Phylogenetic analysis

Three groups of alignments were selected, representing the three protein domains determined from the Uniprot cluster analysis. The protein sequence alignments obtained using T-Coffee software were used to determine the conserved residues traced by Evolutionary Trace Server software [http://mordred.bioc.cam.ac.uk/~jiye/evoltrace/evoltrace.html]. They were then manually edited in Jalview and analyzed in ProtTest software (Abascal et al. 2005) [http://darwin.uvigo.es/software/prottest_server.html] to identify which evolutionary model explained each alignment.

Phyml software [http://www.phylogeny.fr/version2_cgi/one_task.cgi?task_type=phyml] was then used for phylogenetic reconstruction using 1,000 bootstrap repeats as statistical support. Njplot software was used to visualize phylogenetic trees, and then, Tajima's test was used to determine which selection worked on the three groups of domains using MEGA 5.05 software (Kumar et al., 2008).

Results

Our sequencing strategy amplified two fragments of 2633 bp and 1590 bp, upstream of the 1,3-PD operon of Clostridium sp. IBUN 13A. The use of PHPH software verified the high quality of the sequences obtained, meaning that after the data depuration, 2,565 bp and 1,508 bp contigs, respectively, could be assembled without having any dissimilarity. Two ORFs were found in the assembled 2,565 bp sequence, called dhaS and dhaA according to Raynaud et al. (2003) 's description in C. butyricum and an ORF was found in the assembled 1,508 bp sequence.

The first ORF identified as dhaS had 1,218 bp. Potential -35 (5'-TTCATA-3') and -10 (5'-TAAAAT-3^ promoter sites were predicted to be upstream of the start codon. The GeneMark software did not produce a predicted RBS; this is why the TGGTGA sequence was proposed as a putative site in this work; however, this site has not been predicted in other Clostridium species. No sequence was identified for this gene that could form a typical rho-independent transcriptional terminator.

The dhaA gene start codon was located 9 bp from the dhaS gene stop codon and consisted of 1,056 bp. The software did not identify RBS; however, the AGGAAG sequence located 5 bp upstream from the start for this gene, is proposed as its RBS. We identified a possible hairpin (characteristic of a rho-independent transcriptional terminator). The obtained sequence was annotated in GenBank as an update of the 1,3-PD operon sequence of Clostridium sp. IBUN 13A (accession code DQ 901408.6).

Upstream of the dhaS gene, a third 1,059 bp ORF was found in the assembled 1,508 bp region that encodes a putative transcriptional regulator. It was named the dhaY gene because the regulator protein encoded by this ORF is not homologous to the DhaR protein described in K. pneumoniae and C. freundii (Sun et al. 2003). Potential -35 (5'-TTCATA-3') and -10 (5'-TTTTAT-3') promoter sites were located upstream of the start codon. GeneMark software did not predict an RBS; therefore, GGAATA sequence has been proposed as being a putative site in this work. However, this site has not been predicted in other Clostridium species. We identified no sequence for this gene, which could form a typical rho-independent transcriptional terminator. The sequence obtained was noted in the GenBank as a new Clostridium sp. IBUN 13A sequence (accession code JF827037).

The dhaY gene encodes a 353 amino acid residue-long protein (DhaY GenBank accession code AEH42432), the dhaS gene encodes a 405 amino acid residue-long protein (DhaS GenBank accession code ADP02232.1), and the dhaA gene encodes a 351 amino acid residue-long protein (DhaA GenBank accession code ACT78697.2). The cytoplasmic locations for the dhaY, dhaS and dhaA proteins were inferred in all applications.

The search for proteins homologous to the predicted dhaY, dhaS and dhaA proteins revealed the existence of domains belonging to two-component signal transduction systems with a modular design (Figure 1). Because we obtained no functional information from experimental data, proteins were annotated as suggested by Galperin (2006), based on their domain composition rather than any given group of protein sequence similarities.

Histidine kinase sensor (N-terminal region, first 179 residues), histidine kinase (amino acids 205 to 287), and HATPase domains (C-terminal region, last 110 residues) have been identified for the dhaS protein, thereby corroborating that this protein is a histidine protein kinase. PSI-BLAST software revealed a 55% similarity of the N-terminal region with the PocR superfamily. The predicted secondary structure did not show any pattern to classify this sensor as bi-functional or mono- functional according to criteria of Alves & Savageau (2003).

The response regulatory domain —REC— (N-terminal region, first 118 residues) and HTH_ AraC DNA binding domain (C-terminal region, amino acids 247 to 345) were identified for the dhaA protein (common in a two-component response regulator protein). The dhaY protein had hybrid dhaS and dhaA protein modular organization, and the PocR sensor domain (N-terminal region) as HTH_ AraC DNA binding domain (C-terminal region) were identified.

Because of the heterogeneity of the superfamilies identified (more than 70,000 reported sequences), UniProt cluster analysis was used to debug the alignments until protein sequences of the same pattern of motifs as the dhaY, dhaS and dhaA proteins were obtained. The 34 sequences homologous to the dhaY or dhaS proteins were classified within the PocR and histidine kinase superfamilies, and the 44 sequences homologous to the dhaY and dhaA proteins were classified within the REC superfamily and the HTH_AraC superfamily (Suppl. 2). The groups of sequences for each superfamily have been referred to as Hiskin, REC and HTH to facilitate the analysis.

The final population of sequences studied was the following: 15 consensus sequences for the histidine kinases, REC/25 consensus sequences, and

Sequences having the PocR domain were not multiply aligned due to the reduced number of homologous sequences found. However, dhaS Hiskin sensor and dhaY sensor domain similarity with the PocR protein was demonstrated by alignment; this lead to the identification of three highly conserved cysteine residues (Suppl. 3). The previous suggests that they likely make up the interior of the surface contacting the ligand (Anantharaman & Aravind 2005).

The HisKin dhaS catalytic domain (corresponding to a class I histidine protein kinase) is grouped within the group 8 HisKin subfamily; this according to the classification by Grebe & Stock (1999). Motifs common by these kinases were identified in the alignment (Figure 1-a).

According to ProtTest software, the WG and LG evolutionary models explained cluster alignments (Table 1). The three phylogenetic trees showed base nodes having significant bootstrap values (77⁰/o HTH, 56% REC and 100% Hiskin) and topology that was correlated to the structural analysis mentioned below (Figure 2).

Discussion

The first characterization study of genes involved in the anaerobic metabolism of glycerol revealed that the dha regulon organization is present in different microorganisms capable of using glycerol as a carbon source. Although heterogeneity has been shown in the organization of these genes, even within organisms belonging to the same genus, a global system of transcriptional regulation controlled by response regulator proteins has been proposed (Sun et al. 2003). The distinctive characteristic of Clostridia is their two-component system regulator organization; this is demonstrated in the C. butyricum 1,3-PD operon characterization (Raynaud et al. 2003, 2011). Characterizing the 1,3-PD operon in the Colombian strain Clostridium sp. IBUN 13A has indicated an organization similar to that of C. butyricum (Montoya 2008, Quilaguy et al. 2010).

It is likely that the genes sequenced in this study (called dhaS and dhaA) comprise an operon, due to the promoter region identified upstream of the first gene, the reduced distance separating them in the same DNA chain (only 9 bp), and the single rho-factor-independent transcriptional terminator downstream of both genes. This hypothesis must be confirmed by primer extension or 5'-RACE, which could identify a two-component system characterized by its structural genes organized in operons (Mascher et al. 2006). A two-component system may regulate the glycerol metabolism in the Colombian strain, inversely to K. pneumoniae or C. freundii (Sun et al. 2003). Clostridia genomes have shown that two-component signal transduction protein-encoding regions frequently occur (Cheung et al. 2005).

Because C. butyricum is the closest species to the Colombian strain Clostridium sp. IBUN 13A (Montoya et al. 2001, Jaimes et al. 2006), the similarity between their 1,3-PD operon regulator genes could suggest an ancestral event involving horizontal transfer of this genomic region. It has been proposed that two-component transcriptional regulation systems become diversified in prokaryotic organisms because of gene duplication (Hoch 2000).

The domains found in the deduced proteins exhibit the versatile modular organization pattern characteristic of signal transduction proteins. This property confers the organism that harbors them great adaptability (West & Stock 2001, Jung et al. 2012). Our phylogenetic analysis supported this evolutionary trend. Our data suggested a functional purifying selection model (Table 1), following the Lego principle; most domains could be easily recognized and associated with particular biochemical functions (Galperin 2004, 2006). There is currently no generalized classification for all response regulator proteins, because they vary widely in their sequence, membrane topology, composition, and domain arrangement (Mascher et al. 2006).

According to the predictions, the PocR domain binds to intracellular ligands, thereby inducing a conformational change that is then transmitted to a catalytic signal transduction domain, such as the HisKin domain (Anantharaman & Aravind 2005). It has been experimentally shown that PocR protein activity is regulated in vitro by 1,2-propanediol molecules (Rondon & Escalante 1996). In turn, this has led to the proposal that PocR should recognize simple hydrocarbon derivatives such as 1,2-propanediol or acetate, and, therefore, plays a role in the detection of the substrates required for microbial growth (Anantharaman & Aravind 2005).

Due to its importance in 1,3-PD operon transcriptional regulation, it is probable that dhaY and dhaS sense the presence of glycerol (ligand) as a substrate in the cytoplasm, and can activate the promoter transcription because of a two-component system of regulation of nutrient uptake and its metabolism that frequently occurs (Tetsch & Jung 2009). However, little information is available on complementary regulation between a two-component system, and a transcriptional regulator made up of homologous domains (Townsend et al. 2013), which may be the case with a dhaY protein and dhaS/A system. Future studies are required to experimentally determine whether different ligands such as glycerol, 1,2-propanediol or even 1,3-propanediol can generate a conformational change in the HisKin dhaS and dhaY sensor domains of the Colombian strain.

Evidence shows that the sensors could be coupled to transport proteins acting as co-sensors and be responsible for substrate translocation through the membrane (Tetsch & Jung 2009, Kobir et al. 2011). For that reason, further experimental assays must establish whether HisKin dhaS is coupled to the glycerol GlpF transport facilitator in Clostridium sp. IBUN 13A, because of its cytoplasmic location.

The study of the histidine kinase sensor and the response regulator effector domains should be highlighted in sequence analysis since catalytic and REC domains are highly conserved and, as a result, do not provide conclusive information (Mascher 2006). Nevertheless, the analysis of the catalytic domain in dhaS allowed us to predict the absence of the F-box. The H-box also revealed the substitution of the canonical histidine residue by lysine (K-216) (Figure 1-a residue 464); this has been previously identified in C. butyricum (Raynaud et al. 2003). Even though a histidine is not the phosphorylated residue in different bacterial protein homologues (Foussard et al. 2001), only two proteins were identified in our review in which histidine was substituted by arginine (slr1414_ Synechocystis sp. and mth1260_ Methanobacterium sp.).

The identification of this substitution in multiple alignments suggests that the lysine could be phosphorylated to transfer the phosphate group to the response regulator, making the histidine residue substitutable by another residue, this can be observed in the study of conserved traces (ETS) (Figure 1-a). The P and F residues surrounding the histidine residue seemed to have a greater conservation because, perhaps, they play an important role in phosphoryl group interaction with the phosphorylatable residue.

The thermodynamic differences in phosphoryl group transfers from phosphorylated lysine to aspartic residues from the response regulator should be studied. The phosphorylated histidine phosphoimidazole bond seemed to be chemically ideal for this reaction (West & Stock 2001). Trace analysis did not otherwise show glycine residues as being the most conserved in the G-boxes (Figure 1-a). As this motif plays an important role in phosphotransfer (Grebe & Stock 1999), perhaps arginine and aspartic acid residues could have a more direct interaction with adenine nucleotide phosphates.

Even though the residues located in the X-box were not as conserved as in the other boxes, because this motif plays a structural role (Grebe & Stock 1999), four trace residues were identified in the alignment within the group 8 HQ family (L-492, R-497, E-513, R-527) (Figure 1-a).

The dhaA protein was confirmed as being the two-component system response regulator because the characteristic REC and HTH domains were identified. Five conserved residues were identified in the REC domain: D-14, D-15 (located after the first β/a loop), D-62, K-114 and Y-111 (Figure 1-b). However, trace analysis and multiple alignments revealed another difference in the REC's domain: the most conserved residues were located in the protein's N-terminal region; this demonstrates the particularity of the proteins of Clostridium sp. IBUN 13A. The threonine residue located in the active region, important in propagating conformational change following HisKin phosphorylation (West & Stock 2001), was not observed (I-92). Also, the formation of four a-helices interleaved between β- sheets (Alves & Savageau 2003, Casino et al. 2010) was not observed due to the lack of β5 (Figure 1- b).

The conserved threonine hydroxyl group moves away from its position when proteins are phosphorylated; this allows the space left by such movement to become filled by a conserved aromatic residue (F/Y) moving from an exposed position to a buried position on the a4/β5/a5 surface, thereby inducing conformational change (Stock & Da Re 2000). Threonine substitution for isoleucine in the dhaA protein might not affect its functionality, despite the absence of a hydroxyl group. Consequently, these residues were not observed in the trace analysis, contrarily to the three aspartic residues and the lysine residue, without which signal transduction could be deleteriously affected.

There was no discrepancy in conserved trace residues in multiple araC type proteins in the dhaY and dhaA transcriptional regulator domains. The presence of the G-hydrophobic residue in the small-residue triad could be observed in the HTH DNA-binding domain (Aravind et al. 2005), located in the a4/a5 loop (G-349, F-350, S-351); comparable, to the canonical hydrophobic residues in a3 (L-334) and a5 (F-356) (Figure 1-c). DNA binding motifs, from the HTH domain, were organized to facilitate DNAprotein interface binding to a3 residues (recognition helix) and the presence of hydrophobic residues in a1 and a3, stabilizing the domain (Iyer & Aravind 2012). It was also confirmed that these domains presented a tetra-helicoidal conformation, characterized by an additional C-terminal helix (Aravind et al. 2005).

The phylogenetic analysis presented a defined topology for multiple genetic duplication events that were correlated to structural analysis. Such gene duplication-based distribution defines structural evolutionary domains for this highly promiscuous type of protein. The separation of the clade formed by the Colombian strain IBUN 13A and C. butyricum was observed in the group of Hiskin sequences (protein dhaS) (Figure 2), possibly due to the particular organization of residues from the conserved H-box motif in which canonical histidine had been substituted for the lysine residue. This substitution was not found in any other histidine kinase and suggested the probability of a constituted ancestral domain.

It is likely that the domains developed according to a strong purifying selection model (Table 1) suggest that current populations have precise defined functional niches for each domain without having polymorphism. This is intriguing because of the enormous number of current representatives for the groups.

Conclusion

Our study has reported the sequencing, identification and in silico characterization of 1,3-propanediol operon transcriptional regulators. The Clostridium sp. IBUN 13A strain dhaS and dhaA genes were identified, leading to the proposal of a two-component signal transduction system regulation mechanism as described previously for C. butyricum. However, the most important finding was the identification of a third gene named dhaY, which encodes a putative regulator protein. Annotating and studying these deduced protein domains has led to the description of their molecular evolution and to the elucidation of such signal transduction system physiological roles. According to these findings, we propose that the dhaY protein is implicated in the regulation of glycerol metabolism with the dhaS/dhaA two-component system. The identification of these three genes constitutes a first approach to overall glycerol metabolism regulation and will prompt genetic manipulation strategies to improve the fermentation process for Clostridium sp. 1,3-PD production.

Acknowledgments

This study was carried out within the '1,3-propanediol production from glycerol obtained during biodiesel production, using Colombian Clostridium sp. strains: research on the operon, fermentation parameters and its economic viability' project financed by COLCIENCIAS (Departamento Administrativo de Ciencia, Tecnología e Innovación) (project code 1101-1217848) and the Universidad Nacional de Colombia (project codes 20101007337 and 20101006947). We would like to thank Jason Garry (Instituto de Biotecnología, Universidad Nacional de Colombia) for translating the manuscript and José David Montoya for his contributions to improve this document.

Conflict of Interest

The authors declare that they have no conflict of interest regarding this publication. All the authors participated in the research and article preparation, and the final version has been approved by all of them.

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