BACKGROUND
gene expression is regulated at multiple levels, from accessibility of dna through the steps of transcription, post-transcriptional modification, translation and mrna degradation.
transcriptional regulation is a particularly important stage for the control of phenotypic variation in response to internal or external signals.
transcriptional regulation is largely enacted by transcription factors  that bind to sites in genomes and, either alone or in combination with other tfs, thereby activate or repress the production of mrna from one or more target genes.
in escherichia coli k <dig>   <dig> tfs which constitute more than 4% of the protein-coding gene complement have at least one annotated gene-regulatory interaction  <cit> .
many tfs fall within one of two broad classes, global or local, depending on the number of genes they regulate, the cellular processes in which they participate and their chromosomal location relative to their target genes  <cit> .
local regulators  regulate a restricted number of genes that are in close physical proximity on the chromosome, whereas global regulators coordinately target large numbers of genes at multiple locations along the chromosome.
alternative criteria for defining global regulators have been presented  <cit> .
like other types of cellular interactions, the transcriptional regulatory interactions of an organism can be abstracted as a network in which molecules  are represented as nodes  and regulatory interactions as edges.
there are many ways in which transcriptional regulatory networks  evolve, including via modifications to cis-regulatory regions of gene promoters  <cit> , to the tf proteins themselves and to other trans-acting regulatory factors  <cit> .
here we focus on modifications to bacterial trns that have arisen via lateral genetic transfer .
genomic studies leave no doubt that lgt has played a pervasive role in the evolution of prokaryotic genomes and is a significant source of phenotypic innovation among bacteria  <cit> .
successful lgt comprises a succession of steps: transfer and physical uptake of foreign dna into a host new cell; recombination into the main chromosome, or maintenance on an extrachromosomal element; integration into genetic regulatory and biomolecular interaction networks; and finally, establishment in the host population  <cit> .
cellular networks, including trns, necessarily change and evolve as new genetic material appears and existing genetic material is overwritten or lost.
in particular, newly introgressed lateral genes must recruit transcriptional regulators to become better integrated into host-regulatory networks and ensure appropriate stoichiometric and condition-dependent expression  <cit> .
recent analyses indicate that in e. coli, global regulators have mostly evolved vertically, whereas many local regulators have been acquired by lgt, often concurrently with the gene they regulate  <cit> .
in addition, e. coli genes of lateral origin exhibit more-complex regulation  than genes which have been inherited vertically  <cit> .
a major limitation of these and other previous studies of lgt and the evolution of transcription regulation is that they have taken whole genes as the unit of analysis, i.e.
assumed that genes are transferred intact during lgt.
in doing so, they have overlooked the potential significance of the transfer of within-gene fragments in the construction of genomic regions encoding trans-acting regulatory proteins and their targets.
lgt does not necessarily involve genomic regions delineated by exact gene boundaries  <cit> .
chan et al.
<cit>  reported clear evidence of one or more recombination breakpoints in at least  <dig>  of  <dig> sets of orthologous genes across prokaryotes,  <dig> of which did not show strong topological incongruence with the reference tree and, taking entire genes as the unit of analysis, would not be identified as lateral.
a similar proportion of cryptic lgt was found among  <dig> staphylococcus genomes  <cit> .
assigning entire genes as either vertical or lateral can thus significantly undervalue the contribution of lgt to the evolution of cellular networks  <cit> .
although lgt involving clusters of neighbouring genes and operons has been a focus of previous work  <cit> , lateral transfer clearly need not involve genomic regions delineated by exact gene boundaries  <cit> .
are neighbour tf genes and their nearby target genes transferred and integrated all at once into the host regulatory networks, as has been suggested, or have these regions been built up via a succession of transfers?
estimates of the frequency of lgt can be significantly affected by the number of genomes being compared and their relative genetic relatedness.
comparisons among strains of the same species can therefore be particularly informative  <cit> .
the e. coli - shigella clade has been well-sampled and annotated, with complete genome sequences of multiple diverse strains now available.
here we have analyzed  <dig> sets of single-copy, putatively orthologous genes from  <dig> strains of e. coli and shigella to determine the extent to which intact genes and within-gene fragments have been transferred and recombined within this clade.
we report the frequencies of within- and whole-gene transfer in neighbour and global regulator gene sets, and examine evidence for propensity toward co-transfer of neighbour regulators and their nearby target genes.
finally we explore the regulation of lateral genes, comparing the regulation of genes which have been transferred and recombined intact with the regulation of genes within which gene fragments have been recombined in lgt.
RESULTS
lateral genetic transfer in e. coli and shigella
we began our investigation of the impact of within-species lgt on the evolution of the e. coli trn with a systematic study of genetic transfer more broadly among strains belonging to the e. coli - shigella clade.
we analyzed  <dig> completely sequenced genomes:  <dig> e. coli genomes and seven genomes belonging to the closely related genus shigella.
while shigella species have historically been classified within a separate genus, it is now generally accepted that the shigella phenotype evolved multiple times from different e. coli clones and therefore are part of the e. coli species  <cit> .
e. coli strains have previously been divided into five distinct ecor phylogenetic groups  based on genetic markers  <cit> , and our dataset includes representative from each of these five groups.
we extracted  <dig> positionally homologous gene sets of size n ≥  <dig> from a whole-genome alignment of the  <dig> e. coli and shigella which was generated using the progressivemauve program included in mauve version  <dig> .
<dig>  <cit> .
positional homology is implied among aligned regions of the genomes, and the alignment can therefore be used to extract sets of putative positional homologs  <cit> .
nucleotide regions in any given genome are aligned to any other genome only once.
the  <dig> gene sets are therefore restricted to single-copy gene families and range in size from  <dig> to  <dig> members.
using this approach, paralogous sequences are separated into distinct gene sets, thereby reducing the complications of paralogy for our lgt inference.
families of size n <  <dig> were excluded from further analysis as they do not contribute to meaningful phylogenetic inference; as a consequence, we are not able to identify within-clade lgt affecting families of size n <  <dig>  figure  <dig> shows the size distribution of the  <dig> gene sets, and the representation of each of the  <dig> genomes among these gene sets is shown in figure  <dig>  the genome represented in the smallest number of the gene sets is that of the commensal e. coli crooks strain .
a number of pathogenic e. coli strains are also represented in relatively few of these gene sets: umn <dig>  apec <dig>  s <dig>  ut <dig>  ed1a and  <dig> are represented in fewer than  <dig> families.
with the exception of the group d strain umn <dig>  all these strains belong to phylogenetic group b <dig>
we inferred bayesian phylogenetic trees  <cit>  separately for each of the  <dig> e. coli protein sets and subsequently aggregated all adequately supported bipartitions  ≥  <dig> ) to generate an e. coli - shigella reference tree using matrix representation with parsimony   <cit> .
of the  <dig>   <dig> internal bipartitions in these  <dig> individual protein trees,  <dig>   <dig>  have posterior probability  ≥  <dig>  and were used to compute the mrp tree.
the resulting phylogeny  is our reference hypothesis about the vertical evolutionary relationships among the  <dig> e. coli and shigella.
we manually rooted the mrp tree based on the findings of touchon et al.
<cit>  who reconstructed the phylogenetic history of  <dig> e. coli and shigella strains, all of which were included in our work, using escherichia fergusonii as an outgroup.
our mrp tree is remarkably concordant with the e. coli - shigella phylogeny reported by touchon et al.
<cit> , which was inferred using a maximum likelihood approach based on  <dig> concatenated e. coli - shigella core gene sequences .
like the touchon et al.
tree, our mrp tree reconstructs four of the five ecor phylogenetic groups as monophyletic, with only group d recovered as polyphyletic .
three group d strains are represented in our mrp tree: two  are recovered as closely related to the group b <dig> strains, while the third d strain  forms a clade with shigella and the strains belonging to phylogroups a, b <dig> and e. only three bipartitions of our mrp tree are discordant with the phylogeny recovered by touchon et al.
<cit> , all of which occur within the b <dig> subtree .
next, using a rigorous two-phase approach  <cit>  we examined each of the  <dig> aligned gene sets for evidence of genetic recombination.
as the second phase of this approach is based on phylogenetic discrepancy, inference of an internal recombination breakpoint constitutes prima facie evidence of within-gene genetic transfer in a lineage leading to one or more of these genes.
following the classification system proposed by chan et al.
<cit> , we found clear evidence  of recombination breakpoints in  <dig> gene sets .
we also compared each of the  <dig> bayesian protein trees to the mrp reference tree.
of the  <dig> gene sets,  <dig>  were found to yield protein trees discordant with the mrp tree.
these protein trees do not share the common phylogenetic signal in the mrp reference tree and are interpreted as providing putative evidence of lgt within the e. coli - shigella lineage.
we found evidence for lgt in  <dig>  gene sets:  <dig>  show evidence of one or more observable recombination breakpoints within the boundaries of the gene, while a further  <dig>  yield protein trees that are topologically discordant with the reference tree but do not contain one or more observable recombination breakpoints .
using the terminology introduced by chan et al.
<cit> , we refer to these gene sets as observable recombination breakpoint positive  and observable recombination breakpoint negative  respectively.
the latter represent putative instances of the lateral transfer of the entire open reading frame  within the e. coli - shigella lineage.
of the  <dig> gene sets that yielded observable recombination breakpoints,  <dig> were found to be not topologically discordant with the mrp tree.
subtree prune-and-regraft  operations can be applied to reconcile topological discordance between two phylogenetic trees.
in an spr operation, any edge of a binary tree t  is cut, thereby giving two subtrees t' and t''.
the subtree t'' is then regrafted by annealing the same cut edge to a new vertex in t' created by the annealing operation.
each spr operation can be interpreted as equivalent to an lgt event involving a donor and recipient lineage.
the regrafted edge corresponds to the donor taxon and the cut edge corresponds to the recipient taxon.
individual protein trees may be more or less discordant with the mrp reference topology, i.e.
require different numbers of spr operations on the reference tree to reconcile the observed discordance  <cit> .
the minimum number of spr operations required to reconcile observed discordance between a test tree and the reference tree is referred to as its edit distance.
unfortunately, computing the edit distance between two unrooted trees is a nondeterministic polynomial-time -hard problem  <cit> , which in practice means that for sufficiently large data  it is impossible to know whether a globally optimal solution has been found.
we were able to recover an edit distance for  <dig>  of the  <dig> test trees.
these distances range between  <dig>  and  <dig> ; however, more than 70% of the discordant test trees have an edit distance ≤  <dig>  indicative of two or fewer transfer events .
although the extent of discordance is variable across gene sets, instances of discordance implying more-complex patterns of lgt are in a clear minority.
orb+ gene sets have a higher edit distance than orb- gene sets .
our results  demonstrate that within-species lgt plays an important part in the evolution of e. coli - shigella genomes.
as in the 144-genome  <cit>  and staphylococcus studies  <cit> , a solely whole-gene approach would have overlooked a major proportion of lgt.
our next goal was to use these lgt data to assess the contribution of within-species genetic exchange to trn network evolution.
lateral genetic transfer and the transcriptional regulatory network
a list of e. coli k <dig> genes encoding tfs, and the corresponding target genes, were extracted from regulondb  <cit>  and used to construct a e. coli k <dig> trn.
in this network, the genes are represented as nodes and regulatory interactions as directed edges.
the network has  <dig> nodes and  <dig> edges which encompass  <dig> unique tf-encoding genes enacting regulation of  <dig> target genes; as tf-encoding genes are themselves targets of other tfs, 'target genes' and 'tfs' are not mutually exclusive categories.
among the  <dig> tf-encoding genes in the reconstructed trn,  <dig>  were identified as lateral and another  <dig>  as vertical.
a further nine remained unclassified, as it was not possible to carry out phylogenetic analysis on the corresponding gene sets .
we calculated the out-degree and betweenness of the tf-encoding nodes to assess whether lgt and non-lgt nodes occupy different positions in the trn.
out-degree is the number of target genes regulated by a given tf node, and betweenness measures the frequency at which a given node lies on the shortest path between any pair of nodes in the network  <cit> .
we found that vertical tf nodes have higher out-degree than lateral tf nodes  and also have higher betweenness .
thus, vertical tf nodes regulate larger numbers of target genes and are found at more-central positions in the trn than are lateral tf nodes.
this suggests that high centrality may be a barrier to intra-clade transfer of tf genes; however, these results need to be interpreted cautiously, as the out-degree distribution for tf nodes is skewed.
the out-degrees of the nodes of trns have previously been shown to follow a scale-free distribution, characterized by a small proportion of highly connected hub nodes and a relative large proportion of weakly connected nodes  <cit> .
for example, in the reconstructed e. coli k <dig> trn, the  <dig> tfs that regulate the largest number of genes enact  <dig>  of the  <dig> regulatory interactions represented in the network.
given the large influence of so few regulators, we have followed the classification system used by price et al.
<cit>  to assign regulatory genes into two major categories on the basis of their corresponding number of target genes: global regulators, which comprise the  <dig> tfs that regulate the largest number of genes, and neighbour regulators, which regulate adjacent genes in the genome.
eight tf genes which regulate adjacent genes were excluded from the list of neighbour regulators because they are global regulators.
of the  <dig> unique regulatory genes represented in the network,  <dig> were assigned as global regulators and  <dig> as neighbour regulators.
a further  <dig> regulatory genes fell within neither of these two categories and were treated as a third category which we refer to as other  regulators.
in the following sections, we examine the evolutionary histories of regulatory genes in each of these three categories separately to determine if lgt has affected these different modes of regulation differently.
most global regulators evolve vertically within the e. coli - shigella clade
we found that gene sets coding for global regulators are less likely than other protein-coding genes to yield discordant protein trees  and are also less likely to be orb+ .
among the  <dig> global regulatory gene sets , only three were found to yield protein trees that are discordant with our mrp reference tree , and none showed evidence of one or more internal recombination breakpoints.
this indicates that global regulators have mostly evolved vertically within the e. coli - shigella clade.
global regulators are the  <dig> tfs that regulate the largest number of genes in the e. coli k <dig> trn.
here we have assigned regulators as global on the basis of their corresponding number of target genes, but this definition excludes tfs that are encoded by genes that are less well-connected in the network but are nonetheless important for cell fitness.
martínez-antonio and collado-vides  <cit>  have identified an alternative set of e. coli global tfs based on expanded criteria which included, among others, the variety of conditions under which the regulator exerts control.
seven tfs in e. coli satisfy all global tf criteria outlined by martínez-antonio and collado-vides  <cit>  .
a second tier consisting of nine e. coli regulators was also identified that, while less-connected in the network, were considered important for cell fitness , cspa, soxr).
this set of regulators includes five tfs , cspa, soxr) that were not included in the set of global regulators we identified on the basis of their number of target genes.
among all  <dig> e. coli global tfs identified by martínez-antonio and collado-vides  <cit> , only three yield protein trees that are discordant with mrp reference tree, and none shows evidence of one or more internal recombination breakpoints .
thus, even under an alternative definition of 'global', these regulators have mostly evolved vertically and are not subject to within-gene transfer.
these findings confirm and extend a previous report that in e. coli global regulators are less susceptible to lgt than other regulatory genes  <cit> .
definition of a global regulator as given in  <cit> .
neighbour regulators have frequently been transferred both intact and as within-gene fragments between strains of e. coli and shigella
in contrast to global regulators , we found evidence for lgt in at least  <dig>  of the  <dig> neighbour regulator gene sets of size n ≥ 4:  <dig>  of these were found to be orb+, while a further  <dig>  yielded protein trees that are topologically discordant with the reference tree but are orb- .
these sets of neighbour regulator genes are more likely than other protein-coding genes to have experienced within-clade lgt .
the proportion of neighbour regulators that yield discordant protein trees  is comparable to the proportion for all protein-coding genes  ; however, neighbour regulator gene sets are more likely to be orb+ than are other protein-coding genes .
orb- instances of putative lgt involving neighbour regulator gene sets are almost twice as common as orb+ instances.
thus the incorporation of intact neighbour regulators is more frequent than recombination involving within-gene fragments.
neighbour regulator gene sets were more likely to show evidence of lgt than other non-global regulatory gene sets   and were also more likely to be orb+ .
the proportion of neighbour regulator gene sets that yield discordant protein trees  is comparable to that of other non-global regulators  .
lgt of global, neighbour and other non-global regulators is summarised in figure  <dig>
other regulators are the regulatory gene sets which fall within neither the neighbour or global regulator categories.
function of the gene product  is correlated with relative susceptibility, or resistance, to lgt.
in particular, genes involved in dna replication, transcription and translation  have been shown to be less susceptible to transfer than are genes participating in housekeeping functions such as controlling energy metabolism and the biosynthesis of nucleotides and amino acids   <cit> .
to examine possible functional bias pertaining to the high frequency of lgt among neighbour regulator protein sets, we used annotations from the j. craig venter institute  comprehensive microbial resource http://cmr.jcvi.org/ to assign a functional category to all protein-coding genes in e. coli k <dig>  we then compared the functions of genes regulated by global, neighbour and other  regulators.
figure  <dig> shows, for each functional category, the proportion of genes regulated by each type of regulator  compared to its frequency among the full set of  <dig> e. coli k <dig> protein-coding genes.
genes regulated by at least one neighbour regulatory gene are significantly over- or underrepresented in ten  of the jcvi functional categories .
four of these ten functional categories are correspondingly over- or underrepresented for the sets of genes regulated by global and other  regulators .
we did not observe any functional categories to be overrepresented for genes regulated by neighbour regulators but underrepresented in genes regulated by global or other regulators.
biosynthesis of cofactors, prosthetic groups, and carriers is the only functional category underrepresented for genes regulated by neighbour regulators but overrepresented for genes regulated by other regulators; however, given the relatively small proportion of e. coli k <dig> genes that fall within this category it is not likely to be a significant source of functional bias.
these results suggest that the differences between the transfer frequencies of global, neighbour and other  regulatory genes are not due to the function of the genes they regulate.
neighbour regulators and their target genes evolve independently via lgt within the e. coli - shigella clade
we next examined the phylogenetic histories of neighbour regulators and their nearby target genes to determine if there is a propensity toward co-transfer of the elements involved in neighbour regulatory interactions .
we introduce the term regulatory neighbourhood to describe the set of adjacent target genes whose expression is directly modulated by a given neighbour regulator.
each regulatory neighbourhood encodes a set of collinear genes which includes a tf-encoding gene that regulates all other genes in the neighbourhood.
tf genes may be upstream or downstream of their regulatory targets.
we consider the neighbour regulator itself part of the regulatory neighbourhood even if autoregulation is not present.
the number of regulated genes in the regulatory neighbourhoods investigated here varies widely.
while many tf-encoding genes regulate only one adjacent target gene, others regulate many: the gene encoding tf fhla, for example, regulates  <dig> collinear target genes adjacent on the e. coli k <dig> chromosome .
if a neighbour regulator and its nearby target genes have been co-transferred within the e. coli - shigella lineage, we would expect that each of the gene sets in the regulatory neighbourhood will yield protein trees that are topologically discordant with the mrp reference tree and that support the same evolutionary history of e. coli and shigella strains.
in the case of recent co-transfer we would expect all genes in the neighbourhood to be orb-.
alternatively, a more-ancestral co-transfer could be followed by successive within-region lgt events which over time superimpose dna onto the previously incorporated region.
in such cases, we would expect regulatory neighbourhoods to be frequently interrupted by internal recombination breakpoints.
in total, there are  <dig> neighbour regulator gene sets corresponding to  <dig> unique regulatory neighbourhoods .
among the  <dig> neighbour regulators found to be orb-, only seven belong to regulatory neighbourhoods composed exclusively of orb- genes; of these seven, none contains gene sets that all support the same  evolutionary history.
moreover,  <dig>  of these  <dig> regulatory neighbourhoods contain at least one observable recombination breakpoint.
this suggests that regulatory neighbourhoods are probably not co-transferred as genomic stretches of collinear intact genes, or if they are, co-transfer has often been followed by successive layering of lgt events within the region.
one possible scenario is lgt of the regulatory neighbourhood into the e. coli - shigella lineage from an external clade, followed by transfer of within-neighbourhood gene fragments within the lineage.
only four of the  <dig> neighbourhoods investigated show no evidence of transfer within the e. coli - shigella lineage, suggesting that lgt has been very important in the evolution of these regions.
price et al.
<cit>  reported that in e. coli, many neighbour regulatory genes have been acquired by lgt, often simultaneously with the gene they regulate.
in contrast, we found no evidence to suggest that genes participating in neighbour regulatory interactions are constrained to co-transfer within the e. coli - shigella.
these authors used genes as the unit of analysis; here we find  <dig>  of  <dig> neighbour regulator gene sets examined to be orb+.
our finding that regulatory neighbourhoods have been frequently interrupted by internal recombination breakpoints suggests the dna has been successively superimposed onto these regions.
such layering of lgt events may defy analysis, as signatures of ancestral co-transfer of neighbour regulators and their target genes have been overwritten.
our findings extend an earlier report that bacterial tfs do not usually co-evolve with their regulated genes  <cit> .
regulation of lgt genes is no more complex than regulation of non-lgt genes
network representations of transcriptional regulation have previously revealed that, on average, each target gene is regulated by two tfs  <cit> .
we examined the regulation of lgt genes to determine if they are more or less likely than other genes to be regulated by multiple tfs.
we found genes transferred within the e. coli - shigella clade, and genes which have evolved vertically within the lineage, have a comparable number of regulators  .
this suggests that, at least in this case, genetic transfer among closely related strains is not constrained by relative complexity of regulation.
this result is in contrast to a previous report by price et al.
<cit>  that genes of lateral origin exhibit more-complex regulation  than non-lgt genes.
the authors hypothesized that many lgt genes are niche-specific and therefore require more-complex regulation.
to account for these contrasting results, we note a number of differences between the price et al.
study and ours.
price et al.
<cit>  focused on the evolutionary period since the divergence of e. coli from shewanella, and included lateral transfers into the lineage from external clades, whereas we focus only on transfers within e. coli and shigella.
as mentioned above, price et al.
<cit>  took whole genes as their units as analysis for lgt identification.
we examined the regulation of orb+ genes to determine if they are any more or less likely than genes which have been transferred intact to be regulated by multiple regulators, and found no difference in the relative complexity of regulation of orb+ and orb- lateral gene sets  .
discussion
gene regulation at the level of transcriptional control involves a complex network of regulatory interactions, one aspect of which is clearly demonstrated by the combinatorial control of individual genes by multiple tfs .
how lateral genes connect into such complex networks has remained an open issue  <cit> , although some general network properties of laterally transferred genes have been identified by intersecting public network data with lists of genes detected as lateral based on methods which assume genes are transferred as intact units  <cit> .
here we inferred  <dig>  of  <dig> e. coli - shigella orthologous protein-coding gene sets to be orb+:  <dig>  of these yielded protein trees that were concordant with the e. coli - shigella mrp tree.
thus, had we carried out our phylogenetic analysis of lgt based on entire genes only, we would have missed more than 30% of the lateral signal introduced by within-gene lgt and more than 8%  of all lateral signal within the clade.
the importance of accounting for within-gene transfer is even more-striking in the subset of protein-coding e. coli - shigella gene sets that code for tfs, where almost 40% of orb+ gene sets yield a protein tree concordant with the reference tree.
among the  <dig> tf genes for which we could construct a phylogenetic tree,  <dig> were inferred to be orb+, of which  <dig>  were recovered as topologically concordant with the reference.
clearly, a complete picture of how lgt genes connect into trns requires consideration of the impact of both whole- and within-gene transfer on individual regulators and their target genes.
although detection of recombination breakpoints within gene sets has provided a more-comprehensive view of the lateral signal in e. coli - shigella, certain caveats are in order.
foremost, we identified lgt only among lineages of e. coli and shigella, i.e.
our analysis does not identify foreign genes or gene fragments acquired laterally from outside the clade.
second, we did not attempt to identify donor or recipient lineages for genes identified as whole- or within-gene lateral; here, this would have required the identification of all possible spr operations that resolve observed discordance between a discordant protein tree and the mrp reference tree.
in many cases, multiple unique minimum spr paths are possible and the actual historical one cannot be determined  <cit> .
it is even more complicated to match donor and recipient lineages where there has been successive layering of lgt events, as seems likely where we infer multiple recombination breakpoints within a gene set or neighbourhood.
global and neighbour regulators play distinct regulatory roles.
our results confirm that these different roles imply specific constraints on the evolvability of these regulators.
most global regulators were found to evolve vertically.
in the rare case that a global regulator exhibited evidence of transfer within the e. coli - shigella clade, it appeared to have been transferred intact.
in contrast,  <dig>  of the  <dig> neighbour regulator gene sets for which we could construct phylogenetic trees showed evidence of lgt, and  <dig>  of these  <dig> showed evidence of within-gene transfer.
it is apparent that these different modes of regulation  evolve in a very different manner, at least with respect to lateral transfers within the e. coli - shigella clade.
if laterally acquired genetic material is to avoid being silenced and become fixed in its new host, its expression must be appropriately regulated and the gene products it encodes must interact successfully with host systems.
in the case of genes encoding proteins which act as regulators, this includes establishing regulation of appropriate targets.
it is striking that neighbour regulators have undergone more-frequent lateral genetic transfer than other non-global regulators.
this suggests that lateral regulators that have introgressed near their target genes have a higher probability to become fixed within the recipient.
a possible explanation is that physical proximity to target genes, in the immediate term, provides an advantage for the establishment of regulatory interactions following introgression of lateral regulators.
co-transfer of tf genes with their targets might alleviate at least some of the difficulties associated with integration into trns, as this would provide a means for efficient transcriptional regulation in the short term following introgression  <cit> ; however, we found no evidence that neighbour regulators are transferred with their nearby target genes.
in fact, neighbour regulators and their target genes appear to evolve independently via lgt within the e. coli - shigella clade.
regulatory neighbourhoods are frequently interrupted by internal recombination breakpoints, and consist of genomic regions with conflicting evolutionary histories.
this is most readily explained by these regions having been constructed through successive superimposed lgt events which overwrite more-ancestral lateral signal and thereby complicate, or even defy, analysis.
by the same token, we cannot rule out the possibility that ancestral co-transfers have been overwritten by subsequent lgt within regulatory neighbourhoods.
we found no difference in the relative complexity of regulation  of lateral versus vertical genes: relative complexity of regulation does not appear to be a barrier to lateral transfer within the e. coli - shigella clade.
although homologous replacement of a gene fragment, rather than transfer of an intact gene, might avoid some of the problems associated with recruitment of transcriptional regulators following introgression into the recipient genome  <cit> , we found no evidence to suggest a difference in the relative complexity of regulation  of whole-gene lgt genes and within-gene lgt genes.
these results are important because they suggest that relative complexity of regulation does not govern the impact of within-species lgt on trns.
CONCLUSIONS
our results demonstrate that the transfer of both intact genes and within-gene fragments has been frequent within the e. coli - shigella clade.
as more than 30% of orb+ gene sets do not yield discordant trees, the frequency of lgt within the clade would have been significantly underestimated by a phylogenetic study that assumed intact genes as the unit of genetic transfer.
we assessed the relative contribution of within- and whole- gene transfer to the evolution of individual regulators and their target genes as a necessary step toward understanding how lateral genes connect into trns and contribute to overall trn evolution.
it is clear that a substantial proportion of the individual gene components of trns have been subject to lgt within the e. coli - shigella clade, and that different modes of regulation are impacted by lgt differently.
we found that global regulators have mostly evolved vertically, and neighbour regulators have frequently been subject to genetic transfer within the e. coli - shigella clade.
comparing genetic transfer in neighbour regulator and other non-global regulator genes sets, we observed a lower frequency in the latter.
given these findings, future work is well positioned to extend the questions asked here to consider how the lateral transfer of genetic material among more distantly related organisms contributes to trn evolution and if this differs from the impact of lgt among strains of the same species.

 gene expression is regulated at multiple levels, from accessibility of dna through the steps of transcription, post-transcriptional modification, translation and mrna degradation. transcriptional regulation is a particularly important stage for the control of phenotypic variation in response to internal or external signals. transcriptional regulation is largely enacted by transcription factors that bind to sites in genomes and, either alone or in combination with other tfs, thereby activate or repress the production of mrna from one or more target genes. in escherichia coli k tfs which constitute more than 4% of the protein-coding gene complement have at least one annotated gene-regulatory interaction. many tfs fall within one of two broad classes, global or local, depending on the number of genes they regulate, the cellular processes in which they participate and their chromosomal location relative to their target genes. local regulators regulate a restricted number of genes that are in close physical proximity on the chromosome, whereas global regulators coordinately target large numbers of genes at multiple locations along the chromosome. alternative criteria for defining global regulators have been presented. like other types of cellular interactions, the transcriptional regulatory interactions of an organism can be abstracted as a network in which molecules are represented as nodes and regulatory interactions as edges. there are many ways in which transcriptional regulatory networks evolve, including via modifications to cis-regulatory regions of gene promoters, to the tf proteins themselves and to other trans-acting regulatory factors. here we focus on modifications to bacterial trns that have arisen via lateral genetic transfer. genomic studies leave no doubt that lgt has played a pervasive role in the evolution of prokaryotic genomes and is a significant source of phenotypic innovation among bacteria. successful lgt comprises a succession of steps: transfer and physical uptake of foreign dna into a host new cell; recombination into the main chromosome, or maintenance on an extrachromosomal element; integration into genetic regulatory and biomolecular interaction networks; and finally, establishment in the host population. cellular networks, including trns, necessarily change and evolve as new genetic material appears and existing genetic material is overwritten or lost. in particular, newly introgressed lateral genes must recruit transcriptional regulators to become better integrated into host-regulatory networks and ensure appropriate stoichiometric and condition-dependent expression. recent analyses indicate that in e. coli, global regulators have mostly evolved vertically, whereas many local regulators have been acquired by lgt, often concurrently with the gene they regulate. in addition, e. coli genes of lateral origin exhibit more-complex regulation than genes which have been inherited vertically. a major limitation of these and other previous studies of lgt and the evolution of transcription regulation is that they have taken whole genes as the unit of analysis, i.e. assumed that genes are transferred intact during lgt. in doing so, they have overlooked the potential significance of the transfer of within-gene fragments in the construction of genomic regions encoding trans-acting regulatory proteins and their targets. lgt does not necessarily involve genomic regions delineated by exact gene boundaries. chan et al. reported clear evidence of one or more recombination breakpoints in at least of sets of orthologous genes across prokaryotes, of which did not show strong topological incongruence with the reference tree and, taking entire genes as the unit of analysis, would not be identified as lateral. a similar proportion of cryptic lgt was found among staphylococcus genomes. assigning entire genes as either vertical or lateral can thus significantly undervalue the contribution of lgt to the evolution of cellular networks. although lgt involving clusters of neighbouring genes and operons has been a focus of previous work, lateral transfer clearly need not involve genomic regions delineated by exact gene boundaries. are neighbour tf genes and their nearby target genes transferred and integrated all at once into the host regulatory networks, as has been suggested, or have these regions been built up via a succession of transfers? estimates of the frequency of lgt can be significantly affected by the number of genomes being compared and their relative genetic relatedness. comparisons among strains of the same species can therefore be particularly informative. the e. coli - shigella clade has been well-sampled and annotated, with complete genome sequences of multiple diverse strains now available. here we have analyzed sets of single-copy, putatively orthologous genes from strains of e. coli and shigella to determine the extent to which intact genes and within-gene fragments have been transferred and recombined within this clade. we report the frequencies of within- and whole-gene transfer in neighbour and global regulator gene sets, and examine evidence for propensity toward co-transfer of neighbour regulators and their nearby target genes. finally we explore the regulation of lateral genes, comparing the regulation of genes which have been transferred and recombined intact with the regulation of genes within which gene fragments have been recombined in lgt.  lateral genetic transfer in e. coli and shigella we began our investigation of the impact of within-species lgt on the evolution of the e. coli trn with a systematic study of genetic transfer more broadly among strains belonging to the e. coli - shigella clade. we analyzed completely sequenced genomes: e. coli genomes and seven genomes belonging to the closely related genus shigella. while shigella species have historically been classified within a separate genus, it is now generally accepted that the shigella phenotype evolved multiple times from different e. coli clones and therefore are part of the e. coli species. e. coli strains have previously been divided into five distinct ecor phylogenetic groups based on genetic markers, and our dataset includes representative from each of these five groups. we extracted positionally homologous gene sets of size n ≥ from a whole-genome alignment of the e. coli and shigella which was generated using the progressivemauve program included in mauve version.. positional homology is implied among aligned regions of the genomes, and the alignment can therefore be used to extract sets of putative positional homologs. nucleotide regions in any given genome are aligned to any other genome only once. the gene sets are therefore restricted to single-copy gene families and range in size from to members. using this approach, paralogous sequences are separated into distinct gene sets, thereby reducing the complications of paralogy for our lgt inference. families of size n < were excluded from further analysis as they do not contribute to meaningful phylogenetic inference; as a consequence, we are not able to identify within-clade lgt affecting families of size n < figure shows the size distribution of the gene sets, and the representation of each of the genomes among these gene sets is shown in figure the genome represented in the smallest number of the gene sets is that of the commensal e. coli crooks strain. a number of pathogenic e. coli strains are also represented in relatively few of these gene sets: umn apec s ut ed1a and are represented in fewer than families. with the exception of the group d strain umn all these strains belong to phylogenetic group b we inferred bayesian phylogenetic trees separately for each of the e. coli protein sets and subsequently aggregated all adequately supported bipartitions ≥ ) to generate an e. coli - shigella reference tree using matrix representation with parsimony. of the internal bipartitions in these individual protein trees, have posterior probability ≥ and were used to compute the mrp tree. the resulting phylogeny is our reference hypothesis about the vertical evolutionary relationships among the e. coli and shigella. we manually rooted the mrp tree based on the findings of touchon et al. who reconstructed the phylogenetic history of e. coli and shigella strains, all of which were included in our work, using escherichia fergusonii as an outgroup. our mrp tree is remarkably concordant with the e. coli - shigella phylogeny reported by touchon et al., which was inferred using a maximum likelihood approach based on concatenated e. coli - shigella core gene sequences. like the touchon et al. tree, our mrp tree reconstructs four of the five ecor phylogenetic groups as monophyletic, with only group d recovered as polyphyletic. three group d strains are represented in our mrp tree: two are recovered as closely related to the group b strains, while the third d strain forms a clade with shigella and the strains belonging to phylogroups a, b and e. only three bipartitions of our mrp tree are discordant with the phylogeny recovered by touchon et al., all of which occur within the b subtree. next, using a rigorous two-phase approach we examined each of the aligned gene sets for evidence of genetic recombination. as the second phase of this approach is based on phylogenetic discrepancy, inference of an internal recombination breakpoint constitutes prima facie evidence of within-gene genetic transfer in a lineage leading to one or more of these genes. following the classification system proposed by chan et al., we found clear evidence of recombination breakpoints in gene sets. we also compared each of the bayesian protein trees to the mrp reference tree. of the gene sets, were found to yield protein trees discordant with the mrp tree. these protein trees do not share the common phylogenetic signal in the mrp reference tree and are interpreted as providing putative evidence of lgt within the e. coli - shigella lineage. we found evidence for lgt in gene sets: show evidence of one or more observable recombination breakpoints within the boundaries of the gene, while a further yield protein trees that are topologically discordant with the reference tree but do not contain one or more observable recombination breakpoints. using the terminology introduced by chan et al., we refer to these gene sets as observable recombination breakpoint positive and observable recombination breakpoint negative respectively. the latter represent putative instances of the lateral transfer of the entire open reading frame within the e. coli - shigella lineage. of the gene sets that yielded observable recombination breakpoints, were found to be not topologically discordant with the mrp tree. subtree prune-and-regraft operations can be applied to reconcile topological discordance between two phylogenetic trees. in an spr operation, any edge of a binary tree t is cut, thereby giving two subtrees t' and t''. the subtree t'' is then regrafted by annealing the same cut edge to a new vertex in t' created by the annealing operation. each spr operation can be interpreted as equivalent to an lgt event involving a donor and recipient lineage. the regrafted edge corresponds to the donor taxon and the cut edge corresponds to the recipient taxon. individual protein trees may be more or less discordant with the mrp reference topology, i.e. require different numbers of spr operations on the reference tree to reconcile the observed discordance. the minimum number of spr operations required to reconcile observed discordance between a test tree and the reference tree is referred to as its edit distance. unfortunately, computing the edit distance between two unrooted trees is a nondeterministic polynomial-time -hard problem, which in practice means that for sufficiently large data it is impossible to know whether a globally optimal solution has been found. we were able to recover an edit distance for of the test trees. these distances range between and; however, more than 70% of the discordant test trees have an edit distance ≤ indicative of two or fewer transfer events. although the extent of discordance is variable across gene sets, instances of discordance implying more-complex patterns of lgt are in a clear minority. orb+ gene sets have a higher edit distance than orb- gene sets. our results demonstrate that within-species lgt plays an important part in the evolution of e. coli - shigella genomes. as in the 144-genome and staphylococcus studies, a solely whole-gene approach would have overlooked a major proportion of lgt. our next goal was to use these lgt data to assess the contribution of within-species genetic exchange to trn network evolution. lateral genetic transfer and the transcriptional regulatory network a list of e. coli k genes encoding tfs, and the corresponding target genes, were extracted from regulondb and used to construct a e. coli k trn. in this network, the genes are represented as nodes and regulatory interactions as directed edges. the network has nodes and edges which encompass unique tf-encoding genes enacting regulation of target genes; as tf-encoding genes are themselves targets of other tfs,'target genes' and'tfs' are not mutually exclusive categories. among the tf-encoding genes in the reconstructed trn, were identified as lateral and another as vertical. a further nine remained unclassified, as it was not possible to carry out phylogenetic analysis on the corresponding gene sets. we calculated the out-degree and betweenness of the tf-encoding nodes to assess whether lgt and non-lgt nodes occupy different positions in the trn. out-degree is the number of target genes regulated by a given tf node, and betweenness measures the frequency at which a given node lies on the shortest path between any pair of nodes in the network. we found that vertical tf nodes have higher out-degree than lateral tf nodes and also have higher betweenness. thus, vertical tf nodes regulate larger numbers of target genes and are found at more-central positions in the trn than are lateral tf nodes. this suggests that high centrality may be a barrier to intra-clade transfer of tf genes; however, these results need to be interpreted cautiously, as the out-degree distribution for tf nodes is skewed. the out-degrees of the nodes of trns have previously been shown to follow a scale-free distribution, characterized by a small proportion of highly connected hub nodes and a relative large proportion of weakly connected nodes. for example, in the reconstructed e. coli k trn, the tfs that regulate the largest number of genes enact of the regulatory interactions represented in the network. given the large influence of so few regulators, we have followed the classification system used by price et al. to assign regulatory genes into two major categories on the basis of their corresponding number of target genes: global regulators, which comprise the tfs that regulate the largest number of genes, and neighbour regulators, which regulate adjacent genes in the genome. eight tf genes which regulate adjacent genes were excluded from the list of neighbour regulators because they are global regulators. of the unique regulatory genes represented in the network, were assigned as global regulators and as neighbour regulators. a further regulatory genes fell within neither of these two categories and were treated as a third category which we refer to as other regulators. in the following sections, we examine the evolutionary histories of regulatory genes in each of these three categories separately to determine if lgt has affected these different modes of regulation differently. most global regulators evolve vertically within the e. coli - shigella clade we found that gene sets coding for global regulators are less likely than other protein-coding genes to yield discordant protein trees and are also less likely to be orb+. among the global regulatory gene sets, only three were found to yield protein trees that are discordant with our mrp reference tree, and none showed evidence of one or more internal recombination breakpoints. this indicates that global regulators have mostly evolved vertically within the e. coli - shigella clade. global regulators are the tfs that regulate the largest number of genes in the e. coli k trn. here we have assigned regulators as global on the basis of their corresponding number of target genes, but this definition excludes tfs that are encoded by genes that are less well-connected in the network but are nonetheless important for cell fitness. martínez-antonio and collado-vides have identified an alternative set of e. coli global tfs based on expanded criteria which included, among others, the variety of conditions under which the regulator exerts control. seven tfs in e. coli satisfy all global tf criteria outlined by martínez-antonio and collado-vides. a second tier consisting of nine e. coli regulators was also identified that, while less-connected in the network, were considered important for cell fitness, cspa, soxr). this set of regulators includes five tfs, cspa, soxr) that were not included in the set of global regulators we identified on the basis of their number of target genes. among all e. coli global tfs identified by martínez-antonio and collado-vides, only three yield protein trees that are discordant with mrp reference tree, and none shows evidence of one or more internal recombination breakpoints. thus, even under an alternative definition of'global', these regulators have mostly evolved vertically and are not subject to within-gene transfer. these findings confirm and extend a previous report that in e. coli global regulators are less susceptible to lgt than other regulatory genes. definition of a global regulator as given in. neighbour regulators have frequently been transferred both intact and as within-gene fragments between strains of e. coli and shigella in contrast to global regulators, we found evidence for lgt in at least of the neighbour regulator gene sets of size n ≥ 4: of these were found to be orb+, while a further yielded protein trees that are topologically discordant with the reference tree but are orb-. these sets of neighbour regulator genes are more likely than other protein-coding genes to have experienced within-clade lgt. the proportion of neighbour regulators that yield discordant protein trees is comparable to the proportion for all protein-coding genes; however, neighbour regulator gene sets are more likely to be orb+ than are other protein-coding genes. orb- instances of putative lgt involving neighbour regulator gene sets are almost twice as common as orb+ instances. thus the incorporation of intact neighbour regulators is more frequent than recombination involving within-gene fragments. neighbour regulator gene sets were more likely to show evidence of lgt than other non-global regulatory gene sets and were also more likely to be orb+. the proportion of neighbour regulator gene sets that yield discordant protein trees is comparable to that of other non-global regulators. lgt of global, neighbour and other non-global regulators is summarised in figure other regulators are the regulatory gene sets which fall within neither the neighbour or global regulator categories. function of the gene product is correlated with relative susceptibility, or resistance, to lgt. in particular, genes involved in dna replication, transcription and translation have been shown to be less susceptible to transfer than are genes participating in housekeeping functions such as controlling energy metabolism and the biosynthesis of nucleotides and amino acids. to examine possible functional bias pertaining to the high frequency of lgt among neighbour regulator protein sets, we used annotations from the j. craig venter institute comprehensive microbial resource http://cmr.jcvi.org/ to assign a functional category to all protein-coding genes in e. coli k we then compared the functions of genes regulated by global, neighbour and other regulators. figure shows, for each functional category, the proportion of genes regulated by each type of regulator compared to its frequency among the full set of e. coli k protein-coding genes. genes regulated by at least one neighbour regulatory gene are significantly over- or underrepresented in ten of the jcvi functional categories. four of these ten functional categories are correspondingly over- or underrepresented for the sets of genes regulated by global and other regulators. we did not observe any functional categories to be overrepresented for genes regulated by neighbour regulators but underrepresented in genes regulated by global or other regulators. biosynthesis of cofactors, prosthetic groups, and carriers is the only functional category underrepresented for genes regulated by neighbour regulators but overrepresented for genes regulated by other regulators; however, given the relatively small proportion of e. coli k genes that fall within this category it is not likely to be a significant source of functional bias. these results suggest that the differences between the transfer frequencies of global, neighbour and other regulatory genes are not due to the function of the genes they regulate. neighbour regulators and their target genes evolve independently via lgt within the e. coli - shigella clade we next examined the phylogenetic histories of neighbour regulators and their nearby target genes to determine if there is a propensity toward co-transfer of the elements involved in neighbour regulatory interactions. we introduce the term regulatory neighbourhood to describe the set of adjacent target genes whose expression is directly modulated by a given neighbour regulator. each regulatory neighbourhood encodes a set of collinear genes which includes a tf-encoding gene that regulates all other genes in the neighbourhood. tf genes may be upstream or downstream of their regulatory targets. we consider the neighbour regulator itself part of the regulatory neighbourhood even if autoregulation is not present. the number of regulated genes in the regulatory neighbourhoods investigated here varies widely. while many tf-encoding genes regulate only one adjacent target gene, others regulate many: the gene encoding tf fhla, for example, regulates collinear target genes adjacent on the e. coli k chromosome. if a neighbour regulator and its nearby target genes have been co-transferred within the e. coli - shigella lineage, we would expect that each of the gene sets in the regulatory neighbourhood will yield protein trees that are topologically discordant with the mrp reference tree and that support the same evolutionary history of e. coli and shigella strains. in the case of recent co-transfer we would expect all genes in the neighbourhood to be orb-. alternatively, a more-ancestral co-transfer could be followed by successive within-region lgt events which over time superimpose dna onto the previously incorporated region. in such cases, we would expect regulatory neighbourhoods to be frequently interrupted by internal recombination breakpoints. in total, there are neighbour regulator gene sets corresponding to unique regulatory neighbourhoods. among the neighbour regulators found to be orb-, only seven belong to regulatory neighbourhoods composed exclusively of orb- genes; of these seven, none contains gene sets that all support the same evolutionary history. moreover, of these regulatory neighbourhoods contain at least one observable recombination breakpoint. this suggests that regulatory neighbourhoods are probably not co-transferred as genomic stretches of collinear intact genes, or if they are, co-transfer has often been followed by successive layering of lgt events within the region. one possible scenario is lgt of the regulatory neighbourhood into the e. coli - shigella lineage from an external clade, followed by transfer of within-neighbourhood gene fragments within the lineage. only four of the neighbourhoods investigated show no evidence of transfer within the e. coli - shigella lineage, suggesting that lgt has been very important in the evolution of these regions. price et al. reported that in e. coli, many neighbour regulatory genes have been acquired by lgt, often simultaneously with the gene they regulate. in contrast, we found no evidence to suggest that genes participating in neighbour regulatory interactions are constrained to co-transfer within the e. coli - shigella. these authors used genes as the unit of analysis; here we find of neighbour regulator gene sets examined to be orb+. our finding that regulatory neighbourhoods have been frequently interrupted by internal recombination breakpoints suggests the dna has been successively superimposed onto these regions. such layering of lgt events may defy analysis, as signatures of ancestral co-transfer of neighbour regulators and their target genes have been overwritten. our findings extend an earlier report that bacterial tfs do not usually co-evolve with their regulated genes. regulation of lgt genes is no more complex than regulation of non-lgt genes network representations of transcriptional regulation have previously revealed that, on average, each target gene is regulated by two tfs. we examined the regulation of lgt genes to determine if they are more or less likely than other genes to be regulated by multiple tfs. we found genes transferred within the e. coli - shigella clade, and genes which have evolved vertically within the lineage, have a comparable number of regulators. this suggests that, at least in this case, genetic transfer among closely related strains is not constrained by relative complexity of regulation. this result is in contrast to a previous report by price et al. that genes of lateral origin exhibit more-complex regulation than non-lgt genes. the authors hypothesized that many lgt genes are niche-specific and therefore require more-complex regulation. to account for these contrasting results, we note a number of differences between the price et al. study and ours. price et al. focused on the evolutionary period since the divergence of e. coli from shewanella, and included lateral transfers into the lineage from external clades, whereas we focus only on transfers within e. coli and shigella. as mentioned above, price et al. took whole genes as their units as analysis for lgt identification. we examined the regulation of orb+ genes to determine if they are any more or less likely than genes which have been transferred intact to be regulated by multiple regulators, and found no difference in the relative complexity of regulation of orb+ and orb- lateral gene sets. discussion gene regulation at the level of transcriptional control involves a complex network of regulatory interactions, one aspect of which is clearly demonstrated by the combinatorial control of individual genes by multiple tfs. how lateral genes connect into such complex networks has remained an open issue, although some general network properties of laterally transferred genes have been identified by intersecting public network data with lists of genes detected as lateral based on methods which assume genes are transferred as intact units. here we inferred of e. coli - shigella orthologous protein-coding gene sets to be orb+: of these yielded protein trees that were concordant with the e. coli - shigella mrp tree. thus, had we carried out our phylogenetic analysis of lgt based on entire genes only, we would have missed more than 30% of the lateral signal introduced by within-gene lgt and more than 8% of all lateral signal within the clade. the importance of accounting for within-gene transfer is even more-striking in the subset of protein-coding e. coli - shigella gene sets that code for tfs, where almost 40% of orb+ gene sets yield a protein tree concordant with the reference tree. among the tf genes for which we could construct a phylogenetic tree, were inferred to be orb+, of which were recovered as topologically concordant with the reference. clearly, a complete picture of how lgt genes connect into trns requires consideration of the impact of both whole- and within-gene transfer on individual regulators and their target genes. although detection of recombination breakpoints within gene sets has provided a more-comprehensive view of the lateral signal in e. coli - shigella, certain caveats are in order. foremost, we identified lgt only among lineages of e. coli and shigella, i.e. our analysis does not identify foreign genes or gene fragments acquired laterally from outside the clade. second, we did not attempt to identify donor or recipient lineages for genes identified as whole- or within-gene lateral; here, this would have required the identification of all possible spr operations that resolve observed discordance between a discordant protein tree and the mrp reference tree. in many cases, multiple unique minimum spr paths are possible and the actual historical one cannot be determined. it is even more complicated to match donor and recipient lineages where there has been successive layering of lgt events, as seems likely where we infer multiple recombination breakpoints within a gene set or neighbourhood. global and neighbour regulators play distinct regulatory roles. our results confirm that these different roles imply specific constraints on the evolvability of these regulators. most global regulators were found to evolve vertically. in the rare case that a global regulator exhibited evidence of transfer within the e. coli - shigella clade, it appeared to have been transferred intact. in contrast, of the neighbour regulator gene sets for which we could construct phylogenetic trees showed evidence of lgt, and of these showed evidence of within-gene transfer. it is apparent that these different modes of regulation evolve in a very different manner, at least with respect to lateral transfers within the e. coli - shigella clade. if laterally acquired genetic material is to avoid being silenced and become fixed in its new host, its expression must be appropriately regulated and the gene products it encodes must interact successfully with host systems. in the case of genes encoding proteins which act as regulators, this includes establishing regulation of appropriate targets. it is striking that neighbour regulators have undergone more-frequent lateral genetic transfer than other non-global regulators. this suggests that lateral regulators that have introgressed near their target genes have a higher probability to become fixed within the recipient. a possible explanation is that physical proximity to target genes, in the immediate term, provides an advantage for the establishment of regulatory interactions following introgression of lateral regulators. co-transfer of tf genes with their targets might alleviate at least some of the difficulties associated with integration into trns, as this would provide a means for efficient transcriptional regulation in the short term following introgression; however, we found no evidence that neighbour regulators are transferred with their nearby target genes. in fact, neighbour regulators and their target genes appear to evolve independently via lgt within the e. coli - shigella clade. regulatory neighbourhoods are frequently interrupted by internal recombination breakpoints, and consist of genomic regions with conflicting evolutionary histories. this is most readily explained by these regions having been constructed through successive superimposed lgt events which overwrite more-ancestral lateral signal and thereby complicate, or even defy, analysis. by the same token, we cannot rule out the possibility that ancestral co-transfers have been overwritten by subsequent lgt within regulatory neighbourhoods. we found no difference in the relative complexity of regulation of lateral versus vertical genes: relative complexity of regulation does not appear to be a barrier to lateral transfer within the e. coli - shigella clade. although homologous replacement of a gene fragment, rather than transfer of an intact gene, might avoid some of the problems associated with recruitment of transcriptional regulators following introgression into the recipient genome, we found no evidence to suggest a difference in the relative complexity of regulation of whole-gene lgt genes and within-gene lgt genes. these results are important because they suggest that relative complexity of regulation does not govern the impact of within-species lgt on trns.  our results demonstrate that the transfer of both intact genes and within-gene fragments has been frequent within the e. coli - shigella clade. as more than 30% of orb+ gene sets do not yield discordant trees, the frequency of lgt within the clade would have been significantly underestimated by a phylogenetic study that assumed intact genes as the unit of genetic transfer. we assessed the relative contribution of within- and whole- gene transfer to the evolution of individual regulators and their target genes as a necessary step toward understanding how lateral genes connect into trns and contribute to overall trn evolution. it is clear that a substantial proportion of the individual gene components of trns have been subject to lgt within the e. coli - shigella clade, and that different modes of regulation are impacted by lgt differently. we found that global regulators have mostly evolved vertically, and neighbour regulators have frequently been subject to genetic transfer within the e. coli - shigella clade. comparing genetic transfer in neighbour regulator and other non-global regulator genes sets, we observed a lower frequency in the latter. given these findings, future work is well positioned to extend the questions asked here to consider how the lateral transfer of genetic material among more distantly related organisms contributes to trn evolution and if this differs from the impact of lgt among strains of the same species. 
we then used these results to examine the evolutionary histories of tf genes, as well as the transcriptional regulation of lateral genes. rather, the frequency of recombination breakpoints, and conflicting evolutionary histories among neighbour regulators and their target genes, suggest that the genomic regions encoding these genes have been constructed through successive layering of lgt events within the clade. we found evidence of lgt in gene sets: show evidence of recombination breakpoints within the gene boundaries. at least of the neighbour regulator gene sets examined show evidence of lgt, of which have internal recombination breakpoints. the prevalence of recombination breakpoints within regulatory and target gene sets indicates that within-gene transfer has had a significant cumulative effect on the evolution of regulatory interactions in e. coli and shigella. we found that unlike global regulators, which have mostly evolved vertically, neighbour regulators have frequently been subject to transfer within the e. coli - shigella clade. neighbour regulators show no evidence of co-transfer with their nearby target genes. neighbour regulators show higher frequencies of transfer than other types of regulatory genes. here we apply phylogenetic and network-based methods to investigate the relationship between units of lateral transfer and recombination within the escherichia coli - shigella clade and the topological properties of genes in the e. coli transcriptional regulatory network. we carried out a systematic phylogenetic study of genetic transfer among sets of putatively orthologous genes from strains belonging to the e. coli - shigella clade. thus, within- and whole- gene lateral transfer is widespread among strains of e. coli and shigella. we find no difference in the relative complexity of regulation of lateral versus vertical genes. this implicates the topological properties of regulatory genes in the trn, and their physical proximity to targets on the chromosome, as contributing to successful lgt. changes in transcriptional regulation underlie many of the phenotypic differences observed within and between species of bacteria. lateral genetic transfer can significantly impact the transcription factor genes which drive these transcriptional changes. although much emphasis has been placed on lgt of intact genes, the units of transfer and recombination do not necessarily correspond to regions delineated by exact gene boundaries.
 changes in transcriptional regulation underlie many of the phenotypic differences observed within and between species of bacteria. lateral genetic transfer can significantly impact the transcription factor genes which drive these transcriptional changes. although much emphasis has been placed on lgt of intact genes, the units of transfer and recombination do not necessarily correspond to regions delineated by exact gene boundaries. here we apply phylogenetic and network-based methods to investigate the relationship between units of lateral transfer and recombination within the escherichia coli - shigella clade and the topological properties of genes in the e. coli transcriptional regulatory network.  we carried out a systematic phylogenetic study of genetic transfer among sets of putatively orthologous genes from strains belonging to the e. coli - shigella clade. we then used these results to examine the evolutionary histories of tf genes, as well as the transcriptional regulation of lateral genes. we found evidence of lgt in gene sets: show evidence of recombination breakpoints within the gene boundaries. thus, within- and whole- gene lateral transfer is widespread among strains of e. coli and shigella. we found that unlike global regulators, which have mostly evolved vertically, neighbour regulators have frequently been subject to transfer within the e. coli - shigella clade. at least of the neighbour regulator gene sets examined show evidence of lgt, of which have internal recombination breakpoints. neighbour regulators show no evidence of co-transfer with their nearby target genes. rather, the frequency of recombination breakpoints, and conflicting evolutionary histories among neighbour regulators and their target genes, suggest that the genomic regions encoding these genes have been constructed through successive layering of lgt events within the clade. we find no difference in the relative complexity of regulation of lateral versus vertical genes.  neighbour regulators show higher frequencies of transfer than other types of regulatory genes. this implicates the topological properties of regulatory genes in the trn, and their physical proximity to targets on the chromosome, as contributing to successful lgt. the prevalence of recombination breakpoints within regulatory and target gene sets indicates that within-gene transfer has had a significant cumulative effect on the evolution of regulatory interactions in e. coli and shigella. 