according to previously described methods (14).Selection of conjugants was performed at 30°C on LB plates containing 0.5 µg mL −1 tetracycline.Successful transfer of plasmids into P. denitrificans was verified by colony PCR.
Transfer of gene deletion plasmids into P. denitrificans was performed in the same way.Selection of conjugants was performed at 30°C on LB agar plates containing 25 µg mL −1 kanamycin.The respective gene deletion was verified by colony PCR and DNA sequencing (Eurofins Genomics, Ebersberg, Germany), and the deletion strain was propagated in selective LB medium.In each case, the gene to be deleted was replaced by a kanamycin resistance cassette either in the same direction or the opposite direction to exclude polar effects.

High-throughput growth and fluorescence assays with P. denitrificans strains
Cultures of P. denitrificans DSM 413 WT and its derivatives were pre-grown at 30°C in LB medium containing 25 µg mL −1 kanamycin or 0.5 µg mL −1 tetracycline, when neces sary.Cells were harvested, washed once with minimal medium containing no carbon source, and used to inoculate growth cultures of 180 µL minimal medium containing an appropriate carbon source as well as 25 µg mL −1 kanamycin or 0.5 µg mL −1 tetracycline, when necessary.Growth and fluorescence in 96-well plates (Thermo Fisher Scientific, Waltham, MA, USA) were monitored at 30°C at 600 nm in a Tecan Infinite M200Pro plate reader (Tecan, Männedorf, Switzerland).Fluorescence of mCherry was measured at an emission wavelength of 610 nm after excitation at 575 nm.The resulting data were evaluated using GraphPad Prism 8.1.1.To determine whether differences in growth rate or substrate uptake rate are significant, unpaired t-tests with Welch's correction were used.

Whole-cell shotgun proteomics
To acquire the proteome of P. denitrificans WT and ΔcceR (OD 600 ~ 0.4) in minimal medium supplemented with 60 mM glyoxylate, four replicate cultures were grown for each strain.Main cultures were inoculated from precultures grown in the same medium in a 1:1,000 dilution.Cultures were harvested by centrifugation at 4,000 × g and 4°C for 15 min.Supernatant was discarded, and pellets were washed in 40 mL phosphate buffered saline (PBS; 137 mM NaCl, 2.77 mM KCl, 10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , pH 7.4).After washing, cell pellets were resuspended in 1 mL PBS, transferred into Eppendorf tubes, and repeatedly centrifuged.Cell pellets in Eppendorf tubes were snap-frozen in liquid nitrogen and were stored at −80°C until they were used for the preparation of samples for LC-MS analysis and label-free quantification.
For protein extraction, bacterial cell pellets were resuspended in 4% sodium dodecyl sulfate (SDS) and lysed by heating (95°C, 15 min) and sonication (Hielscher Ultrasonics GmbH, Teltow, Germany).Reduction was performed for 15 min at 90°C in the presence of 5 mM tris(2-carboxyethyl)phosphine followed by alkylation using 10 mM iodoacetamide at 25°C for 30 min.The protein concentration in each sample was determined using the BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer's instructions.Protein cleanup and tryptic digest were performed using the SP3 protocol as described previously (63) with minor modifications regarding protein digestion temperature and solid phase extraction of peptides.SP3 beads were obtained from GE Healthcare (Chicago, IL, USA).Trypsin (1 µg) (Promega, Fitchburg, MA, USA) was used to digest 50 µg of total solubilized protein from each sample.Tryptic digest was performed overnight at 30°C.Subsequently, all protein digests were desalted using C18 microspin columns (Harvard Apparatus, Holliston, MA, USA) according to the manufac turer's instructions.
LC-MS/MS analysis of protein digests was performed on a Q-Exactive Plus mass spectrometer connected to an electrospray ion source (Thermo Fisher Scientific, Waltham, MA, USA).Peptide separation was carried out using an Ultimate 3000 nanoLCsystem (Thermo Fisher Scientific, Waltham, MA, USA), equipped with an in-house packed C18 resin column (Magic C18 AQ 2.4 µm; Dr. Maisch, Ammerbuch-Entringen, Germany).The peptides were first loaded onto a C18 precolumn (preconcentration setup) and then eluted in backflush mode with a gradient from 94% solvent A (0.15% formic acid) and 6% solvent B (99.85% acetonitrile, 0.15% formic acid) to 25% solvent B over 87 min, continued with 25% to 35% of solvent B for an additional 33 min.The flow rate was set to 300 nL/min.The data acquisition mode for the initial label-free quantification (LFQ) study was set to obtain one high-resolution MS scan at a resolution of 60,000 (m/z 200) with scanning range from 375 to 1,500 m/z, followed by MS/MS scans of the 10 most intense ions.To increase the efficiency of MS/MS shots, the charged state screening modus was adjusted to exclude unassigned and singly charged ions.The dynamic exclusion duration was set to 30 s.The ion accumulation time was set to 50 ms (both MS and MS/MS).The automatic gain control (AGC) was set to 3 × 10 6 for MS survey scans and 1 × 10 5 for MS/MS scans.Label-free quantification was performed using Progenesis QI (version 2.0).MS raw files were imported into Progenesis, and the output data (MS/MS spectra) were exported in mgf format.MS/MS spectra were then searched using MASCOT (version 2.5) against a database of the predicted proteome from P. denitrificans downloaded from the UniProt database (www.uniprot.org;download date 26 January 2017), containing 386 common contaminant/background proteins that were manually added.The following search parameters were used: full tryptic specificity required (cleavage after lysine or arginine residues); two missed cleavages allowed; carbamidomethylation (C) set as a fixed modification; and oxidation (M) set as a variable modification.The mass tolerance was set to 10 ppm for precursor ions and 0.02 Da for fragment ions for high energycollision dissociation (HCD).Results from the database search were imported back to Progenesis, mapping peptide identifications to MS1 features.The peak heights of all MS1 features annotated with the same peptide sequence were summed, and protein abundance was calculated per LC-MS run.Next, the data obtained from Progenesis were evaluated using the SafeQuant R-package version 2.2.2 (64).

Electrophoretic mobility shift assays
Fluorescently labeled DNA fragments for EMSAs were generated by PCR from the genomic DNA of P. denitrificans DSM 413.For the Pbhc regulatory region, primers Pbhc_fw and Pbhc_rev-dye were used to generate a 238-bp fragment containing the putative Pbhc promoter.The primers bhcA_fw and bhcA_rev-dye were used to generate a 255-bp fragment containing a part of the bhcA gene as negative control.For the Pglc regulatory region, primers Pglc_fw and Pglc_rev-dye were used to generate a 156-bp fragment containing the putative Pglc promoter.The primers glcD_fw and glcD_rev-dye were used to generate a 156-bp fragment containing a part of the glcD gene as negative control.All respective reverse primers were 5′-labeled with the Dyomics 781 fluorescent dye (Microsynth AG, Balgach, Switzerland).Binding reactions were performed in buffer A (20 mM potassium phosphate pH 7.0, 1 mM DTT, 5 mM MgCl 2 , 50 mM KCl, 15 µg mL −1 bovine serum albumin, 50 µg mL −1 herring sperm DNA, 5% vol/vol glycerol, 0.1% Tween20) in a total volume of 20 µL.The respective DNA fragments (0.025 pM) were incubated with various amounts of the purified protein BhcR (0x/400x/2,000x/4,000x/ 10,000x/20,000x/30,000x/40,000x molar excess) or GlcR (0x/ 20x/100x/200x/500x/1,000x/ 1,500x/2,000x molar excess), and protein:DNA complexes were incubated with various concentrations of effector molecules as indicated in the respective figure legends.After incubation of the reaction mixtures at 37°C for 20 min, the samples were loaded onto a native 5% polyacrylamide gel and electrophoretically separated at 110 V for 60 min.BhcR/GlcR:DNA interactions were detected using an Odyssey FC Imaging System (LI-COR Biosciences, Lincoln, NE, USA).Band intensity on EMSA gels was quantified using FIJI, and the intensity of the top band was divided by the intensity of the bottom band to obtain the intensity ratio.

Substrate uptake experiments
Quantitative determination of glycolate in spent medium was performed using an LC-MS/MS.The chromatographic separation was performed on an Agilent Infinity II 1290 HPLC system using a Kinetex EVO C18 column (150 × 1.7 mm, 3-µm particle size, 100 Å pore size, Phenomenex) connected to a guard column of similar specificity (20 × 2.1 mm, 5-µm particle size, Phenomenex) at a constant flow rate of 0.1 mL/min with mobile phase A being 0.1% formic acid in water and phase B being 0.1% formic acid in methanol (Honeywell, Morristown, NJ, USA) at 25°C.
The injection volume was 1 µL.The mobile phase profile consisted of the following steps and linear gradients: 0-4 min constant at 0% B; 4-6 min from 0% to 100% B; 6-7 min constant at 100% B; 7-7.1 min from 100% to 0% B; 7.1-12 min constant at 0% B. An Agilent 6495 ion funnel mass spectrometer was used in negative mode with an electrospray ionization source and the following conditions: ESI spray voltage 2,000 V, nozzle voltage 500 V, sheath gas 400°C at 11 L/min, nebulizer pressure 50 psig, and drying gas 80°C at 16 L/min.The target compound was identified based on its mass transitions and retention time compared to standards.Chromatograms were integrated using MassHunter software (Agilent, Santa Clara, CA, USA).Absolute concentrations were calculated based on an external calibration curve prepared in fresh medium.Mass transitions, collision energies, cell accelerator voltages.and dwell times were optimized using chemically pure standards.Parameter settings for glycolate were as follows: quantifier 75→75; collision energy 0; qualifier 75→47; collision energy 6; dwell 20; cell accelerator voltage 5.
Glucose concentrations in spent medium were quantified using the Glucose-Glo Assay kit (Promega, Walldorf, Germany).Luminescence measurements of diluted medium samples were performed in white 384-well plates (Greiner BioOne, Kremsmün ster, Austria) in a Tecan Infinite M200Pro plate reader (Tecan, Männedorf, Switzerland) according to the instructions of the kit.

Phylogenetic analyses
Sequences of BhcR homologs and other transcriptional regulators of the IclR family were downloaded from the NCBI Protein database (https://www.ncbi.nlm.nih.gov/protein/) and were aligned using MUSCLE (65).A maximum likelihood phylogenetic tree of the aligned sequences was calculated with MEGA X (66) using the Le-Gascuel model (67) with 100 bootstraps.The resulting tree was visualized using iTOL (68).The phylogenetic tree for GlcR homologs and other transcriptional regulators of the FadR subfamily was generated in the same way.

Visualization and statistical analysis
Data were evaluated and visualized using GraphPad Prism 8.1.1., and results were compared using an unpaired t-test with Welch's correction in GraphPad Prism 8.1.1.

ACKNOWLEDGMENTS
We gratefully acknowledge the expert support of Peter Claus in performing small molecule mass spectrometry measurements and Jörg Kahnt in performing mass spectrometry measurements for proteomics.
This study was funded by the Max-Planck-Society (T.J.E.) and the German Research Foundation (E.B. and T.J.E.; SFB987 "Microbial diversity in environmental signal response").
L.S.V.B., E.B., and T.J.E.conceptualized the project and designed and supervised the experiments.L.S.V.B. performed genetic and biochemical experiments, growth assays, substrate uptake experiments, and phylogenetic analysis, and analyzed the data.L.H. performed electrophoretic mobility shift assays.K.K. generated and character ized promoter reporter strains.S.B. generated MBP-GlcR and performed fluorescence polarization assays.B.P. generated P. denitrificans gene deletion strains.N.P. performed

FIG 1
FIG 1 DNA-binding properties of BhcR.(a) Top, a fluorescently labeled 238-bp DNA fragment carrying the putative promoter region of the bhc gene

FIG 2
FIG 2 Characterization of P. denitrificans ΔbhcR.(a and b) Growth curves of wild-type P. denitrificans DSM 413 (gray) and bhcR deletion strains (orange + red) grown in the presence of 60 mM glycolate (a) or 60 mM glyoxylate (b).Deletion of bhcR is sufficient to abolish growth in the presence of these carbon sources.These experiments were repeated three times (Continued on next page)

FIG 2 (
FIG 2 (Continued)independently with similar results.(c) Growth rates (μ) of wild-type P. denitrificans DSM 413 (gray) and bhcR deletion strains (orange + red) grown in the presence of 60 mM acetate, 30 mM succinate, 40 mM pyruvate, or 20 mM glucose.The growth rates of the bhcR deletion strains were either not significantly changed or only slightly decreased on these substrates when compared to the wild-type (ns = not significant; * = significant change, P < 0.05).The results of n = 6 independent experiments are shown, and the black lines represent the mean.(d) Growth and fluorescence of promoter reporter strains ΔbhcR (orange), ΔbhcABCD (brown), and WT (gray) with pTE714-P bhc on different carbon sources.These experiments were repeated three times independently with similar results.Growth and fluorescence of negative control strains are shown in Fig.S6.

FIG 3
FIG 3 DNA-binding properties of GlcR.(a) A fluorescently labeled 156-bp DNA fragment carrying the putative promoter region of the glc gene cluster (P glc )

8 FIG 4
FIG 4 Characterization of P. denitrificans ΔglcR.(a and b) Growth curves of wild-type P. denitrificans DSM 413 (gray) and glcR deletion strains (light + dark blue) grown in the presence of 60 mM glycolate (a) or 60 mM glyoxylate (b).These experiments were repeated three times independently with similar results.(c) Growth rates (μ) of wild-type P. denitrificans DSM 413 (gray) and glcR deletion strains (light + dark blue) grown in the presence of 60 mM glycolate, 60 mM glyoxylate, 30 mM succinate, or 60 mM acetate.When compared to the wild-type, the growth rates of the glcR deletion strains were slightly decreased in the presence of glyoxylate, succinate, and acetate (ns = not significant; * = significant change, P < 0.05).The results of n = 6 independent experiments are shown, and the black lines represent the mean.(d) Growth and fluorescence of promoter reporter strains ΔglcR (light blue), ΔglcDEF (purple), and WT (gray) with pTE714-P glc on different carbon sources.These experiments were repeated three times independently with similar results.Growth and fluorescence of negative control strains are shown in Fig. S6.

FIG 6
FIG 6 Characterization of P. denitrificans ΔcceR.Growth rates (μ) of wild-type P. denitrificans DSM 413 (gray) and cceR deletion strains (light + dark green) grown in the presence of various carbon sources (final carbon concentration, 120 mM).When compared to the wild-type, the growth rates of the ΔcceR strains were significantly decreased in the presence of all substrates, except for the glycolytic carbon sources D-glucose, D-gluconate, D-sorbitol, glycerol, and D-glycerate (highlighted in red in the bottom row; ns = not significant; * = significant change, P < 0.05).The results of n ≥ 3 independent experiments are shown, and the black lines represent the mean.2-OG: 2-oxoglutarate.

FIG 7
FIG 7 Proteome analysis of P. denitrificans DSM 413 ΔcceR.(a) Analysis of the proteome of glyoxylate-grown ΔcceR compared to WT.All proteins that were quantified by at least three unique peptides are shown.The proteins in carbon metabolism that showed the strongest decrease or increase in abundance are marked in red or blue in the volcano plot, respectively.X-axis represents log 2 fold change of the groups' means, Y-axis indicates the -log 10 q value.(b) The log 2 fold change of these proteins, sorted by locus name (in brackets).(c) The role of these up-and downregulated proteins in the carbon metabolism of P. denitrificans DSM 413.Altered enzyme production levels in key metabolic routes, such as the Entner-Doudoroff pathway, the C3-C4 node, and the 2-methylcitrate cycle, demonstrate marked changes upon deletion of cceR.

FIG 8
FIG 8 Substrate uptake during growth of P. denitrificans DSM 413 WT and ΔcceR.The WT (a) and ΔcceR (b) strains were grown on glycolate, glucose, or mixtures of the two carbon sources.At seven time points during growth, glycolate concentrations (light blue) were determined via LC-MS, and glucose concentrations (orange) were determined via a luminescence-based assay.The results of n = 6 independent experiments are shown; the dots represent the mean, and the error bars represent the standard deviation.Biomass-specific substrate uptake rates were calculated for glycolate (c) and glucose (d).Empty bars denote the WT strain, striped bars denote the ΔcceR strain (ns = not significant).

TABLE 1
Ligand-binding residues of IclR family transcriptional regulators a (33,34nd-binding residues of E. coli IclR that were previously described(33,34) are compared to their counterparts in E. coli AllR and P. denitrificans BhcR.Numbering is based on the sequence of E. coli IclR.