The bacterial dcw gene cluster: an island in the genome

Chapter 13 The bacterial dcw gene cluster: an island in the genome? JESÚS MINGORANCE1* and JAVIER TAMAMES2 1 Centro Nacional de Biotecnología (CSIC) Campus de Cantoblanco 28049 Madrid 2 ALMA Bioinformática. Ronda de Poniente, 4. Tres Cantos. 28760 Madrid Spain. *corresponding author 1. Introduction The bacterial dcw cluster is a group of genes (16 in Escherichia coli, Fig. 1) involved in the synthesis of peptidoglycan precursors and cell division (Ayala et al., 1994). A prominent feature of this cluster is that it is conserved in many bacterial genomes over a broad taxonomic range (Vicente et al., 1998; Nikolaichik and Donachie, 2000; Tamames et al., 2001). A finding that is further underscored by the fact that in the prokaryotes genome organization is very dynamic in evolutionary terms, meaning that the gene content as well as the gene order within operons and gene clusters diverge quickly with increasing evolutionary distance (Tamames et al., 1997; Itoh et al., 1999; Tamames, 2001; Xie et al., 2003; Chapter 11). An analysis of gene order conservation in the genomes of several bacteria and archaea found eighteen highly conserved groups including nearly 120 genes. This means that less than 3% of the E. coli genes have a genome organization highly conserved across the prokaryotes (Tamames, 2001). Nine of the groups included ribosomal protein genes and other genes related with the processes 1 2 Mingorance and Tamames Figure 1. Schematic representation of the dcw cluster in several bacterial genomes. Adjacent boxes represent contiguous genes. White boxes correspond to genes that are not known to be related with division or cell wall synthesis. Groups of genes separated by spaces represent groups that are separated in the genome. Isolated genes are not represented but most are present in all the genomes (Chapter 8). of transcription and translation. Among these, there was a large cluster of ribosomal protein genes that is the most conserved, and is found in bacterial, archaeal, and organelle genomes (Wächtershäuser, 1998; Hansmann and Martin, 2000; Tamames, 2001). Other conserved groups included the dcw cluster, the atp operon (coding for the subunits of ATP-synthase), and the his operon (coding for the enzymes involved in the biosynthesis of histidine). In most cases it is obvious that the clustered genes are functionally related (Tamames et al., 1997), what is not evident at all is which are the selective forces that hold these genes together in a precise order over large evolutionary distances. Comparison of the dcw cluster from several bacterial genomes using phylogenetic analysis tools has shown that there is a strong relationship between the organization of this gene cluster and the morphology of the cells (Tamames et al., 2001). This correlation suggests that the organization of these genes in the chromosome is related to the processes of cell growth and division. We postulate that for the rod-shaped bacteria the mechanism linking gene order in the chromosome with cell shape is the co-translational The dcw gene cluster 3 assembly of the products of the mur and fts genes to form the multiprotein complexes responsible for cell wall growth and cell division. 2. The dcw cluster: structure and function Part of the dcw cluster was initially identified in Escherichia coli as a region containing genes related to the synthesis of murein precursors, and was called mra region (Miyakawa et al., 1972) or mra operon (Donachie, 1993). Later several cell division genes were found to map adjacent to this region, and therefore the name dcw cluster, standing for division and cell wall, was proposed to include the whole set of linked genes (Ayala et al., 1994). In the context of comparative genomics the term cluster seems more appropriate than operon, because in most organisms the transcriptional structure of this region is not known. And in fact in some organisms, like Neisseria ghonorrhoeae, the cluster might not be an operon but be composed of several independent transcriptional units (Francis et al., 2000; but see also Snyder et al., 2003). In E. coli the cluster contains 16 tightly packed genes (Fig. 1), some overlapping, and all of them transcribed in the same direction (Vicente et al., 1998; Dewar and Dorazi, 2000). There is one initial promoter upstream of mraZ (mraZ1p) several internal promoters, and a single transcription terminator downstream of envA (lpxC), the gene after ftsZ. There are four known promoters in the 5’ end of the cluster and six in the 3’ end, in the region upstream of ftsZ (Vicente et al., 1998). The first promoter, mraZ1p, is essential for transcription of at least the first nine genes of the cluster (Mengin-Lecreulx et al., 1998). Six internal promoters have been characterized in the region containing the genes ddlB-ftsQ-ftsA-ftsZ, contributing to about one third of the total expression of the ftsZ gene (Flärdh et al., 1998). The remaining two thirds are derived from promoters located upstream of ddlB (de la Fuente et al., 2001). The absence of internal transcription terminators suggests that in E. coli there might exist a large transcript arising from mraZp1 and spanning the whole cluster, but experimental evidence for the existence of this transcript is still lacking. In any case this hypothetical transcript could be differentially processed to yield shorter transcripts due to the presence of RNase targets at specific regions of the sequence, for example the ftsZ transcript (Cam et al., 1996). A high-resolution transcript analysis of the whole cluster using DNA microarrays has shown two peaks with higher steady-state transcript levels just downstream of the promoters mraZ1p and ftsQ2p1p, and a central region with lower levels. In addition it was found that the transcripts from this 4 Mingorance and Tamames central region are more stable than those from the lateral ones, which might have a faster turnover (Selinger et al., 2003). The transcriptional pattern may be different in other organisms, for example in Bacillus sp. there are two ftsI homologs (pbpB and spoVD) separated by a transcriptional terminator (Daniel et al., 1996) and responsible respectively for the synthesis of peptidoglycan during vegetative and sporulation septum formation (Daniel et al., 1994). The function of the first gene of the cluster, mraZ (yabB), is not known. The next gene, mraW (yabC), codes for an S-adenosylmethioninedependent methyltransferase of unknown substrate and unknown function (Carrión et al., 1999). The mur genes, together with mraY and ddlB code for the enzymes responsible for the synthesis of the murein precursors (van Heijenoort, 1996). These genes are considered to be unique and essential (Boyle and Donachie, 1998; Kobayashi et al., 2003). The gene ddlB has an additional copy in the E. coli chromosome, ddlA, and therefore although it is not essential in itself in E. coli, the activity of the enzyme coded by these two genes, D-alanine:D-alanine ligase, is essential for cell wall synthesis (Zawadzke et al., 1991; van Heijenoort, 1996). The cluster contains six fts genes (ftsL, ftsI, ftsW, ftsQ, ftsA and ftsZ), all of them are essential for cell division, and all of them code for proteins that have been shown to localize to the division site during septation (Chapter 5). Downstream of ftsZ there are some genes that are specific of some bacterial groups, like the envA gene found in the -proteobacteria, or a cluster of up to six additional genes, namely ylmD, ylmE, ylmF, ylmG, ylmH and divIVA, found in the Gram-positive bacteria (Massidda et al., 1998). 3. Conservation of the genes in the dcw cluster Some generalizations can be made from the comparison of the dcw cluster in several genomes (Nikolaichik and Donachie, 2000; Tamames et al., 2001). First, although most of the genes of the cluster are conserved in the majority of the genomes studied (see Chapter 9), they are not invariably found in the cluster, and most of them can be found in other locations in some instances. The frequency with which the genes appear in the cluster is also variable, and there are genes, like murB, that are found more often in other locations, while others, like ftsQ, ftsA and ftsZ only rarely are found isolated. Second, the relative order of the genes is highly conserved, and a hypothetical archetypal, or ancestral (Nikolaichik and Donachie, 2000), cluster can be defined that contains all the genes (Fig. 1). Most of the clusters analyzed can be derived from this archetypal cluster by deletion, and The dcw gene cluster 5 Figure 2. Schematic representation of the dcw cluster from two deep branching bacterial lineages. sometimes insertion of extra genes, without changing the relative order of the remaining genes (by relative order it is meant the ordering of neighbour genes in relation to the direction of transcription). In some organisms, like Caulobacter crescentus, or Neisseria gonorrhoeae the cluster has been split by insertions. In others, like Thermotoga maritima, or Rickettsia prowazekii it is dispersed into two or three separated groups, and even more in Helicobacter pylori and Campylobacter jejuni, but the same relative gene order is almost invariably maintained within each group (Figs. 1, 2 and 3). As a result, a pattern of conservation can be outlined in the hypothetical cluster: the 5’ region is variable, as it contains the mraZ and ftsL genes that are missing in many genomes; then there is a conserved block comprising the genes ftsI to murG; this is followed by another variable region that may contain the genes murC, murB and ddl, though only rarely the three genes are found together; and finally there is another conserved block comprising the division genes ftsQAZ. This pattern was clearly seen when conservation of the cluster was measured as occurrence of gene pairs (Fig. 4 in Tamames et al., 2001). 4. On the origin of the dcw cluster Is the dcw cluster an ancient and conserved trait, or is it of recent origin? It seems very unlikely that a complex gene structure like this, with such precise gene ordering, might be a recent acquisition arisen independently by convergent evolution in phylogenetically distant bacterial groups. Other possibilities are that the cluster might be an ancient trait that was already present in the last common ancestor (LCA) of extant bacteria, or 6 Mingorance and Tamames Figure 3. Hypothetical pathway of assembly of the dcw cluster indicating the four gene groups implicated. The order of the different events shown is arbitrary. The pathway starts with a primordial mur operon containing the genes responsible for the synthesis of the peptidoglycan precursor. An operon containing the rodA/ftsW and the pbpA/ftsI ancestors was duplicated, and then the pathway could diverge into septal and lateral peptidoglycan synthesis pathways. Incorporation of the fts genes might have facilitated the localization of the Mur complex to the septal zone during division. alternatively, that it might have originated after the LCA, and might have been later spread through the bacterial domain by lateral gene transfer. The phylogenetic analysis of the sequences of individual genes shows trees that are congruent with the standard 16S rRNA tree (Faguy and Doolittle, 1998). There are a few exceptions but these correspond to different genes in different organisms showing that in general the genes have evolved in a manner consistent with that of the organisms to which they belong, and arguing against extensive lateral gene transfer after the LCA (Fig. 8). The cluster is found in Thermotoga maritima, and in a very reduced version in Aquifex aeolicus (Fig. 2), both belonging to the deepest branches of the bacterial phylogenetic tree (Bocchetta et al., 2000). In T. maritima the cluster is split in two groups plus several isolated genes. One of the groups contains some mur genes and the other contains the ftsA-ftsZ pair. In A. aeolicus the cluster is reduced to the murB and ddlB genes clustered with the ftsA-ftsZ pair. The fact that both organisms have clusters but these do not overlap might be explained in several ways, but the most parsimonious explanation The dcw gene cluster 7 is that the genomic organization of the cluster in these two bacteria is derived secondarily by reduction from a large cluster that contained all the genes, and that this has happened also in other groups. A corollary of this is that the LCA had a large dcw cluster that has been maintained in some lineages and has been reduced in others (Fig. 1). How did this cluster arise? Although this is rather speculative, examination of gene function and gene order suggests that it might have arisen from the blending of four gene subsets (Fig. 3): i) the mur genes (including mraY and ddlA/B), responsible for the synthesis of peptidoglycan precursors; these genes code for the enzymes of a single biosynthetic pathway, and might have been maintained together forming an operon before the LCA; ii) the ftsI and ftsW genes that participate in the specialized peptidoglycan synthesis at the septum, and have counterparts in the pbpA and rodA genes, involved in the synthesis of lateral wall peptidoglycan; these two pairs of genes must have arisen also by duplication from a single ancestor pair; iii) the ftsL and ftsQAZ genes, that code for proteins that localize at the division ring; iv) the mraZ and mraW genes, that are of unknown function, and might belong to some of the other groups. The order in which the different rearrangements shown in Fig. 3 occurred is unknown. 5. Phylogenetic distribution of the dcw cluster and bacterial shape A phylogenetic analysis of the clusters from several completed genomes, based not on gene sequences, but on gene content and gene order, produced a tree different from the standard 16S rRNA-based tree (Tamames et al., 2001). A complete description of the procedure for coding gene order information is given in Tamames et al. (2001). Briefly, a set of vectors is created, one vector for every genome. The vectors contain as many positions as possible pairs of genes (only dcw cluster genes), and each position codes for a given pair of genes, being set to one if the pair exists, and to zero if it does not. A pair of genes might be defined as a pair of adjacent genes (distance 1), or a pair of genes separated by n intervening genes (distance n+ 1). The resulting set of vectors contains information about the gene pairs that are found in the genomes analyzed. If a given position is zero in all vectors, meaning that the corresponding gene pair is never found, it is considered uninformative, and it is removed. These sets of vectors allow the building of phylogenetic trees by several methods. Two different approaches were used: a parsimony analysis (Felsenstein, 1996) (using the "mix" program in the Phylip package), and a hierarchical clustering algorithm, 8 Mingorance and Tamames Figure 4. Schematic representation of the dcw cluster of bacteria with different shapes belonging to two different lineages. T. thermophilus (rod) and D. radiodurans (coccus) belong to the Thermus/Deinococcus group, while B. subtilis (rod) and S. aureus (coccus) belong to the high GC Gram-positives, but regarding the composition of the dcw cluster the two rod-shaped species are clearly more similar, and the two cocci have in common a reduced cluster. SOTA (Herrero et al., 2001). Both methods produce very similar results, with minimal disagreements. The tree showed two main branches, one that included organisms with a compact cluster, and the other formed by organisms in which the genes are more dispersed through the genome and the cluster is reduced, or does not exist at all. The distribution of bacterial species in the tree was found to correlate with their cell morphologies. Bacilli and filamentous bacteria fell in the group that had a large dcw gene cluster, while cocci, helicobacteria and spirochetes formed the branch in which the dcw cluster genes are more dispersed or lost altogether. In some instances the trees separated organisms that belong to the same phylogenetic group but have different shapes, like the low GC Gram-positive bacilli and cocci, the rod-shaped Thermus thermophilus and the coccus Deinococcus radiodurans, or the helicobacteria (Campylobacter and Helicobacter, -proteobacteria) and the rest of the proteobacteria (Fig. 4; Tamames et al., 2001). To test the robustness of the conclusions drawn in the original work with twenty-six sequenced genomes, the same study has been repeated including The dcw gene cluster 9 Figure 5. Dendrogram of the dcw cluster based on gene content and gene order. The tree shown in this figure was obtained with distance 2 as explained in the text. Names in black correspond to rod-shaped or filamentous bacteria, while those in grey color correspond to bacteria with other shapes. twenty-four additional genomes. The results of this new study fully confirmed the previous results, the rod-shaped and filamentous bacteria tend to branch together, and separate from cocci, spirochetes and helicobacteria, although there are a few exceptions. The pattern was less clear (there were more exceptions) when the analysis was done with adjacent gene pairs, probably because even in the organisms in which the cluster is reduced or dispersed several gene pairs are conserved (see for example H. pylori, Fig. 1). When the analysis was done with a distance 2 (Fig. 5) or a distance 3 (Fig. 6) between genes (meaning that there are respectively one and two intervening genes between the pair being considered) the pattern was more pronounced. The fact that increasing the distance between genes improves the pattern of separation of rod-shaped cells from other cell types supports the conclusion that the pattern found is due to the clustering of the genes, and not to the individual genes. The branching pattern observed might be due to differences in gene content between genomes, and independent of the positions of the genes in their genomes. To test this possibility an analysis was done taking into 10 Mingorance and Tamames Figure 6. Distance 3 dendrogram of the dcw cluster. The tree was obtained as described in the legend to Fig. 5 but using distance 3 (two intervening genes between each pair of genes). Names in black correspond to rod-shaped or filamentous bacteria, while those in grey color correspond to bacteria with other shapes. account only the presence or absence of the genes from the archetypal cluster, independently of their positions in the genome. In this case the correlation with cell morphology was not found (Fig. 7). The tree obtained was poorly informative, there were some groups consistent with the standard bacterial phylogeny, but several others were mixed and no trends could be discerned, probably because the tree is based on few and highly conserved (uninformative) characters. This shows that the correlation between the organization of the dcw gene cluster and the cell morphology is not an artifact due to the different gene contents of the genomes, but is due to the fact that these genes are clustered and that the degree of clustering is different in different genomes. Sequence-based phylogenies were done for the individual genes and were found to be fairly similar to the standard 16S rRNA phylogeny (Fig. 8). Most groups were correctly identified, although the relations between them were sometimes inaccurate. Only the helicobacteria, C. jejuni and H. pylori, were found to branch consistently apart from the other proteobacteria, suggesting that in this group at least some of the genes of the cluster might have been The dcw gene cluster 11 received by horizontal gene transfer. The differences between the individual sequence-based gene trees and the tree of the cluster indicate that while individual genes have evolved at the pace of the chromosomes in which they are contained, there have been genomic rearrangements within the dcw cluster that have affected differently to different bacterial lineages. Assuming that the ancestor of extant bacteria had a dcw cluster similar to the archetypal cluster represented in Fig. 1, it can be concluded that the cluster has been maintained in most rod-shaped bacteria and tends to be lost from bacteria with other morphologies. This agrees with the inference that coccoid, helical, and other cell shapes have arisen several times independently from a rod-shaped ancestor (Siefert and Fox, 1998; Chapter 1). 6. How is the dcw cluster maintained? Figure 7. Dendrogram based on the dcw gene content in the different bacterial genomes. For each genome the presence or absence of the dcw genes (those from the hypothetical ancestor cluster shown in Fig. 1) were recorded independently of their chromosomal positions, and the resulting vectors where used to construct a tree using the methods described in the text. 12 Mingorance and Tamames Figure 8. FtsZ sequence tree. Tree based on the FtsZ predicted protein sequence using a neighbour-joining method. The -proteobacteria H. pylori and C. jejuni branch in an anomalous position, the rest of the tree agrees fairly well with the standard 16S rRNA phylogeny. If the cluster originated in the ancestor of extant bacteria then what are the forces that have maintained the cluster for billions of years? And why in some lineages these forces are relaxed or do not operate anymore? We have shown that there is a relation between the genomic structure of the dcw cluster and the shape of the cells, but it is very unlikely that the cluster in itself has any morphogenetic properties. Transferring the cluster from a rodshaped bacterium into a coccus would not transform it into a bacillus because most cocci lack several other genes, like mreB, also involved in the determination of the cell morphology (Chapter 9). Furthermore, breaking the continuity of the cluster in E. coli has several effects, and may cause filamentation, but does not convert E. coli cells into cocci (Palacios et al., 1996; Mengin-Lecreulx et al., 1998). There are currently several models to explain the clustering of genes and the persistence of clusters for long evolutionary periods (reviewed in Lawrence and Roth, 1996). These models are based on different mechanisms, and therefore do not exclude each other, but might operate simultaneously. The dcw gene cluster 13 The natal model postulates that clusters originate by gene duplication and subsequent sequence divergence. In this case the order of genes in the cluster simply reflects the order in which duplications occurred. This model might apply to the origins of part of the dcw cluster (Fig. 3), as four of the genes (murC, murD, murE and murF) are paralogues that must have arisen by gene duplication in the ancestor of all eubacteria, but it cannot explain the presence in the cluster of other, unrelated, genes, the long persistence of the cluster, or its relation with cell morphology. In the selfish operon model clustering benefits the cluster itself, and conservation of gene order reflects the spreading of the cluster by lateral gene transfer. This model applies successfully to weakly selected functions, but it has difficulties with housekeeping genes. To apply the selfish operon model to the dcw cluster it should be assumed that 1) a full cluster is acquired via LGT, and 2) the old (endogenous) division genes, initially dispersed through the genome, are immediately lost, since in most cases they will interfere with the new (exogenous) genes (for example, exogenous ftsZ genes are often toxic for E. coli). A simple mechanism to effect the rapid and simultaneous loss of the dispersed genes is difficult to envisage, and the model is useless if it assumes that they were already clustered. Moreover, the selfish operon model might explain why the cluster has such a broad phylogenetic distribution, but not why it is related to cell shape. The co-regulation model proposes that clustering facilitates the coordinate expression and regulation of the clustered genes (Lawrence and Roth, 1996). Contrary to this, there are many instances of known genes that are co-regulated, and are in different chromosomal locations, or are found together in some organisms and not in others (Tamames et al., 1997; Itoh et al., 1999). Therefore, although gene grouping contributes to the coordinate regulation of gene expression at the DNA level, it is clear also that these mechanisms are very flexible in evolutionary terms, and do not require conservation of gene order (Xie et al., 2003). An alternative to this model proposes that clustering might be related to the co-regulation of gene expression at the RNA level (Siefert et al., 1997). This might apply to the cluster of ribosomal protein genes and other clusters that are known to be regulated at the level of mRNA translation in at least some organisms, but so far this level of regulation has not been described in the dcw cluster, and it is not clear at all why such a strict conservation of gene order would be required. Finally, the Fisher model postulates that clustering is the result of selection acting on a co-adapted set of genes to maintain them together because physical proximity decreases the probability of recombination occurring between them. This model is well suited to sexual organisms, where recombination is frequent. The discovery that prokaryotes may 14 Mingorance and Tamames undergo extensive horizontal gene transfer (Nelson et al., 1999) made the application of this model to bacteria more plausible, although the frequency of gene exchange in most species is not known. It has been postulated that the conservation of gene order in the dcw cluster might be explained by this model as the result of the lateral exchange of a set of linked genes that code for a co-adapted set of proteins (Nikolaichik and Donachie; 2000). This argument has two parts: first, that the linked genes are co-adapted; and second that the linkage is maintained by selection against the mixture of genes (“alleles”) from different sources. When applied to the dcw gene cluster, the first part of the argument is likely to be correct, as co-adapted proteins are those that interact either physically or functionally, which is the case in this cluster. On the other hand, it is unlikely that the cluster might have been maintained by horizontal gene transfer. It is true that exogenous cell division genes are usually toxic, at least for E. coli, and lateral gene transfer is certainly more frequent in prokaryotes than previously thought, but it is doubtful that the frequency of lateral gene transfer has been so high as to spread the cluster throughout the entire bacterial domain. Moreover, the division and cell wall genes are ancient genes likely to belong to the group of orthologous genes that are not likely to participate in lateral gene transfer (Daubin et al., 2003). The sequence-based phylogenetic trees obtained for the individual genes are congruent with the standard 16S rRNA tree topology (Fig. 8), indicating that the cluster has not been subjected to extensive lateral gene transfer after the LCA. Therefore, none of these models is able to explain satisfactorily the existence of the dcw gene cluster, and although some of them might be partially correct, a new mechanism should be invoked that takes into account the properties of the cluster. 7. A model on the relation of bacterial cell shape and the dcw gene cluster 7.1 Gene order reflects protein-protein interactions It has been found that conservation of gene order in several gene pairs is a predictor of protein-protein interactions (Dandekar et al., 1998; Overbeek et al., 1999; Huynen et al., 2000; Chapter 11). As gene order is maintained during transcription and translation, it has been suggested that this correlation results from an interdependence of the folding of the proteins involved (co-translational folding; Dandekar et al., 1998). This interdependence should be weak in the case of the dcw cluster, as some of The dcw gene cluster 15 the genes have been successfully expressed in isolation from plasmids. Instead, we postulate that the maintenance of gene order is related to the cotranslational assembly and localization of protein complexes, and that the order might be important to promote the correct interactions among the different components of the complexes. Co-translational assembly and localization might be especially important for complexes that are in low copy numbers in the cells. It has been already pointed that some of the genes of the cluster have been expressed from plasmids and have been found to be functional and able to rescue conditional mutations in the chromosome, therefore clustering is not essential for the correct folding of the proteins produced, and is not essential for cell growth and division. Yet it has to be remembered that in most cases the cloned genes were in multicopy plasmids, and therefore the number of protein molecules produced was probably much higher than in the wild type cells. The high local concentrations of the polypeptide chains that may be reached in polysomes might favour assembly even if only a few molecules of some of the components exist in the cell (for example, in E. coli FtsQ is present at ca. 50 molecules/cell; Carson et al., 1991). In addition, as some of the products of the genes in the cluster are membrane proteins, the coupled transcription-translation-insertion of these proteins might direct the localization of the protein complexes to specific domains in the cell membrane (Norris et al., 1996). A co-translation mechanism would not necessarily mean that the order of the genes should be essential, only that it should have a significant effect on fitness. It might work on housekeeping genes, and it might be operative over large evolutionary distances, as far as there is some selective pressure to maintain the function of the protein complexes involved. The products of the fts genes of the cluster are good candidates for such mechanism, as they are known to localize in a ring at the septation site during division, and it is thought that there they form a protein complex responsible for septation (Nanninga, 1991; Rothfield et al., 1999). Moreover, several interactions among the products of ftsI, ftsQ, ftsA and ftsZ genes have been described (Dai and Lutkenhaus, 1992; Descoteaux and Drapeau, 1987; Weiss et al., 1999). Among the mur genes, on the other hand, some code for soluble and others for membrane proteins, all of them involved in the same biochemical pathway. Most of these proteins have been purified and characterized, and they are active in vitro in isolation (Duncan et al., 1990; Michaud et al., 1990; Pratviel-Sosa et al., 1991; Liger et al., 1995). However analysis of the pool levels of peptidoglycan precursors in E. coli has shown that the cellular concentrations of the metabolites in the pathway are below the Km values for the corresponding enzymes (van Heijenoort, 1996). The pools of lipids I and II in particular are very low (700 and 1000-2000 molecules/cell 16 Mingorance and Tamames respectively), and seem to be a limiting factor for the synthesis of peptidoglycan. It has been suggested that as these enzymes are not saturated by substrate, the efficiency and the control of the pathway could be greatly improved if they were part of a multiprotein complex and could transfer sequentially the products of every enzyme to the next enzyme of the pathway (van Heijenoort, 1996). This improvement might provide a selective force favouring the presence of a cluster of mur genes if the grouping of genes facilitates complex assembly. 7.2 Connecting the mur and fts complexes: the two-competing sites model for peptidoglycan synthesis The dcw cluster contains both mur and fts genes interspersed, therefore the co-translational assembly model requires that additional interactions between the mur complex(es) and the fts complex(es) should be operative and physiologically advantageous to explain the persistence of the cluster and its broad taxonomic range. We propose that the genomic structure of the dcw cluster, as well as its phylogenetic distribution result from the simultaneous operation of co-translational assembly of Mur and Fts proteins, and the existence of two-competing sites for peptidoglycan synthesis in rodshaped cells (Satta et al., 1994). In these, peptidoglycan synthesis involves different precursors and multienzyme complexes during elongation and during septation (Canepari et al., 1997; Höltje, 1998; de Pedro et al., 1997). Synthesis of new peptidoglycan during elongation occurs in patches, bands and areas of diffuse insertion in the sidewall of the cell (de Pedro et al., 2003), while the insertion of new peptidoglycan during septation occurs in a narrow zone at the division site. The two-competing sites model for peptidoglycan synthesis states that these two processes are in fact two competing reactions (or pathways), and that the balance between them regulates cell shape in rod-shaped bacteria, while in the cocci the lateral-wall elongation reaction is severely reduced or absent (Satta et al., 1994). Penicillin-binding proteins have been found to be part of two large multienzyme complexes in E. coli (Bhardwaj and Day, 1997), B. subtilis (Simon and Day, 2000) and Haemophilus influenzae (Alaedini and Day, 1999). We propose that in rod-shaped bacteria, prior to cell division, cotranslational folding and assembly of the products of the mur and fts genes results in the assembly and localization of a Mur-Fts complex able to synthesize murein precursors and channel them towards septal peptidoglycan synthesis. After cell division this complex disassembles, and the Mur moiety might diffuse through the membrane, and then interact with the lateral wall The dcw gene cluster 17 Figure 9. Schematic representation of the co-translational assembly and localization of multiprotein complexes involved in the elongation of the lateral cell wall, and in division. Coupled transcription-translation together with the insertion of membrane proteins, a process called “transertion” (Norris et al., 1996), facilitates the localized assembly of protein complexes at the division site. After division the Mur moiety of this complex might migrate through the membrane to interact with the elongation complex. elongation machinery, again to channel the murein precursors towards the synthesis of lateral peptidoglycan (Fig. 9). The localization of a Mur-Fts complex that synthesizes the murein precursors at the division site provides a mechanism to channel the flux of precursors (that provide matter and energy) towards the synthesis of septal peptidoglycan during division. This is an intense, and highly localized process that would be substrate-limited if the precursors were synthesized and used throughout the whole cell surface. This mechanism might be even more important in filamentous cells that grow apically to avoid diffusion of the precursors through the long cell body. On the contrary, the channeling of murein precursors might not be as important in cocci, in which there is one narrow zone of murein synthesis either for elongation and for division (Morlot et al., 2003; Pinho and Errington, 2003). The localization of the complex responsible for the synthesis of the peptidoglycan precursors would not be necessary in the cocci, and therefore the selective pressure to maintain 18 Mingorance and Tamames the clustering would be relieved, and as a consequence the dcw genes have often been dispersed. In other organisms, like the spirochetes, or the helicobacteria the lack of a compact cluster might also be related to their modes of cell growth and division. The localization of a Mur-Fts complex might need assistance because it involves the concentration of few molecules in a narrow area to form protein complexes that are predicted to be weak and transient, as the Z-ring is not a static protein assembly, but a highly dynamic structure (Stricker et al., 2002; Rueda et al., 2003). Therefore, gene clustering and co-translational assembly and localization of protein complexes might help rod-shaped cells to grow and divide more efficiently. However these are not by themselves morphogenetic mechanisms. Other genes have been associated to the morphogenesis of E. coli (Chapter 9). The mreB gene seems to have a key role, as it is found in prokaryotes (both bacteria and archaea) with rod or similar shapes, but not in the genomes of several cocci (Jones et al., 2001). As more genomes are sequenced exceptions arise, for example Methylococcus capsulatus and Magnetococcus sp., are cocci but both contain complete dcw clusters, and mreB homologs. These exceptions do not invalidate the model, that assumes that clustering may be present in all kind of cells, but it is a favourable trait only for the rod-shaped, and is dispensable in others. Some predictions can be made from our model that might be experimentally tested. For example, it would be expected that the dcw mRNA transcript (or part of it) and the products of the mur genes should colocalize with the Z-ring during septation in rod-shaped bacteria. Moreover, several weak interactions between the proteins coded by the genes of the cluster should be expected, and these should occur between protein pairs in a manner consistent with the order of the genes in the cluster. This last prediction is partially supported by the intriguing observation that in the Chlamydia the genes murC and ddlA are fused (Chopra et al., 1998), as it has been argued that the fusion of two genes in one genome indicates that the protein products of these two genes may interact and form complexes, even in other organisms in which the genes are not fused (Enright et al., 1999). Although the Chlamydia lack both peptidoglycan, and the ftsQAZ genes, they have the other genes of the cluster, and are sensitive to penicillin, suggesting that the pathway is active in these organisms (Ghuysen and Goffin, 1999; McCoy et al., 2003). The dcw gene cluster 8. 19 Conclusion It has been shown that extant bacterial lineages descend from rod-shaped, peptidoglycan containing bacteria (Siefert and Fox, 1998; Koch, 2003). It seems likely that those bacteria already contained a dcw cluster that had evolved in parallel with the peptidoglycan synthesizing machinery to allow an efficient co-ordination between growth and division by alternatively channelling the precursor synthesis towards the division site or to the lateral wall. This co-ordination might still favour gene clustering in the bacilli, but might become useless when the shape of the cells or their life cycles change during evolutionary history making the cluster susceptible to dispersal, reduction or reorganization. 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