Distinct co-evolution patterns of genes associated to DNA polymerase III DnaE and PolC

  • Stefan Engelen1, 2,

    Affiliated with

    • David Vallenet2,

      Affiliated with

      • Claudine Médigue2 and

        Affiliated with

        • Antoine Danchin1, 3Email author

          Affiliated with

          BMC Genomics201213:69

          DOI: 10.1186/1471-2164-13-69

          Received: 20 June 2011

          Accepted: 14 February 2012

          Published: 14 February 2012

          Abstract

          Background

          Bacterial genomes displaying a strong bias between the leading and the lagging strand of DNA replication encode two DNA polymerases III, DnaE and PolC, rather than a single one. Replication is a highly unsymmetrical process, and the presence of two polymerases is therefore not unexpected. Using comparative genomics, we explored whether other processes have evolved in parallel with each polymerase.

          Results

          Extending previous in silico heuristics for the analysis of gene co-evolution, we analyzed the function of genes clustering with dnaE and polC. Clusters were highly informative. DnaE co-evolves with the ribosome, the transcription machinery, the core of intermediary metabolism enzymes. It is also connected to the energy-saving enzyme necessary for RNA degradation, polynucleotide phosphorylase. Most of the proteins of this co-evolving set belong to the persistent set in bacterial proteomes, that is fairly ubiquitously distributed. In contrast, PolC co-evolves with RNA degradation enzymes that are present only in the A+T-rich Firmicutes clade, suggesting at least two origins for the degradosome.

          Conclusion

          DNA replication involves two machineries, DnaE and PolC. DnaE co-evolves with the core functions of bacterial life. In contrast PolC co-evolves with a set of RNA degradation enzymes that does not derive from the degradosome identified in gamma-Proteobacteria. This suggests that at least two independent RNA degradation pathways existed in the progenote community at the end of the RNA genome world.

          Keywords

          replication degradosome LUCA phylogenetic profiling nanoRNase

          Background

          Future developments of Synthetic Biology require that patterns of gene organization in genomes are carefully taken into account [1]. Following the pioneering work of Sueoka [2], Lobry and co-workers identified a replication-linked bias in the nucleotide distribution in bacterial chromosomes. Subsequently, a bias in favor of genes transcribed in the same direction as that of the movement of the replication fork was observed in most bacterial genomes [3, 4]. The bias was correlated with the presence in the genome of a single origin of replication. Taken together, these observations led to the construction of algorithms meant to identify in silico the origins of replication [5]. The cause of the bias has been a matter of speculation until it was observed that Firmicutes displayed the strongest bias [6, 7] reaching 87% in organisms such as Thermoanaerobacter tengcongensis [8]. A first hypothesis proposed that the bias was favoring genes requiring high expression [9]. Yet, there was no correlation between gene expressivity and transcription from the leading strand. Indeed, many genes of the replications machinery are expressed at a low level, and they are transcribed from the leading strand [10]. The leading strand bias had therefore to be accounted for by physical constraints: transcription of key genes must avoid head-on collision with the replication machinery to prevent formation of truncated transcripts. The latter are known to be toxic for the cell, in particular when they code for polypeptides belonging to protein complexes (see [11] for a general description of the process).

          A further observation noticed that organisms that were strongly biased in the leading vs lagging strand replication encoded two DNA polymerases III, DnaE and PolC, rather than a single one [12]. DnaE was originally identified in Escherichia coli [13] whereas PolC was identified in Bacillus subtilis [14]. Yet, in contrast with the situation in Eukarya where the presence of two polymerases is the norm, most bacterial species listed in genome reference databases code for only one DNA polymerase III. In E. coli, the same structural type of DnaE replicase acts on both the leading and lagging strand. Two identical replicase molecules are held together in a complex with the replicative helicase and subunits with priming activities, allowing two identical alpha catalytic subunits to assume different functions on the two strands of the replication fork [15]. In contrast, in B. subtilis, asymmetric DNA synthesis requires replicative DNA polymerases with two distinct structures, DnaE and PolC. In contrast to PolC, DnaE, which replicates the lagging strand, is devoid of 3' --> 5'-proofreading exonuclease activity and has a low processivity (1-75 nucleotides), requiring additional factors to fulfill its role in replication [16].

          DnaE and PolC differ both in structure and activity [15]. This prompted us to explore whether their genes co-evolved with consistent groups of genes, allowing us to propose scenarios of the origins of the replication machineries. In particular the cell manages compartmentalization via the cell's envelope, appendages, but also nanomachines such as the ribosome, ATP synthase, the degradosome and many others [17]. We present here a phylogenetic profile analysis focused on the bacterial dnaE and polC genes and show that proteins co-evolving with PolC have distinct features, and may form a specific kind of degradosome. The consequences in terms of the origin of bacteria are discussed.

          Methods

          To separate between the history of DnaE and that of PolC, we established a heuristics meant to identify genes that co-evolved in bacterial genomes. The approach is straightforward: we first identify orthologous genes by pairwise comparison to compute phylogenetic profiles; we subsequently compare them after statistical validation, taking into account the phylogenetic distances between organisms with co-occurring genes; finally, we combine phylogenetic profiling with other methods that take into account the genomic context.

          Pairwise gene comparison and phylogenetic profiles

          Phylogenetic profiling uses binary vectors that, taking genes one by one, identify in which organisms an ortholog is present (resp. absent). To this aim, Tatusov introduced the notion of "occurrence vector" for groups of orthologous proteins [18]. Here we used the complete RefSeq NCBI database of bacterial genomes [19] comparing the cognate proteomes using BlastP (all genes of a proteome against all proteomes). We subsequently identified orthologs using bidirectional best hits (BBH) as described by Koonin and co-workers [20]. Next, we retained orthologs according to the distribution of a gene similarity scores, s, designed to take into account biological constraints other than orthology, using relevant thresholds (see equation 4 for definition of the thresholds).

          Lacking an evolutionary model common to all genomes, we chose the simplest model: the similarity score we used is the direct convolution of identity (i) and coverage (c: length of the BlastP alignment divided by the length of the longest protein) of the BlastP hits:
          http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-13-69/MediaObjects/12864_2011_3981_Equ1_HTML.gif
          (1)
          We computed the values of s for each gene ortholog present in the target organism. As an example, Figure 1 (left panel) displays the behavior of sorted s for 8 genes of the B. subtilis histidine pathway. The x axis indicates the number of orthologs of the target gene having a s score below x. The curves displayed in Figure 1 show that if we used a common threshold (score of the y axis) for all genes, we would find inconsistent levels of orthology. For example, fixing the threshold at 40 (arbitrary units, dashed black line) for the gene hisF retained almost all hisF orthologs but only some hisC orthologs (selected orthologs have similarity scores above the threshold). Ths is because there is no common molecular evolutionary clock [21] for the genes in the pathway: hisC orthologs genes maintain their function despite a high rate of mutation, generating BlastP hits with lower similarity scores. This constrasts with the evolution of hisF orthologs. Had we used the same threshold for hisC and hisF genes we would not have found them to be correlated.
          http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-13-69/MediaObjects/12864_2011_3981_Fig1_HTML.jpg
          Figure 1

          Curves of sorted similarity score s (equation 1) for 8 genes of the B. subtilis histidine pathway. The x axis represents the number of orthologs of the target gene having a s score below y. Left panel: The same threshold (dashed black line at 40) for all genes is used. Right panel: Different thresholds chosen according to the average of s are used for each gene.

          We therefore selected orthologous genes verifying the following formula:
          http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-13-69/MediaObjects/12864_2011_3981_Equ2_HTML.gif
          (2)

          This allowed us to select orthologous genes with a similarity score s above the average of s, avg(s) (see Figure 1 right panel). Now, the number of orthologous genes selected for the genes hisF and hisC is almost the same. As a consequence, if these orthologs belong to the same organisms, the two genes will be found to be correlated, a functionally relevant observation.

          Next, we included in the model the fact that orthologous genes must have a similar homology score because positive selection pressure tends to retain only a limited number of mutations among those that are constantly created. This implies that a high density of similarity score should correspond to families of organisms that keep the function of a gene with no alteration. To this aim, we first computed the distribution of s values, d(s), for each ortholog of target genes.

          Figure 2 shows how d(s) varies for the histidine pathway's genes of B. subtilis. We subsequently retained the orthologs genes verifying the following formula:
          http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-13-69/MediaObjects/12864_2011_3981_Fig2_HTML.jpg
          Figure 2

          Distribution d(s) of orthologs s values (equation 1) for the histidine pathway's genes of B. subtilis.

          http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-13-69/MediaObjects/12864_2011_3981_Equ3_HTML.gif
          (3)
          Finally, we took into account the fact that proteins may keep their function while suffering different mutation rates (a different threshold for each gene) and that families of organisms that keep the function of a gene have similar homology scores because they undergo positive selection pressure (high densities of homology score). To this aim we selected orthologs with a similarity score s above the average and otherwise orthologs genes with a s density above the average, combining (2) and (3).
          http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-13-69/MediaObjects/12864_2011_3981_Equ4_HTML.gif
          (4)
          Figure 3 shows an example of this selection (selected orthologs genes have their s values indicated in grey) for hisI orthologs.
          http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-13-69/MediaObjects/12864_2011_3981_Fig3_HTML.jpg
          Figure 3

          Distribution d(s) of orthologs s values (equation 1) for gene hisI of B. subtilis. Orthologs of hisI which have their s values indicated in grey correspond to orthologs selected applying equation 4.

          Relevant selection of orthologs has been computed for each gene of the target organism. This allowed the building up of binary vectors of presence/absence for these genes to explore the hypothesis that functionally linked genes have the same occurrence vectors. The next step was then to compare together these occurrence vectors to underscore functional relationships that group together genes of the target organism.

          Comparison of phylogenetic profiles

          As previous authors, we assumed that in the course of evolution functionally-related genes tend to be gained or lost together. This results in a correlation of their occurrence vectors. The first exploration of this hypothesis compared co-occurrence profiles using Hamming's distance [22]. Subsequently, many different statistical approaches to compare phylogenetic profiles have been used, such as mutual information [23], Pearson correlation coefficient [24] and Fisher's test [25]. Here we used the phi coefficient to compare phylogenetic profiles of two genes X and Y. This measure is similar to the Pearson correlation coefficient. In fact, a Pearson correlation coefficient estimated for two binary variables will return the phi coefficient:
          http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-13-69/MediaObjects/12864_2011_3981_Equ5_HTML.gif
          (5)
          With n11, the number of organisms in which X and Y are present; n00, the number of organisms in which X and Y are absent; n10, the number of organisms in which X is absent and Y is present; n01, the number of organisms in which X is present and Y is absent. The formula is symmetric:
          http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-13-69/MediaObjects/12864_2011_3981_Equ6_HTML.gif
          (6)

          Measure of phylogenetic distances

          The Pearson correlation, as other statistical methods, ignores that organisms are phylogenetically related and that the phylogenetic kinships may be biased. This must be taken into account, as the genome samples that have been sequenced is considerably biased, in terms of relative phylogenetic proximity. This may have a negative influence on our predictions. To reduce the effect of this phylogenetic skew, we modified the formula, taking into account the phylogenetic distance between organisms in which the genes co-occur. The idea was to give a larger weight to genes co-occurring in distant organisms than to those present/absent simultaneously in closely related organisms. As we do not have a detailed model that would account for the sampling bias in genome data, we used a plausible straightforward phenomenological measure of the proximity between two organisms, A and B, D(A,B):
          http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-13-69/MediaObjects/12864_2011_3981_Equ7_HTML.gif
          (7)

          with N(A,B) the number of genes occurring in organisms A and B, N(A) the number of genes in organism A and N(B) the number of genes in organism B.

          In the absence of a model describing genome evolution, and knowing that there is a considerable anthropomorphic bias in the choice of the organisms that have been sequenced we chose a somewhat arbitrary non-linear model to increase the weight of distant organisms. Our formula to measure the functional link between two genes X and Y is now:
          http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-13-69/MediaObjects/12864_2011_3981_Equ8_HTML.gif
          (8)

          with A and B the most distant organisms in which genes X and Y co-occur.

          Coupling with genomic context methods

          Further biologically-relevant factors must be taken into account to construct a plausible heuristics. A great many methods use the genomic context of genes to predict functional links between proteins. For example, functional links are suggested by conservation of gene neighbourhood and gene order [26], gene fusion events [27], correlation of the genes' evolutionary rate [28] and correlation of genes' occurrence in organisms (phylogenetic profiles). In [23] an evaluation of the methods that emphasized these factors showed that conservation of the gene neighborhood and the gene order covered 45% of the functional interaction between genes of Mycoplasma genitalium. In the present work, the gene neighborhood was measured using the Syntonizer software (implemented in the MicroScope platform [29]), which is based on an exact graph-theoretical approach to measure synteny [30]. A factor K was used: K(X,Y) = 1 if the two genes X and Y are found at least once in synteny and 0.9 otherwise.

          The final formula to compute the co-evolution score C between two genes X and Y is then:
          http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-13-69/MediaObjects/12864_2011_3981_Equ9_HTML.gif
          (9)

          with ϕ(X,Y) the correlation score between phylogenetic profiles of the genes X and Y, D(A,B) the distance between the most distant organisms A and B in which the genes X and Y co-occur and K(X,Y) a factor measuring the conservation of genes neighborhood.

          This phylogenetic profile method (PhyloProfile) has been integrated in the MicroScope platform [29]. It is directly available in the gene editor and allows the users to compute dynamically co-evolution scores of the target gene against all genes of the organism of interest.

          Construction of clusters of co-evolution

          Finally, following computation of the relevant phylogenetic profiles, we constructed co-evolution clusters. To this aim, we computed co-evolution scores, C, for all genes of B. subtilis, E. coli and Acinetobacter baylyi. If n is the number of genes of an organism, we obtained n*(n-1) scores. These scores were used to build up networks in which nodes correspond to genes and edges correspond to scores of co-evolution between genes. Applying a clustering method on these networks will allow construction of partitions of these genomes into clusters of co-evolving genes. Here we used as a clustering method the Markov Cluster algorithm (MCL; Van Dongen 2000; http://​micans.​org/​mcl/​) that is designed to cluster large numbers of relationships in a similarity space. The MCL algorithm is a fast and scalable unsupervised cluster algorithm for networks, based on simulation of flows in graphs. It has successfully been applied for clustering large sets of protein sequences [31, 32]. In the present work, clusters corresponding to functionally relevant processes, such as metabolic pathways (histidine biosynthesis...) or global functions (degradosome ...) were obtained, depending on the threshold used in the clustering procedure.

          As a validation of our approach we verified that molybdopterin biosynthesis and use have disappeared in a concerted way, as observed in Pseudoalteromonas haloplanktis [33].

          Results

          The contrasted replication-associated gene orientation bias uncovered between most bacterial clades and Firmicutes prompted us to explore the underlying phylogenetic constraints that support this discrepancy. To this aim we meant to uncover the functions that co-evolved with either DnaE or PolC.

          To get a first crude view of the processes underlying this replication-associated bias we used the JMP® software (SAS Institute, Cary, NC) to compute a hierarchical clustering of the B. subtilis essential genes [34] according to their occurrence proportion in different Bacteria clades (Figure 4). Three main clusters were obtained. Unexpectedly, they were all related to DNA replication. This substantiated the conjecture that the way replication is organized was indeed at the core of some important functional variation specific to bacteria forming a given clade.
          http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-13-69/MediaObjects/12864_2011_3981_Fig4_HTML.jpg
          Figure 4

          Hierarchical clustering of occurrence proportion vectors of B. subtilis essential genes in clades of the domain Bacteria. Squares show the proportion of a phylum (ascending from white to black) having an occurrence of a gene. The dendogram depicts distances between the proportion vectors. This dendogram separates B. subtilis essential genes into three clusters. The first one (red) contains dnaE and corresponds to genes occurring in the majority of bacterial clades. The second one (blue) contains dnaA and spans the whole domain of bacteria. The last one (green) contains polC and is composed of genes essentially specific to Firmicutes. The computing and visualization of the clusters was performed using the JMP® software (SAS Institute, Cary, NC).

          We observed the following, from top to bottom in Figure 4: A first cluster corresponded to genes present in almost all bacterial clades. This cluster contains dnaE, which, however, seems to be absent from the Deinococcus-Thermus and Chlamydia-Verrucomicrobia clades. It also comprises the bulk of the translation machinery (ribosome protein genes), including a limited number of tRNA synthetases, RNA polymerase genes, core iron-sulfur metabolism, and the core genes required to synthesize an envelope (Additional file 1). The second cluster (in blue) corresponded to genes spanning the whole domain Bacteria. It comprizes most genes of tRNA synthetases and the remaining set of ribosomal protein genes. Remarkably it clusters with dnaA as well as the gene for DNA primase. Finally, the third cluster corresponded to genes mostly specific to Firmicutes. This cluster co-evolves with polC, genes involved in cell division and genes involved in RNA degradation.

          While already revealing, this first analysis cannot give us a detailed view of gene co-evolution as it is based on a considerably biased sample of genomes. Indeed, genomes have been chosen to be sequenced as a function of the history of biological studies in the academic world (and particularly oriented towards bacteria of medical interest), and not based on a view spanning the whole tree of life, with equal weight for all extant species. In general, finding the same frequency of gene occurrence in a clade does not imply that these genes are simultaneously present or absent in the same organisms. This is particularly true when clades are made of only a few organisms (Thermotogae, Acidobacteria, Chloroflexi, Deinococcus have less than twelve organisms). Naturally, this is much less so in clades that are well represented (Firmicutes and gamma-Proteobacteria comprize more than 250 organisms). Furthermore this preliminary analysis was only based on the essential genes of B. subtilis, and it is well established that functional ubiquity does not equate to gene ubiquity: a same function could be essential in other clades, but performed by a gene of a completely different descent.

          In order to investigate more accurately gene co-evolution we needed therefore to analyze the occurrence profiles of all B. subtilis genes at the level of several phylogenetically distant model organisms rather than clades. To this aim, we designed a specific phylogenetic profile heuristics (PhyloProfile, see Methods) and based our exploration on the recently re-sequenced and re-annotated genome of B. subtilis [35] in parallel with that of the reference sequence and annotation of E. coli [36], an organism phylogenetically distant from B. subtilis. The method allowed us to identify clusters of genes that specificallly co-evolved with dnaE and polC.

          Genes co-evolving with dnaE

          As a possibly ancestral protein, DnaE co-evolves with a very large number of genes. We used different co-evolution scores thresholds (0.50 to 0.90) to investigate how the proteins of the whole proteome clustered with DnaE. When the threshold was higher than 0.75, the number of genes co-evolving with DnaE stabilized to approximately 250 genes. As an example, using 0.77 as a threshold, we listed the genes co-evolving with DnaE from B. subtilis (Additional file 1). This list matched remarkably well with the key genes coding for the persistent functions that are required to allow reproduction and replication [37], substantiating the validity of the heuristics.

          Most of the genes coding for the translation and transcription machineries, including factors and enzymes involved in modification and maturation of basic components of the machineries, were found in the list. This list also comprized the genes coding for the major metabolic pathways allowing construction of the cell: pyrimidine and purine biosynthesis and salvage, the core of the glycolytic/gluconeogenesis pathway (eno, gapA, pgk, tpiA...) and ATP synthase, as well as the secretion machinery. A number of components of the replication apparatus (DnaA, DnaC, DnaG, DnaN, GyrAB, LigA, PcrA, RnhB, SsbA) and genes involved in recombination and repair (Nth, Obg, PolA, RadA, RecA, RecG, RecN, RecO, RuvAB) were present as well in this set of proteins co-evolving with DnaE. Remarkably, a set of functions was missing from this list, that which corresponds to many components of RNA degradation (see below) [38]. Indeed, we found only a very limited set of RNA maturation and catabolism genes: rnc (coding for RNase III), rnhB (already mentioned, allowing degradation of RNA/DNA hybrid sequences during replication of the DNA lagging strand) and pnpA, a gene coding for an enzyme degrading RNA while preserving phosphate bond energy, polynucleotide phosphorylase.

          Beside genes coding for known functions, a consistent set of genes coding for unknown functions was present in most bacterial clades. The corresponding list of the corresponding 33 « y » B. subtilis genes within the larger DnaE-related gene set is displayed in Table 1. These genes were further analyzed as they correspond to unknown or poorly identified functions that, if understood, would enhance considerably our understanding of bacteria. In the course of this exploration, we benefited from continuous re-annotation of the updated genome sequence [35]. Using the most recent publications, this limited the unknowns to at most 18 genes. Interestingly, the majority of the newly identified functions was involved in ribosomal RNA metabolism (rsmI(yabC), rsmD(ylbH), rlmN(yloN), rimP(ylxS), hflX(ynbA), cpgA(yloQ)), further substantiating the DnaE/translation connection. We also found in this set the hibernation promoting factor (hpf(yvyD)) and further genes involved in recombination and repair (uup(yfmR), recD(yrrC), rarA(yrvN)). All of these genes have a counterpart in E. coli. A gene, shorter in B. subtilis than in E. coli, codes for a function important for mRNA turnover, rpsA in E. coli and ypfD (renamed rpfA) in B. subtilis. This gene codes for ribosomal protein S1 in E. coli and there is a clear indication, in this case, that the ribosomal protein function has been superimposed on the general function of mRNA presentation to the degradosome [39]. This is a first indication that there is divergence between proteins functionally related to DnaE polymerase III in E. coli and B. subtilis, in particular at the level of RNA degradation.
          Table 1

          Genes of unknown function co-evolving with Bacillus subtilis dnaE

          Label

          B. subtilis

          old name

          E. coli

          Function

          BSU00200

          yaaK

          yaaK

          ybaB

          DNA binding protein1

          BSU00360

          rsmI

          yabC

          yraL

          ribosomal RNA small subunit methyltransferase I2

          BSU00390

          yabD

          yabD

          ycfH

          metal-dependent DNase1

          BSU00480

          yabJ

          yabJ

          yjgF

          putative enzyme resulting in alteration of gene expression

          BSU05910

          ydiB

          ydiB

          yjeE

          putative ATPase or kinase UPF0079

          BSU05920

          ydiC

          ydiC

          yeaZ

          putative chaperone or protease

          BSU05950

          ydiF

          ydiF

          ybiT

          putative ABC transporter (ATP-binding protein)3

          BSU07370

          uup

          yfmR

          ycbH

          putative ABC protein involved in RecA-independent precise excision of transposons1

          BSU07900

          rbn

          yfkH

          yihY

          putative ribonuclease BN2

          BSU09240

          yhcW

          yhcW

          yniC

          putative phosphoglycolate phosphatase

          BSU14350

          yknX

          yknX

          ybjY (macA)

          putative efflux permease3

          BSU15010

          rsmD

          ylbH

          yhhF

          ribosomal RNA small subunit methyltransferase D2

          BSU15380

          ylmE

          ylmE

          yggS

          conserved hypothetical protein

          BSU15750

          rlmN

          yloN

          yfgB

          23S rRNA m2A2503 methyltransferase2

          BSU15780

          cpgA

          yloQ

          rsgA (yjeQ)

          GTPase involved in ribosome and sacculus morphogenesis2

          BSU16590

          rimP

          ylxS

          yhbC

          ribosome maturation factor2

          BSU17430

          hflX

          ynbA

          hflX

          ribosome associating GTPase2

          BSU21450

          yolJ

          yolJ

          "-"

          putative glycosyltransferase

          BSU22880

          rpfA

          ypfD

          "-" rpsA

          RNA degradation presenting factor (ribosomal protein S1 homolog)2

          BSU24790

          yqgX

          yqgX

          ycbL

          putative metal-binding hydrolase

          BSU24890

          folN

          yqgN

          ygfA

          5-formyltetrahydrofolate cyclo-ligase

          BSU25320

          yqfG

          yqfG

          ybeY

          putative metal-dependent hydrolase

          BSU25620

          yqeL

          yqeL

          ybeB

          putative ribosomal maturation protein2

          BSU27370

          yrrL

          yrrL

          yceG

          conserved hypothetical protein

          BSU27390

          yrrK

          yrrK

          yqgF

          putative Holliday junction resolvase1

          BSU27480

          recD

          yrrC

          recD

          exodeoxyribonuclease V alpha chain1

          BSU27490

          yrrB

          yrrB

          yciM

          putative tetratricopeptide repeat family protein

          BSU27530

          rarA

          yrvN

          ycaJ

          DNA-dependent ATPase1

          BSU27700

          yrbF

          yrbF

          yajC

          component of the preprotein translocase

          BSU28360

          rdgB

          ysnA

          yggV (rdgB)

          inosine/xanthosine triphosphate pyrophosphatase (subunit A)

          BSU30680

          ytjA

          ytjA

          yidD

          conserved hypothetical protein

          BSU35310

          hpf

          yvyD

          yhbH

          ribosome-associated sigma 54 modulation protein2

          BSU36950

          tamT

          ywlC

          yrdC

          tRNA threonylcarbamoyladenosine biosynthesis protein2

          1: DNA metabolism

          2: RNA metabolism

          3: transport

          Starting with the B. subtilis nomenclature, we grouped the 18 remaining genes of unknown function according to common features, either extracted from the literature or using the neighborhood software STRING [40], with the hope to further uncover some of the associated functions. Despite its consistently valid outcomes we refrained from using our PhyloProfile to avoid circular validation. Interestingly, all but one (yolJ, a gene belonging to bacteriophage SPbeta, that disappeared from PhyloProfile when the threshold was increased) of these co-evolving genes have at least one clear ortholog in E. coli. yaaK belongs to an operon involved in DNA replication and repair, its STRING pattern further subtantiates this functional association. yabD and yrrK yrrL are connected together and relate to genes of the translation machinery (metG, ksgA, serS, rsmG) as well to genes involved in DNA metabolism and aromatic compounds biosynthesis. yabJ (yjgF in E. coli) codes for a putative enzyme with a 3D known structure conserved in all three domains of life [41]. Here, it is related to intermediary metabolism via aminotransferase PatB, the control of purine metabolism (purR, relA, gmk, rdgB), isoleucine biosynthesis and to proteolysis (via ClpX). In contrast, in E. coli, it is related to catabolism of threonine, pyrimidine metabolism and to a network of genes of unknown function. ydiB (coding for a putative shikimate dehydrogenase [42]), ydiC and ydiF connected to ribosome maturation and sulfur metabolism. yhcW connects to ilvA and translation; yknX, in an operon coding for a putative efflux permease, down regulated in the presence of benzoate [43]; ylmE to cell division and proline and purine/polyphosphate metabolism; yqgX to translation via aspartyl and histidyl tRNA synthetases, as well as D-tyrosine deacylase, transformylase and RelA; yqfG to translation and phosphate metabolism; yqeL and ytjA to translation and tRNA modification; finally, yrrB and yrbF are connected to several genes involved in tRNA modification.

          In summary, DnaE co-evolves with most of the replication, recombination, translation, transcription and secretion machineries, and with some of the core metabolic biosynthetic pathways. We noted however the absence of a consistent RNA degradation pathway as well as most enzymes of the envelope biosynthetic pathways. This suggested that both RNA degradation and envelope biosynthesis, while functionally essential, might derive from different descent in different bacterial clades.

          Genes co-evolving with polC

          The list of the 162 genes co-evolving with polC differed considerably from that co-evolving with dnaE (Additional file 1). In particular, among those, 69 were genes of unknown function, a considerable proportion (Table 2). Interestingly, when analyzing their neighborhoods with STRING, we observed that there was seldom any connection with the dnaE-related networks, as if polC had evolved from a completely different origin. We also noticed that almost all genes in the list, while present in B. subtilis, do not have an ortholog in E. coli. This is exactly the opposite of what we found with genes co-evolving with dnaE. As for DnaE, the PolC-related proteins could be clustered into functionally significant groups (Tables 2 and 3). Remarkably, many of these clusters correspond to some aspect of RNA metabolism. Using STRING, these genes could be further clustered into seven groups (Table 3), the other ones remaining isolated.
          Table 2

          Genes of unknown function co-evolving with Bacillus subtilis polC

          label

          B. subtilis

          E. coli

          Function

          Functional connection

          String groups

          BSU00030

          yaaA

          ybcJ

          putative RNase or phosphorylase; conserved in yeast; very weak in E. coli 2

          RNA/DNA metabolism

          group 1

          BSU00330

          yabA dnaH

          "_

          subunit of the DNA replication complex1

          DNA polymerase and "y" network

          group 1

          BSU00340

          yabB

          "_

          putative RNA methyltransferase2

          RNA metabolism

          group 1

          BSU00630

          yabR

          "_

          putative RNA degradation protein; polyribonucleotide nucleotidyltransferase or phosphorylase2

          degradosome network with RfaA

          group 2

          BSU31390

          yugI

          "_

          putative RNA degradation protein or phosphorylase or nucleotidyl transferase;

          degradosome network with RfaA

          group 2

          BSU01750

          ybbP

          "_

          homolog of YabR and RpfA2 DAC domain protein present in Archaea

           

          group 3

          BSU01760

          ybbR

          "_

          substrate for Sfp phosphopantetheinyl transferase-catalyzed protein labeling by small molecule-CoA conjugates

           

          group 3

          BSU40510

          yybT

          ".

          phosphodiesterase acting on cyclic dinucleotides; possibly nanornase2

          RNA metabolism

          group 3

          BSU15060

          ylbM

          "_

          conserved hypothetical protein, found in a ribonuleoprotein complex in Mus musculus2

          RNA metabolism

          group 3

          BSU25630

          yqeK

          "_

          putative hydrolase

           

          group 3

          BSU25680

          yqeG

          ".

          putative hydrolase

           

          group 3

          BSU11030

          yitL

          "_

          RNA-binding protein2

          RNA metabolism

          group 3

          BSU19680

          yozE

          ".

          conserved hypothetical protein2

          RNA metabolism

          group 3

          BSU24860

          yqgQ

          "_

          putative single strand nucleic acid binding protein2

          RNA metabolism

          group 3

          BSU14540

          ykzG

          "_

          omega subunit of RNA polymerase2

          RNA metabolism

          group 3

          BSU16610

          ylxR

          "_

          putative RNA binding protein; putative new fold2

          RNA binding

          group 3

          BSU27380

          yrzB

          "_

          putative anti-sigma factor

           

          group 3

          BSU15830

          yloU

          "_

          conserved hypothetical protein

           

          group 3

          BSU15840

          yloV

          dhaL

          putative dihydroxyacetone/glyceraldehyde kinase

           

          group 3

          BSU08680

          ygaC

          "_

          putative factor, domain associated with ribonuclease E and G, possibly involved in Fe-S group formation2

          degradosome network

          group 3

          BSU15970

          ylxM

          "-

          conserved hypothetical protein2

          RNA/DNA metabolism

          group 3

          BSU27950

          ysxB

          "-

          conserved hypothetical protein with ribosomal function2

          ribosome

          group 3

          BSU16000

          ylqC

          "-

          putative RNA binding protein2

          RNA binding

          group 3

          BSU25670

          yqeH

          "-

          GTPase involved in ribosome 30S assembly2

          ribosome

          group 3

          BSU16970

          ymdB

          "-

          putative phosphoesterase

           

          group 3

          BSU27400

          yrzL

          "-

          conserved hypothetical protein functionally linked to alanine tRNA loading2

          RNA metabolism

          group 3

          BSU32310

          yutD

          "-

          conserved hypothetical protein2

          RNA metabolism

          group 3

          BSU14640

          yktA

          "-

          conserved hypothetical protein

           

          group 3

          BSU15000

          ylbG

          "-

          conserved hypothetical protein

           

          group 3

          BSU17880

          ynzC

          "-

          conserved hypothetical protein

           

          group 3

          BSU22190

          ypsA

          "-

          conserved hypothetical protein

           

          group 3

          BSU07420

          yfmM

          "-

          putative polyphosphate-AMP phosphotransferase

           

          group 3

          BSU07430

          yfmL

          "-

          putative ATP-dependent RNA helicase2

          RNA metabolism

          group 3

          BSU00970

          yacP

          "-

          putative ribonuclease with PIN and NYN domains; similar to eukaryotic RNases2

          RNA metabolism

          group 4

          BSU23950

          yqjA

          "-

          conserved hypothetical protein putative

           

          group 4

          BSU32280

          yutG

          "-

          phosphatidyl-glycerophosphatas e A

           

          group 4

          BSU16845

          ymfF

          "-

          putative metalloprotease

          protein metabolism

          group 4

          BSU16860

          ymfH

          "-

          putative processing protease

          protein metabolism

          group 4

          BSU09800

          yheA

          "-

          conserved hypothetical protein

           

          group 4

          BSU09930

          yhaM

          "-

          3'-5' exonuclease2

          DNA/RNA metabolism

          group 4

          BSU01450

          ybxA ecfA

          "-

          energizing coupling factor ABC multiple influx transporter (ATP-binding protein)3

          specific transport

          group 5

          BSU01460

          ybaE ecfB

          "-

          energizing coupling factor ABC multiple influx transporter (ATP-binding protein)3

          specific transport

          group 5

          BSU01470

          ybaF ecfT

          "-

          permease component of the EcfAB influx transporters3

          specific transport

          group 5

          BSU11590

          yjbL

          "-

          putative phosphatase

           

          group 6

          BSU11600

          yjbM

          "-

          (p)ppGpp synthetase

           

          group 6

          BSU11620

          yjbO

          rluD

          pseudouridylate synthase2

          RNA metabolism

          group 6

          BSU29820

          ytpR

          "-

          putative protein with RNA binding domain2

          RNA binding

          group 7

          BSU29840

          ytpP

          "-

          putative thiol-disulfide oxidoreductase with thioredoxin domain5

          sulfur metabolism

          group 7

          BSU29860

          ytoP

          frvX

          glutamyl aminopeptidase; deblocking enzyme (wrong annotation in E. coli)

          protein metabolism

          group 7

          BSU13480

          ykrK

          "_

          conserved hypothetical protein

            

          BSU14100

          ykuJ

          "_

          putative RNA-specific modification enzyme subunit2

          RNA metabolism network of "y" genes

           

          BSU14130

          ykuL

          "_

          conserved hypothetical protein

            

          BSU15410

          ylmH

          "_

          factor involved in shape determination, RNA-binding fold2

            

          BSU17440

          ynbB

          "_

          putative C-S lyase5

          sulfur metabolism

           

          BSU17699

          ynzK

          "_

          putative membrane protein

            

          BSU17910

          yneF

          "_

          conserved hypothetical protein; methionine-rich5

          sulfur metabolism

           

          BSU19620

          yodJ

          "_

          D-alanyl-D-alanine carboxypeptidase lipoprotein4

            

          BSU24260

          yqxC

          "_

          putative methyltransferase with RNA binding domain2

          RNA binding

           

          BSU28650

          ysgA

          "_

          putative RNA methylase2

            

          BSU29780

          ytxG

          pqiB

          homolog involved in DNA repair in Mus musculus 1

          DNA repair

           

          BSU29980

          ytjP

          "_

          putative dipeptidase

          protein metabolism

           

          BSU30460

          ytrA

          "_

          transcriptional regulator (GntR family)

            

          BSU30490

          ytqB

          "_

          putative RNA methylase2

          RNA metabolism

           

          BSU30870

          ytcB

          "_

          putative UDP-glucose epimerase4

            

          BSU36340

          ywpE

          "_

          putative sortase

            

          BSU36910

          ywlG

          "_

          conserved hypothetical protein; present in Archaea and Eukarya

            

          BSU37340

          ywiB

          "_

          putative RNA binding protein possibly involved in aminoacyl-tRNA editing2

          RNA binding

           

          BSU38499

          ywzH

          "_

          conserved hypothetical protein

            

          BSU40939

          yyzM

          "_

          conserved hypothetical protein

            

          1: DNA metabolism

          2: RNA metabolism

          3: transport

          4: envelope metabolism

          5: sulfur metabolism

          Table 3

          Genes groups identified using the STRING software [40]

          Groups

          STRING genes

          group 1

          yazA yabB yaaT yaaR yabC yabA yaaA yadB tmk holB dnaN dnaA dnaD recF

          group 2

          yabR rpfA yugI yabMNOPQ hprT rpmE divIC rpfA(ypfD) alaRT ftsZ gatA ppiB acpS alaS aroD cbsA cca cotD csbA ctsR cysS defB dltC dltD dnaB dnaC dnaD ecsB ffh ftsK ftsL ftsY glcU glmM gluKP glyQS gpsB hcrA infB jag kapB lepA ltaSA lysA mecAB mtnN murB mutSB nadD nrnA nusA obg polC priA prmA rbfA recA recG recU recX rex ribC ribT rimM rmpA rnc rnhB rnjA rplI rplS rplU rpmB rpmF rpoZ rpsP rpsR rsiW

          group 3

          scpAB sdaAA sdaAB sfp sigW smc speA spoIIIAD spoIVFA spoIVFB spoVS suhB sul tkt trmD truB ung uppS yaaQ yaaR yabF yabO yazC ybbP ybbR ydcK yebG yerH yetN yfhJ yfmKLMO ygaBCD yhaL yhcV yheA yisL yitKL yizA yktAB ykzG ylaH ylbFGMNP yloUV ylqC ylxMPQRS ymdAB yneABER ynzC yozE ypeQ ypsA yqeGHIKLM yqfC yqgNQY yqhP yqzF yrdA yrrKLM yrzBL yslB ysxB ytpI ytwI yuiB yutDEM yuzB yvcKL ywfO ywnH yybPRST yycHI

          group 4

          yacOP yueI yazC yvcS ywhD ylaL yozC yetH yqjB artMQ sigH ispDF cysS gltX cysE radA yqjAB mecB yutGH yneR ftsL cinA veg ydaS ypjQ yheA yojF ymxH fabR ymfFGHIJ lgt mreC recFG ctsR ybaF yheAB yqgQ yhaMO ywbD aroCF tyrA cwlJ yhaMNO rpe tmk rsgA ksgA thiN smpB topA cca

          group 5

          ksgA ecfAB ecfT ytlC cbiOA cbiOB rpsI rpoA truA rplQ rplM skfE ykoCD yuiG bioY

          group 6

          ywpF ywhD ymfK ywzC ylaL ywjG ypjB bofC ppnKA ndk rpoZ rpoABCE relA pyk folE yjbLMO yaaC ytzF lspA prfB truAB rluB

          group 7

          yrvN polA msrB pheST metG alaS ispG ytpPQRST ytoPQ ysdC ytzB comEA

          Group 1 clusters a subunit of the DNA replication complex, DnaH(YabA), together with putative RNA binding proteins (YaaA and YabB). Distant homologs exist outside of Firmicutes, in particular in Eukarya.

          Group 2 comprizes proteins involved in the Firmicute degradosome network and possibly involved in stress management (YabR and YugI (general stress protein GSP13 [44], essential in Staphylococcus aureus [45]), both displaying a S1 motif [46]).

          Group 3, the largest group, is organized around several proteins, most of which small or very small. They have often a known 3D structure but do not yet have an idenfied function. They are involved in RNA metabolism (transcription, RNA modification and turnover): YitL, an RNA binding protein, RnpZA(YkzG), a component of the omega subunit of RNA polymerase, YktA (possibly involved in polyamine metabolism, YlbG (possibly involved in activity of a conserved small RNA, CsfG [47]), YozE, of unknown function, YqgQ (putative single-strand nucleic acid binding protein involved in transcription [48]), and finally YrzL (essential in S. aureus [45]) and YutD, unknown proteins that are possibly hydrolases. With STRING we observed that this cluster is further connected to RnjA, the non-orthologous functional equivalent of RNase E in proteobacteria [49]. Many of these proteins are members of UniProt Unknown Protein Families (UPFs), most of which associated to Firmicutes and sometimes Archaea and Eukarya. This cluster further connects via YqgQ to DNA replication and recombination, to energy-dependent proteases, to divalent metal transporters and to proteins involved in shaping the cell. Via YloU (with a paralog, YqhY), YloV (dihydroxyacetone kinase-like), YlxM (conserved in Mycoplasma sp.), YnzC, YfmM (proposed to code for polyphosphate-AMP phosphotransferase in Staphylococcus epidermidis [50]), YmdB (putative phosphoesterase), YqeG (essential in S. aureus [45]), this cluster again comprises a large number of UPFs. It has some connection with the recombination machinery (RecA, RecG) and the envelope (PlsX, Ffh). YloU has similarity to yeast S-adenosylmethionine-dependent tRNA (uracil-5-)-methyltransferase. This may be significant, because in A+T-rich Firmicutes this activity differs from that in most other organisms, TrmFO, the methylating activity, depending on methylene-tetrahydrofolate rather than AdoMet, thus suggesting recruitment for another RNA modification activity [51]. Finally a sub-cluster of this large group associates YlbM (member of a ribonucleoprotein complex), YqeG (hydrolase), YqeK, YqeH (phosphohydrolase), and YybT (a putative phosphodiesterase, with motif GGDQV related to that of cyclic-diGMP synthesis and degradation). It is connected to Cca, RplI and YqeI, a putative RNA binding protein. Again, this cluster has a clear RNA metabolism flavour.

          Group 4 comprises YmfF and YmfG, two peptidyl hydrolases of unknown substrates, perhaps associated to transport of peptidyl siderophores [52] and connects to YheA (related to metabolism of aromatics (YwbD, AroC, TyrA, AroF)). It contains proteins such as YqjA (a putative membrane bound protein), YacP (possibly involved in ribosomal RNA maturation), connected to a network with tRNA synthetases CysS and GltX and with sigma factor SigH. It also comprises YhaM (a 3'-5' oligonucleotidase) and YutG (a putative phosphatidyl-glycerophosphatase A).

          Group 5 has now an identified function: it corresponds to the EcfABT energy-dependent activating part of a multi-substrate transporter [53].

          Group 6 clusters together YjbL, a putative phosphatase, YjbO, similar to RluD pseudouridylate synthase and YjbM, connected to RelA and RNA polymerase (RpoABCZ) as expected from its putative function in ppGpp synthesis and turnover. This homolog of RelA/SpoT seems to be specific to A+T-rich Firmicutes. It is also connected to riboflavin and folate biosynthesis.

          Group 7 associates YtpP and YtpR, connected to tRNA synthetases PheST, AlaS and MetG, as well as DNA polymerase PolA and methionine sulfoxide reductase MsrA. It contains also YtoP (FrvX, a glutamyl aminopeptidase in E. coli).

          Finally, the 20 remaining genes do not belong to clear clusters and do not have obvious functions. Yet, six among them (ykuJ, ylmH, yqxC, ysgA, ytqB, ywlB) code for proteins having RNA binding properties. ynbB and yneF are connected to sulfur metabolism and possibly tRNA modification. Protein YtxG has an homolog involved in DNA repair in Mus musculus. YodJ and YtcB are protein likely to be involved in murein synthesis or turnover.

          Discussion

          Cells encode several DNA polymerases that fulfill a variety of reactions: genome replication, repair and recombination. Eukarya have two types of DNA replicases. In the same way, the genomes of the Firmicutes clade have a unique heterodimeric DNA polymerase III α-subunits, PolC and DnaE [54]. In contrast the E. coli replicase is made of two identical DnaE subunits. The difference between the replicase of E. coli and that of B. subtilis is further reflected by the composition of the other subunits of DNA polymerase III that differ from those in other clades. In E. coli, a single protein, the AAA+ ATPase DnaC (counterpart of DnaI in B. subtilis), is used to load helicase DnaB [55]. In contrast, in B. subtilis and other low-G+C Firmicutes, three different proteins, DnaD, DnaB (no counterpart in E. coli, used for loading to DnaC), and the AAA+ ATPase DnaI, are needed to load the replicative helicase (DnaC in B. subtilis is the counterpart of DnaB in E. coli). DnaA binds first, followed by DnaD and then DnaB, and finally the DnaI-mediated loading of helicase occurs [56].

          The existence of two replicases implies a physical difference in the replication process that should be reflected in a bias in nucleotide strand composition. Analysis of compositional strand asymmetries of prokaryotic genome sequences in terms of the presence or absence of PolC has found not only a correlation with PolC [12], but also with a purine asymmetry [7]. This latter asymmetry is however probably not the result of the physical differences in the leading and lagging strands replication, but, rather, the consequence of the increase in the gene content of the leading strand in organisms with PolC, which alleviates conflicts between the transcription and replication machineries [10, 57]. In particular it was noticed that G seems to favour the leading strand in most bacterial genomes, which fits with an over representation of genes in this strand in Firmicutes, as coding regions are overrepresenting GNN codons [58].

          DNA composition analysis is of limited interest per se, as it does not provide much information about gene functions. The present study showed that while the functional connection of the DnaE subunit spans the whole domain Bacteria, with almost all functions that are expected to make a minimal genome, this is not so of the PolC co-evolving genes. Furthermore, the latter group of genes seems highly specific and related to original features of biological processes, RNA metabolism in particular.

          PolC is the subunit of DNA replicase that replicates the leading strand of the DNA double helix in Firmicutes. Structural data on the PolC family of replicases shows that it evolved separately from DnaE [59]. This process requires in addition to PolC, ten other proteins. In addition to these 11 proteins, lagging strand replication requires DnaE and primase [56]. Progression of the replisome implies that DnaE uses Okasaki fragments as primers, and these fragments need to be removed, so that they do not interfere with progression of PolC-mediated leading strand replication progression [60]. It is even suspected that, because replication of the lagging strand must be slow, bacterial replicases are made of not of two, but three replication subunits, two of them involved in replication of the lagging strand [61].

          In this context, management of the degradation of RNA derived from Okazaki fragments has a central importance. In particular degradation of nanoRNAs, fragments of size smaller than 5 nts, becomes crucial, as they can enter the replication bubble. It is established that PolC discriminates against RNA primers while DnaE uses RNA primers efficiently [54, 56]. As a consequence it can be expected that PolC co-evolved with a particular type of RNA degradosome. Indeed, in the present work, many proteins co-evolving with PolC could be seen as associated to properties of a Firmicutes-specific degradosome (or downstream from it) [39], comprizing nanoRNases such as NrnA [62], an RNase which degrades short oligoribonucleotides (and which is present in the smallest genome of an autonomous living organism, M. genitalium) but also RnmV, MrnC(YazC), YacP, Rny(YmdA), YhaM, YkzG, YlbM, YlmH, RnhC(YsgB) and YybT, proteins that are not present in the E. coli degradosome (Additional file 1). The presence of a particular type of RNase H (RnhC), required to hydrolyze RNAs belonging to RNA-DNA hybrids, essential in B. subtilis [63], is particularly revealing, as it further supports the conjecture that the co-evolution we observed is related to RNA turnover. A further related observation is that there is no global counterpart of the degradosome proteins in those evolving with DnaE, except for the core phosphorylase, polynucleotide phosphorylase, and a specific RNase H (RnhB) of descent different from that of RnhC. NanoRNases (essential for degrading Okazaki fragments) have now been identified from three descents: Orn in gamma-Proteobacteria, NrnA in Firmicutes) and NrnC in alpha-Proteobacteria [64]. This indicates that there may exist more than two degradosomes in the Bacteria domain. Further work similar to the one presented here will tell.

          Finally, we remarked that the genes that co-evolve with polC have often counterparts in the three domains of life. This suggested that, despite their absence from the genes co-evolving with the majority of the essential cellular processes, these genes are of very ancient descent. Interestingly, many of the corresponding functions are related to RNA metabolism but also to phosphate or polyphosphate metabolism.

          Conclusion

          Analysis of the genes co-evolving with the two forms of DNA replicase found in Bacteria, DnaE and PolC revealed that, while DnaE co-evolved with the translation and transcription machineries, PolC co-evolved with proteins that do not belong to the same group. In particular PolC co-evolved with a form of the RNA degradation machinery that is distinct from that characterized in gamma-Proteobacteria, the E. coli degradosome [38]. Among other possibilities, this observation suggests that, while there may exist a last common ancestor to the translation/transcription machinery, this was probably not so for the machineries leading to RNA turnover, which may have appeared independently on several occasions. The RNA world, that developed RNA-centered metabolism [65], predated the RNA genome world, in which RNA synthesis and turnover must have been essential. Our observations are consistent with the discovery of DNA and DNA replication at least twice [66] suggesting that the origin of present living organisms was a community of organisms developing more or less independently from one another (the progenote hypothesis [67, 68]), splitting and fusing as time elapsed until the present domains were more stably defined.

          Declarations

          Acknowledgements

          This work benefited from the support of the European Union Microme programme FP7-KBBE-2007-3-2-08-222886.

          Authors’ Affiliations

          (1)
          AMAbiotics SAS, Bâtiment G1
          (2)
          CEA, DSV, IG, Genoscope & CNRS-UMR8030, Laboratoire d’Analyse Bioinformatiques en Génomique et Métabolisme (LABGeM)
          (3)
          Department of Biochemistry, Faculty of Medicine, Hong Kong University

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