A Conserved Mammalian Protein Interaction Network

<div><p>Physical interactions between proteins mediate a variety of biological functions, including signal transduction, physical structuring of the cell and regulation. While extensive catalogs of such interactions are known from model organisms, their evolutionary histories are difficult to study given the lack of interaction data from phylogenetic outgroups. Using phylogenomic approaches, we infer a upper bound on the time of origin for a large set of human protein-protein interactions, showing that most such interactions appear relatively ancient, dating no later than the radiation of placental mammals. By analyzing paired alignments of orthologous and putatively interacting protein-coding genes from eight mammals, we find evidence for weak but significant co-evolution, as measured by relative selective constraint, between pairs of genes with interacting proteins. However, we find no strong evidence for shared instances of directional selection within an interacting pair. Finally, we use a network approach to show that the distribution of selective constraint across the protein interaction network is non-random, with a clear tendency for interacting proteins to share similar selective constraints. Collectively, the results suggest that, on the whole, protein interactions in mammals are under selective constraint, presumably due to their functional roles.</p> </div

PPI presence and absence at the different nodes in the rooted eutherian phylogenetic tree. A)

<p>At each node, we have shown the predicted percentage of human PPIs present at that node (necessarily 100% at the human tip). The percentages at the other seven tip nodes were inferred by the presence or absence of the orthologs of the two human proteins making up the PPI (<i>Methods</i>). We then inferred the states of the internal nodes under the assumption that a given PPI ortholog pair could appear only once in the phylogeny (<i>Methods</i>). The topology was visualized using FigTree <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052581#pone.0052581-Rambaut1" target="_blank">[61]</a>. Branch lengths are the mean K_s value (e.g., number of synonymous substitutions per synonymous site) found across the genes surveyed for that branch of the tree (See <i>Methods</i>). The five colored branches indicate potential origin points for a PPI under our limited parsimony model (<i>Methods</i>), while the two gray branches were used to estimate the rate of PPI <i>loss</i>. The dashed branches indicate the fact the K_s values could not be distinguished for these two branches because the models used produce unrooted trees. <b>B)</b> There is an association between the age of the branch along which a PPI appears (<i>x-</i>axis; estimated via K_s above) and the average interaction degree of the proteins that make up that interaction (<i>y</i>-axis). Note that the blue distance was estimated as one-half the K_s distance between the rodent-primate and horse-dog-cow clade in the unrooted topology of (<b>A</b>). See <i>Methods</i> for details.</p

Over- and under-represented GO terms of genes present at least once in a primate-specific PPI.

a<p>Observed instances of the GO term. 1675 genes present in primate PPIs vs 7201 genes never observed in primate PPIs.</p>b<p>Expected number of occurrences among an randomly-selected set of genes of the same size.</p>c<p><i>P</i>-values for the test of the hypothesis of no difference between the observed and expected number of occurrences of the term after a Bonferonni multiple-test correction.</p>d<p>Term was <i>under-represented</i> among the primate-specific PPIs.</p

Differences between primate-specific and phylogenetically-distributed interactions. A)

<p>Gene sets used in the GO analyses of primate-specific protein interactions. There are 8876 human genes having at least one interaction (for a total of 32,916 PPIs). Among those genes, 1502 interactions (encoded by 1675 genes) are found only in primates. Of those 1675 genes, 1,521 are also involved in other, nonprimate-specific interactions, and 154 are only involved in primate specific interactions. <b>B)</b> Genes involved in primate-specific interactions have, on average, more total interactions (i.e., the genes involved in these interactions tend to have a higher degree <i>k</i>). The distribution of the difference in degree (<i>k</i>) for each gene in a pair of interaction proteins was compared (here referred to as ‘absolute degree difference’, Δ<i>k</i>; <i>x</i>-axis). In black are the primate-specific interactions (primatePPIs) while red (dashed-line) shows the remainder of the interactions.</p

Over- and under-represented GO terms of genes present in PPIs where proteins in the protein pair have ω>0.5 for both branches vs remaining 4506 genes.

a<p>Observed instances of the GO term. 524 genes with ω>0.5 for both branches vs remaining 4506 genes (of 5030 genes in total from 12472 PPIs for which mirrortrees could be constructed with reliable ML scores).</p>b<p>Expected number of occurrences among an randomly-selected set of genes of the same size.</p>c<p>Uncorrected <i>P</i>-value for the test of the hypothesis of no difference between the observed and expected number of occurrences of the term.</p

Connectivity statistics of genes involved in primate PPIs vs genes part of nonprimate PPIs.

a<p>Set of genes involved <i>only</i> in primate-specific interactions.</p>b<p>All genes not in (<i>a</i>).</p>c<p>Wilcoxon test.</p

Paired cases of relaxed selective constraints for PPI pairs.

<p>For each clade in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052581#pone-0052581-g001" target="_blank">Figure 1</a>, we plot the number of cases where both members have either ρ>1.0 (<b>A</b>) or >0.5 (<b>B</b>). <i>P</i>-values are shown for the test of the hypothesis that there are more such shared cases of relaxed constraint than would be expected by chance (χ² test, <i>Methods</i>). Cases where no <i>P</i>-value is shown had too few observations of ρ>5 for valid statistical conclusions to be drawn.</p

Resistance Determinants and Mobile Genetic Elements of an NDM-1-Encoding <i>Klebsiella pneumoniae</i> Strain

<div><p>Multidrug-resistant <i>Enterobacteriaceae</i> are emerging as a serious infectious disease challenge. These strains can accumulate many antibiotic resistance genes though horizontal transfer of genetic elements, those for β-lactamases being of particular concern. Some β-lactamases are active on a broad spectrum of β-lactams including the last-resort carbapenems. The gene for the broad-spectrum and carbapenem-active metallo-β-lactamase NDM-1 is rapidly spreading. We present the complete genome of <i>Klebsiella pneumoniae</i> ATCC BAA-2146, the first U.S. isolate found to encode NDM-1, and describe its repertoire of antibiotic-resistance genes and mutations, including genes for eight β-lactamases and 15 additional antibiotic-resistance enzymes. To elucidate the evolution of this rich repertoire, the mobile elements of the genome were characterized, including four plasmids with varying degrees of conservation and mosaicism and eleven chromosomal genomic islands. One island was identified by a novel phylogenomic approach, that further indicated the <i>cps-lps</i> polysaccharide synthesis locus, where operon translocation and fusion was noted. Unique plasmid segments and mosaic junctions were identified. Plasmid-borne <i>bla</i>_CTX-M-15 was transposed recently to the chromosome by IS<i>Ecp1</i>. None of the eleven full copies of IS<i>26</i>, the most frequent IS element in the genome, had the expected 8-bp direct repeat of the integration target sequence, suggesting that each copy underwent homologous recombination subsequent to its last transposition event. Comparative analysis likewise indicates IS<i>26</i> as a frequent recombinational junction between plasmid ancestors, and also indicates a resolvase site. In one novel use of high-throughput sequencing, homologously recombinant subpopulations of the bacterial culture were detected. In a second novel use, circular transposition intermediates were detected for the novel insertion sequence IS<i>Kpn21</i> of the IS<i>NCY</i> family, suggesting that it uses the two-step transposition mechanism of IS<i>3</i>. Robust genome-based phylogeny showed that a unified <i>Klebsiella</i> cluster contains <i>Enterobacter aerogenes</i> and <i>Raoultella</i>, suggesting the latter genus should be abandoned.</p></div

Genomic islands.

a<p>Islands integrated into RNA genes are assigned the orientation of the target gene; remaining islands are arbitrarily assigned the genomic orientation.</p>b<p>Putative <i>att</i> sites identified at each flank.</p>c<p>Status in the closely related <i>K. pneumoniae</i> HS11286 chromosome; No/Yes, absent/present in HS11286; Alt, alternative island in HS11286 at the Kpn2146 island site.</p>d<p>The PHAST islands are strongly phage-like; we note where other islands have proteins suggestive of phage function, and other features of interest.</p>e<p>All islands had phyloblocks support.</p>f<p>Damage denotes loss of an acceptor stem nucleotide in the tRNA gene fragment <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099209#pone.0099209-Mantri1" target="_blank">[31]</a>; phage names are the closest relatives determined by PHAST.</p>g<p>CP003200.1/581777-594029; with IS<i>903</i> inserted at position 4245 with 9-bp DR; directly at tRNA-Leu <i>att</i> site with no intervening Kpn21L.</p>h<p>50403-bp alternative island at CP003200.1/1288317-1338720 with same initial 1989 bp as Kpn49R.</p>i<p>CP003200.1/1778319-1808718; 2 mismatches; with 7201 bp deleted from peg.320 to 3′ end of gpII intron.</p>j<p>CP003200.1/2277469-2325549; with IS<i>Ec22</i> at position 15471 with 8-bp DR.</p>k<p>CP003200.1/4502805-4558770; with IS<i>903B</i> insert at position 9777 with 9-bp DR.</p>l<p>CP003200.1/4819193-4835187; no mismatches.</p

Replicon copy numbers.

a<p>Unique 21-mers were identified for each replicon, and counted among MiSeq reads.</p>b<p>Mean 21-mer coverages were very similar to median values, and were normalized to that of the chromosome (which had 68.8 occurrences per 21-mer).</p