Study of duplicated galU genes in Rhodococcus jostii and a putative new metabolic node for glucosamine-1P in rhodococci
Introduction
A key feature of some organisms from the Rhodococcus genus is their ability to accumulate lipids, which is a property that can be exploited to engineer these organisms to become microbial factories for biofuel production [1,2]. During cultivation under nitrogen limitations and with gluconate as the sole carbon source, the oleaginous bacterium Rhodococcus jostii accumulates up to 70% of its dry cell weight as lipids [3]. In contrast, R. fascians does not show oleaginous behavior under the same conditions [4,5], except when grown in glycerol as a carbon supplier [1]. In addition to triacylglycerides, members of this bacterial genus can accumulate other storage compounds, such as glycogen or polyphosphates [3,6]. It has been hypothesized that glycogen functions as an intermediary molecule for temporal carbon allocation since the glucan is accumulated during exponential growth phase and later mobilized for lipid synthesis [3,6].
Recently, the biochemical characterization of the ADP-glucose (ADP-Glc) pyrophosphorylase (EC 2.7.7.27, ADP-GlcPPase) from R. jostii and R. fascians provided evidence for the above hypothesis from a kinetic and regulatory point of view [[7], [8], [9]]. This allosteric enzyme catalyzes the limiting step in glycogen synthesis by activating glucose-1P (Glc-1P) to form ADP-Glc, the specific glucosyl donor to the α-1,4-glucan chain elongation [10,11]. Indeed, the rhodococcal ADP-GlcPPase responds by allosteric regulation to specific intracellular signals of carbon availability, directing Glc-1P to glucan synthesis, via ADP-Glc. Thus, Glc-1P is the precursor for the temporal carbon allocation “metabolite” glycogen in these organisms.
The metabolic destiny of Glc-1P is dictated by actions of several NDP-GlcPPases, including ADP-GlcPPase and UDP-GlcPPase (EC 2.7.7.9, GalU) to produce ADP-Glc and UDP-Glc, respectively. The former is the specific substrate for glycogen elongation in bacteria [10,11] and in some Actinobacteria is related to trehalose synthesis [12,13]. UDP-Glc is generally ascribed to many metabolic fates, such as lipopolysaccharides [14,15], structural capsule/membrane oligosaccharides [16], or intracellular oligo- [17] or poly-saccharides [18]. Studying Glc-1P partitioning in rhodococci is critical to better understand carbon management for glycogen synthesis, carbohydrate metabolism, and other metabolic fates. Therefore, we performed a comparative study of two rhodococci with different metabolic behavior and focused on their UDP-GlcPPases, which are encoded by the gene galU. Interestingly, genomic analysis of rhodococci shows two galU genes (galU1 and galU2) are present in R. jostii [19], whereas R. fascians has only one galU gene (BioProject ID 286803).
Metabolic gene redundancy constitutes the foundation for rhodoccocal catabolic versatility, its functional robustness, and its adaptation to diverse environments, thereby providing enhanced competition capabilities [19,20]. Indeed, some examples of functional redundancy found in Rhodococcus spp. have been linked to mechanisms related to niche adaptation, especially aromatic compound degradative pathways [21]. The genetic redundancy in rhodococci may have originated either from gene duplication or horizontal gene transfer events [21]. Duplicated genes typically evolve to encode (iso)enzymes with different substrate preferences and/or expression profiles. Moreover, some duplications present functional redundancy if they perform the same cellular function or have overlapping substrate ranges [21]. These types of duplications are usually relatively recent or were under low evolutionary pressure. Nevertheless, gene duplication together with protein promiscuity is critical for offering a source of diversity and metabolic innovation in prokaryotes [22,23].
Given the plethora of functions derived from UDP-Glc synthesis, it is imperative to comprehend how UDP-GlcPPase gene duplications are distributed and affect metabolism in a variety of bacteria. Indeed, UDP-GlcPPase duplication and divergence has been briefly described in other organisms. For example, in some enterobacterial species a gene duplication of galU occurred and gave rise to an enzymatically nonfunctional galF gene. The proteins encoded by these genes have diverged and while GalF has lost enzymatic activity [24], it still interacts with GalU [25]. Additionally, in most cyanobacterial species a gene encoding a putative GDP-mannose PPase (EC 2.2.7.13) produces a protein with UDP-GlcPPase activity (named CugP). In some cyanobacteria, CugP occurs simultaneously with GalU proteins with redundant UDP-GlcPPase activities; however, the metabolic impact of such duplication was not analyzed [26]. A recent report presented the molecular cloning of duplicated galU genes in R. opacus [27], but the authors failed to obtain a soluble form of the recombinant GalU1 enzyme and thus it could not be further characterized. Herein we tackle this issue and report the comparative kinetic study of both GalU1 and GalU2 proteins encoded by the respective galU genes from R. jostii. We also characterized the single GalU enzyme present in the related bacterium R. fascians. Our results from the characterization of substrate utilization of rhodococcal GalUs also led us to characterize the critical bifunctional enzyme in amino sugar metabolism, GlmU [28], from R. jostii. Together, the results presented in this work with those recently obtained from studies on rhodococcal ADP-GlcPPases [[7], [8], [9]], strongly support an additional metabolic node for carbon allocation at the level of glucosamine-1P (GlcN-1P), similar to the node for Glc-1P.
Section snippets
Chemicals
Protein standards, antibiotics, IPTG, Glc-1P, GlcN-1P, galactose-1P (Gal-1P), N-acetylglucosamine-1P (GlcNAc-1P), mannose-1P (Man-1P), ATP, UTP, GTP, CTP, and oligonucleotides were from Sigma-Aldrich (Saint Louis, MO, USA). All other reagents were of the highest quality available.
Bacteria and plasmids
Escherichia coli Top 10 F′ cells (Invitrogen) and pGEM®-T Easy vector (Promega, ampicillin resistance) were used for cloning procedures. The galU gene from R. fascians (RfagalU) and glmU from R. jostii (RjoglmU) were
Recombinant expression and structural characterization
From the genomic analysis of R. jostii RHA1 [19], we identified two galU genes, RjogalU1 (903 bp) and RjogalU2 (921 bp). These genes encode putative UDP-GlcPPases, namely RjoGalU1 and RjoGalU2, with theoretical molecular masses of 31.75 kDa and 32.68 kDa, respectively. UDP-GlcPPases catalyze the reaction to transform Glc-1P and UTP to UDP-Glc and pyrophosphate. RjoGalU1 and RjoGalU2 sequences are 78% identical and share 95.7% and 98.4% identity compared tothe GalU1 and GalU2 enzymes recently
Kinetic/structural considerations
Nucleotide sugars are donors of carbohydrate moieties in the synthesis of many molecules, including oligo and polysaccharides, glycosylated secondary metabolites (i.e., antibiotics), and glycoproteins and glycolipids [49,50]. In bacteria, about 70 different nucleotide sugars have been identified. Almost all organisms reportedly produce UDP-Glc [51], but ADP–Glc is limited to bacteria, green algae, and plants [10,11]. Both are built from Glc-1P by the respective NDP-GlcPPases, depending upon the
Conclusion
The results of this study provide new insight into GlcN-1P metabolism and may impact the field at different levels. First, the rhodococcal GalU2-type enzyme constitutes a new enzymatic tool for producing a new molecule (UDP-GlcN) that would be suitable as a substrate for (amino)glucosyl transferases [85,86]. This widens the possibility of obtaining new glycosylated molecules, which could improve tools for glycorandomization [[58], [59], [60], [61]]. Secondly, this newly identified activity may
Author contributions
AEC, MAH and MDA performed the experiments. AEC, MDA, AAI, MAB and HMA conceived the study and designed the experiments. AEC, MLK, AAI, HMA and MDA refined the kinetic models and analyzed the data. AEC, MLK, AAI, HMA and MDA wrote the paper. All authors revised and approved the submitted manuscript.
Declaration of Competing Interest
The authors declare no conflict of interest.
Acknowledgements
This work was supported by grants from ANPCyT (PICT’17 1515 to AAI and PICT’15 0634 to MDAD) and CONICET (PIP2015-2016 0529, PUE 2018-033 to HMA and PUE 2016-0040 to IAL). AEC is a Fellow from CONICET. HMA, MAH, AAI and MDAD are Career Investigator members from the same Institution.
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