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Analisis computacional de los reguladores transcripcionales del operon 1,3-propanodiol: indicios sobre la regulacion del metabolismo del glicerol en Clostridium sp.

Computational analysis of 1,3-propanediol operon transcriptional regulators: insights into clostridium sp. glycerol metabolism regulation.

Analise computacional dos reguladores transcricionais do operon do 1,3-Propanodiol: Panorama da regulacao do metabolismo do glicerol no Clostridium sp.


Biodiesel is a fatty acid methyl-ester obtained from renewable sources such as oil palm, soybean and sunflower seeds; it is one of the most promising alternatives to fossil fuels as it can be used in diesel motors and other combustion systems (Papanikolaou et al. 2008). Crude glycerine is the main subproduct produced during biodiesel synthesis; it can be used in anaerobic fermentation as a carbon source to produce 1,3-propanediol (1,3-PD) (Barbirato et al. 1998, Paulo da Silva et al. 2009, Ayoub & Abdullah 2012). This 1,3-PD is used in the textile industry, in the production of adhesives, lubricants, solvents, resins, antifreeze and pharmaceutical products (Zeng & Biebl 2002) and in the synthesis of polyesters, polyurethanes and especially polytrimethylene terephthalate (PTT), which is more elastic and has better tension recovery properties than other polymers (Nakamura & Whited 2003, Saxena et al. 2009).

Additionally, 1,3-PD is produced by chemical synthesis using two different courses: acrolein hydration or ethylene oxide hydroformylation (Paulo da Silva et al. 2009). However, for the last few years, research efforts have been focused on its biological production as it is a more environmentally, friendly alternative (Saxena et al. 2009, Yao & Shimizu 2013). Biotechnological 1,3-PD production from glycerol as the sole carbon source has been characterized in Clostridium butyricum, Klebsiella pneumoniae and Cytrobacter freundii (Barbirato et al. 1998, Saxena et al. 2009, Kubiak et al. 2012). C. butyricum is considered one of the best producers and is characterized because it does not require coenzyme B12 to be added during fermentation, making this species an ideal biological model for diol research and production (Papanikolaou et al. 2000, Gonzalez-Pajuelo et al. 2005).

The Colombian strain Clostridium sp. IBUN 13A is closely related to C. butyricum (Jaimes et al. 2006, Montoya et al. 2001), and it has shown similar 1,3-PD yields to those obtained by reference strains such as C. butyricum DSM 523 and C. butyricum DSM 2478 (Cardenas et al. 2006). The genes involved in 1,3-PD synthesis have been molecularly characterized recently, and the structure of the 1,3-PD operon for this strain has been determined (GenBank accession code DQ901408) (Montoya 2008, Quilaguy et al. 2010).

Due to the similarity with the C. butyricum VPI1718 operon, it has been postulated that the regulator genes of the 1,3-PD operon of Clostridium sp. strain IBUN 13A are located upstream of the dhaB1 gene as described for C. butyricum strain VPI1718 (Raynaud et al. 2003). The dhaS and dhaA genes are proposed to be involved in the C. butyricum operon transcriptional regulation by means of a two- component signal transduction system whose regulation could have a mechanism similar to the dhaR protein found in other 1,3-PD producing microorganisms (Sun et al. 2003).

This study referred to the in silico prediction of the modular structure of regulator proteins and their phylogenetic history from dhaS and dhaA sequences, and to a putative transcriptional regulator gene identified upstream of the 1,3-P.D operon genes of Clostridium sp. strain IBUN 13A, called dhaY in this research. Consequently, possible transcriptional regulation mechanisms could be proposed for a signal transduction system on the 1,3-PD operon.

Materials & Methods

Sequencing and sequence assembling

Clostridium sp. strain IBUN 13A, obtained from the strain bank of the Institute of Biotechnology of the Universidad Nacional de Colombia (Montoya et al. 2000), and C. butyricum strain, DSM 2478 (positive control), were used in this study. The strains were activated following the methodology described by Montoya et al. (2000). The microorganisms were grown in strict anaerobic conditions at 37 [grados]C in modified TGY medium (16g/L tryptone, 5 g/L glucose, 5g/L yeast extract, 5g/L NaCl, and 0.5g/L L-cysteine) supplemented with 0.05 mg/mL resazurin for chromosomal DNA extraction. Chromosomal DNA was extracted using the methodology described by Jaimes et al. (2006).

GenBank sequences of C. butyricum VPI 1718 (accession codes AY112989.1 and AY138581) were used for designing the primers (Suppl. 1) due to the similarity found between the 1,3-PD operon of Clostridium sp. strain IBUN 13A and C. butyricum VPI1718 (Montoya, 2008).

Amplification reactions were done in 100 [micro]L tubes using 25 [micro]L final volumes at the following concentrations: 0.25 [micro]M primers (synthesized by Integrated DNA Technologies, Inc), 0.07U/[micro]L Taq polymerase (GoTaq, Promega), 2 mM MgCl2, 200 [micro]M dNTPs, 1X buffer and 50 ng chromosomal DNA, and a multigene thermocycler was used (Labnet International, Inc). Amplification products were confirmed using conventional electrophoresis on 1.5% agarose gels with 0.5 X TBE.

Amplified fragments were sent for purification and sequencing to Macrogen Inc. Sequence quality was assessed using PHPH software (Togawa & Brigido 2003) [] and assembled using Cap3 software.

Gene annotation

GeneMark.hmm PROKARYOTIC software (Besemer & Borodovsky 2005) [http://opal.biology. cgi] was used to find open reading frames (ORF) and ribosome binding sites (RBS). Clostridium acetobutylicum was the model organism selected to make predictions. BPROM [ phtml?topic=bprom&group=programs&subgroup] software was used to predict -10 and -35 boxes from the promoter region.

The presence of rho-independent terminators was determined using the EMBOSS Palindrome application, and the parameters defined by Lesnik et al. (2001) for this kind of transcriptional terminator. The sequence predicted by Palindrome software was evaluated by OligoAnalyzer 3.0 to find its respective [DELTA]G value.

Sequence analysis

Using the PSI-BLAST tool from the prediction made by using GeneMark, we performed a search for homologous protein sequences in the SwissProt database; the search was restricted to bacterial taxa and 10-5 E- value. The process was iterated until convergence was obtained. A search was then made in UniProt with the domains identified in SMART [] and SCAN-PROSITE [] (Sigrist et al. 2013) for each protein, obtaining 100% cluster groupings.

The partial and redundant sequences obtained were debugged until we obtained a population of 35 protein clusters homologous with a domain present in the dhaS or dhaY proteins. Similarly, we obtained a population of 44 protein clusters that were homologous with a domain present in the dhaA and dhaY proteins (Suppl. 2). These sequences were classified according to the superfamily to which they belonged; C. butyricum AAM54726.1 and AAM54727.1 sequences were used as a reference.

Secondary structure was predicted using PSIPRED software []. PSORTB 5.0 [ html] software was used to determine possible dhaS, dhaA and dhaY protein cellular localization (Yu et al. 2010), and obtaining prediction for Gram-positive eubacteria proteins. TMpred [http://www.] and TopPred [] software were used to find hypothetical transmembrane regions. T-Coffee software was used for the multiple alignments, using default parameters and manual editing.

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 [ evoltrace/evoltrace.html]. They were then manually edited in Jalview and analyzed in ProtTest software (Abascal et al. 2005) [ software/prottest_server.html] to identify which evolutionary model explained each alignment.

Phyml software [ 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).


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 -0REC-0 (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 HTH_AraC/15 consensus sequences for response regulators. These sequences made up the analysis study population (Figure 1). Considering that most of the sequences selected came from the clusters, alignment included Hiskin/125, REC/566 and HTH/191 individual sequences for the respective groups of related domains.


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 (77% HTH, 56% REC and 100% Hiskin) and topology that was correlated to the structural analysis mentioned below (Figure 2).


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 [beta]/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 [beta]- sheets (Alves & Savageau 2003, Casino et al. 2010) was not observed due to the lack of [beta]5 (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 [alpha]4/[beta]5/[alpha]5 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 DNAbinding domain (Aravind et al. 2005), located in the [alpha]4/[alpha]5 loop (G-349, F-350, S-351); comparable, to the canonical hydrophobic residues in [alpha]3 (L-334) and [alpha]5 (F-356) (Figure 1-c). DNA binding motifs, from the HTH domain, were organized to facilitate DNA-protein interface binding to [alpha]3 residues (recognition helix) and the presence of hydrophobic residues in [alpha]1 and [alpha]3, 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.


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.

doi: 10.11144/Javeriana.SC20-1.capo


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, Tecnologia e Innovacion) (project code 1101-1217848) and the Universidad Nacional de Colombia (project codes 20101007337 and 20101006947). We would like to thank Jason Garry (Instituto de Biotecnologia, Universidad Nacional de Colombia) for translating the manuscript and Jose 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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Computational analysis of 1,3-propanediol operon transcriptional regulators: insights into Clostridium sp. glycerol metabolism regulation
Supplement 1. Designed primers for amplifying and sequencing
chromosomal upstream region of the Clostridium sp. IBUN 13A strain's
1,3-PD operon, using the thermocycler conditions according to each
primers pair. The figure below shows a map of the amplified
chromosomal region using the sequence of C. butyricum as template.
*D-Forward, R-Reverse

Primer            Sequence (5'-->3')          Template position (bp)
                                              (Acc Cod GenBank)

RGS1 -D*          GAGAAAGCAATAAATGCCTG        4857-4876 (AY 138581)
RGS2 -R           TTTGCACTTTGTAACTCACC        1440-1421(AY112989.1)
RGS5 -D           ACTTCCAATAATGTCACTGG        532-551 (AY 112989.1)
RGA3 -D           GCGGAATAGGCATATCAAAGG       1122-1142 (AY112989.1)
RGA4 -R           ATTTCTGTAGGCTGATGGTG        32-13 (DQ 901408.5)
                                                2359-2340 (AY 112989.1)
RGA6 -D           TGGAATTATTGCTCTTATCC        1719-1738 (AY 112989.1)
RGA7 -R           CTCCAATCACATTCTTACTTGG      354-333 (DQ 901408.5)
                                                2681-2660 (AY 112989.1)
RTGli1 D          GTTATATATGAGTATTCAATGGG     15-37 (AY 138581)
RTGli2 -R         AGCTGATTCTCCAAAACTTC        1645-1626 (AY 138581)
RGS1-RGS2:        1 X (94[degrees]C/3 min);   30 X (94[degrees]C/30 s,
RGS5-RGS2:        1 X (94[degrees]C/3 min);   30 X (94[degrees]C/30 s,
RGA3-RGA4:        1 X (94[degrees]C/5 min);   30 X (94[degrees]C/30 s,
RGA6-RGA7:        1 X (94[degrees]C/5 min);   30 X (94[degrees]C/30 s
RTGli1- RTGli2:   1 X (94[degrees]C/5 min);   30 X (94[degrees]C/30 s

Primer            Region

RGS1 -D*          Intergenic dhaK3-dhaS
RGS2 -R           dhaA
RGS5 -D           dhaS
RGA3 -D           dhaS
RGA4 -R           dhaA
RGA6 -D           dhaA
RGA7 -R           Intergenic dhaA-dhaB1
RTGli1 D          putative
                    regulator (dhaY)
RTGli2 -R         dhaD
RGS1-RGS2:        59[degrees]C/30 s,       1 X (72[degrees]C/3 min)
                    72[degrees]C/90 s);
RGS5-RGS2:        59[degrees]C/30 s,       1 X (72[degrees]C/3 min)
                    72[degrees]C/50 s);
RGA3-RGA4:        58[degrees]C/30 s,       1 X (72[degrees]C/3 min)
                    72[degrees]C/70 s);
RGA6-RGA7:        56[degrees]C/30 s,       1 X (72[degrees]C/3 min)
                    72[degrees]C/50 s);
RTGli1- RTGli2:   55[degrees]C/30 s,       1 X (72[degrees]C/3 min)
                    72[degrees]C/105 s);

Supplement 2. Selected sequences for multiple sequence alignment

Sequences have been organized according to domain homology.

* characterised proteins

([dagger]) proteins selected for MSA.

** Number of cluster means number of organisms identified with the
same consensus sequence (

Code         Name


Q05587.2     PocR
D2P159       PocR
B7NC30       Transcriptional regulator: propanediol utilization


O31516.1     YesM ([dagger])
C1HQL6       Histidine kinase ([dagger])
B0K3C6       Histidine kinase ([dagger])
P94513.1     LytS
Q4A009.1     LytS ([dagger])
Q4L8V3.      LytS
Q81JL2 *     LytS ([dagger])
Q814J0.1     LytS
Q82Z75 *     LytS ([dagger])
Q53705 *     LytS
Q8E7H4 *     LytS ([dagger])
P0AA94 *     YpdA ([dagger])
C3NWE4       Autolysin sensor kinase ([dagger])
A1UV04       Histidine kinase ([dagger])
A9R5L7       Sensor histidine kinase ([dagger])
Q0TNM2       Sensor histidine kinase ([dagger])
D1JYM2       Two--component system sensor ([dagger])
B5XP86       Sensor histidine kinase ([dagger])
B0U2X0       Two--component system sensor protein ([dagger])
P58363 *     ArcB
P0AE84 *     Sensor protein CpxA
A7WWQ7 *     Sensor protein kinase WalK
Q57QC4 *     Virulence sensor histidine kinase PhoQ
P0A4I6 *     Sensor protein CiaH
P0A5Z9 *     Sensor--type histidine kinase PrrB
P09384 *     CheA
B1QUC8       Two--component sensor histidine kinase
Q48768 *     CheA
P33639 *     Sensor protein PilS
A5I0M8       Sensor histidine kinase
C7T955       Sensor transduction histidine kinase
C2GVB6       Histidine kinase sensor


O31517.1     YesN ([dagger])
Q4L8V4.1|    LytR
P0AE68 *     CheY ([dagger])
P0AA18 *     OmpR ([dagger])
P0A9Q3       ArcA ([dagger])
Q2G2G2 *     Response regulator SaeR ([dagger])
B2SB45 *     Polar--differentiation RR DivK
P0A2D6 *     CheY
P60611 *     LytR
P0A4H8 *     Transcriptional regulatory CiaR
A4TIX9       Two--component regulatory system ([dagger])
C4KXK1       CheY ([dagger])
C3M734       CheY ([dagger])
Q81JL3       LytT ([dagger])
A9M2D6       Two--component system regulator ([dagger])
P95193 *     Transcriptional regulatory protein DevR ([dagger])
P0A4H6 *     CheY ([dagger])
Q06239 *     Regulatory protein VanR ([dagger])
A0Q7W8       Two--component response regulator ([dagger])
A3M2P4       Two--component regulatory activator OmpR ([dagger])
A5I5I        CheY ([dagger])
P44845 *     Nitrate/nitrite response regulator ([dagger])
B0RXW        Two--component system regulatory protein ([dagger])
P71403       CheY ([dagger])
B1QYY4       Two--component response regulator ([dagger])
A0PYZ2       Two--component response regulator ([dagger])
Q97KQ2       Two--component response regulator ([dagger])
P29369       Glycerol metabolism activator AgmR ([dagger])
P0ACZ6 *     Positive transcription regulator EvgA ([dagger])
B1BNB8       DNA--binding response regulator


A4WG91.1     Transcriptional activator RhaS
B5FPP5.1     Transcriptional activator RhaS
P07642.1 *   AraC ([dagger])
P0A9E1.1 *   AraC ([dagger])
P0A9E4 *     Regulatory protein SoxS ([dagger])
P63202 *     Transcriptional regulator GadW ([dagger])
Q56143 *     Regulatory protein SoxS ([dagger])
Q1C0W2 *     Transcriptional activator RhaR ([dagger])
P68913 *     Transcriptional regulator Rv1395 ([dagger])
B5QWY5 *     RhaR ([dagger])
P45008 *     Transcriptional regulator HI1052 ([dagger])
Q48413 *     Transcriptional activator RamA ([dagger])
P0A2S7 *     Transcriptional regulator MxiE ([dagger])
P72171 *     Ornithine utilization regulator ([dagger])

Code         Microorganism                  Cluster **


Q05587.2     Salmonella typhimurium         8
D2P159       Listeria monocytogenes         7
B7NC30       Escherichia coli               4


O31516.1     Bacillus subtilis              --
C1HQL6       Escherichia coli               4
B0K3C6       Thermoanaerobacter sp          4
P94513.1     Bacillus Subtilis              --
Q4A009.1     Staphylococcus saprophyticus   --
Q4L8V3.      Staphylococcus haemolyticus    --
Q81JL2 *     Bacillus anthracis             9
Q814J0.1     Bacillus cereus                --
Q82Z75 *     Enterococcus faecalis          4
Q53705 *     Staphylococcus aureus          36
Q8E7H4 *     Streptococcus agalactiae       7
P0AA94 *     Escherichia coli               43
C3NWE4       Vibrio cholerae                11
A1UV04       Burkholderia mallei            7
A9R5L7       Yersinia pestis                18
Q0TNM2       Clostridium perfringens        6
D1JYM2       Bacteroides sp.                4
B5XP86       Klebsiella sp.                 4
B0U2X0       Xylella fastidiosa             3
P58363 *     Escherichia coli               13
P0AE84 *     Escherichia coli               53
A7WWQ7 *     Staphylococcus aureus          38
Q57QC4 *     Salmonella enterica            25
P0A4I6 *     Streptococcus pneumoniae       22
P0A5Z9 *     Mycobacterium tuberculosis     16
P09384 *     Salmonella typhimurium         8
B1QUC8       Clostridium butyricum          2
Q48768 *     Listeria monocytogenes         6
P33639 *     Pseudomonas aeruginosa         5
A5I0M8       Clostridium botulinum          4
C7T955       Lactobacillus rhamnosus        4
C2GVB6       Bifidobacterium longum         4


O31517.1     Bacillus subtilis              --
Q4L8V4.1|    Staphylococcus haemolyticus    --
P0AE68 *     Escherichia coli               49
P0AA18 *     Escherichia coli               100
P0A9Q3       Escherichia coli               66
Q2G2G2 *     Staphylococcus aureus          57
B2SB45 *     Brucella abortus               37
P0A2D6 *     Salmonella typhimurium         32
P60611 *     Staphylococcus aureus          30
P0A4H8 *     Streptococcus pneumoniae       27
A4TIX9       Yersinia pestis                25
C4KXK1       Burkholderia pseudomallei      25
C3M734       Vibrio cholerae                24
Q81JL3       Bacillus anthracis             23
A9M2D6       Neisseria sp.                  20
P95193 *     Mycobacterium tuberculosis     19
P0A4H6 *     Listeria monocytogenes         16
Q06239 *     Enterococcus faecalis          13
A0Q7W8       Francisella tularensis         12
A3M2P4       Acinetobacter sp.              11
A5I5I        Clostridium botulinum          10
P44845 *     Haemophilus influenzae         9
B0RXW        Xanthomonas sp.                9
P71403       Helicobacter pylori            8
B1QYY4       Clostridium butyricum          2
A0PYZ2       Clostridium novyi              --
Q97KQ2       Clostridium acetobutylicum     --
P29369       Pseudomonas aeruginosa         5
P0ACZ6 *     Escherichia coli               60
B1BNB8       Clostridium perfringens        5


A4WG91.1     Enterobacter sp.               --
B5FPP5.1     Salmonella enterica            --
P07642.1 *   Erwinia chrysanthemi           --
P0A9E1.1 *   Escherichia coli               --
P0A9E4 *     Escherichia coli               60
P63202 *     Escherichia coli               28
Q56143 *     Salmonella typhimurium         27
Q1C0W2 *     Yersinia pestis                21
P68913 *     Mycobacterium tuberculosis     16
B5QWY5 *     Salmonella sp.                 13
P45008 *     Haemophilus influenzae         8
Q48413 *     Klebsiella pneumonia           7
P0A2S7 *     Shigella sp.                   5
P72171 *     Pseudomonas aeruginosa         4

Supplement 3. Comparison of DhaS and DhaY secondary structure using Psi-Pred with MSA obtained by Anantharaman and Aravind (2005). A. Prediction of secondary structure and consensus sequence made for PocR by Anantharaman and Aravind (2005), the script E represents prediction of [beta]-sheet and H for [alpha]-helix. B. DhaS and DhaY protein's sequences and secondary stricture, in green is shown the conserved G residue and in red boxes are shown the three highly conserved cysteine residues.


Carlos Eduardo Barragan (1, [mail]), Andres Julian Gutierrez-Escobar (2), Dolly Montoya Castano (1)

Edited by Alberto Acosta ([mail])

(1.) Grupo de Bioprocesos y Bioprospeccion, Instituto de Biotecnologia, Universidad Nacional de Colombia, Bogota, Colombia

(2) Grupo de Investigaciones Biomedicas y de Genetica Humana Aplicada, Universidad de Ciencias Aplicadas y Ambientales--UDCA, Bogota, Colombia

Received: 16-06-2014 Accepted: 10-07-2014

Published on line: 26-09-2014

Funding: COLCIENCIAS; Universidad Nacional de Colombia.

Electronic supplementary material: Suppl 1,2,3.
Table 1. Natural Selection Test for HTH, REC and
Hiskin clusters. MEGA5.10 software was used to
estimate Tajima's test. All positions with gaps and
missing data were eliminated from the dataset (complete
deletion option). The abbreviations used were as follows:
m = number of sites, S = number of segregating sites,
Ps = S/m, T = Ps /a1 and [THETA] = nucleotide diversity. D
is Tajima's test score.

Cluster   m     S       Ps         T       [THETA]       D

HTH       14   101   0.980583   0.308346   0.732956   6.144532
REC       26   106   0.972477   0.254845   0.721581   7.174758
HISKIN    15   246   0.964706   0.296690   0.731877   6.498782
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Author:Barragan, Carlos Eduardo; Gutierrez-Escobar, Andres Julian; Montoya Castano, Dolly
Publication:Revista Universitas Scientarum
Date:Jan 1, 2015
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