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. 2016 Sep 7;6:32590. doi: 10.1038/srep32590

Snapshots of a shrinking partner: Genome reduction in Serratia symbiotica

Alejandro Manzano-Marín 1,a, Amparo Latorre 1,2,b
PMCID: PMC5013485  PMID: 27599759

Abstract

Genome reduction is pervasive among maternally-inherited endosymbiotic organisms, from bacteriocyte- to gut-associated ones. This genome erosion is a step-wise process in which once free-living organisms evolve to become obligate associates, thereby losing non-essential or redundant genes/functions. Serratia symbiotica (Gammaproteobacteria), a secondary endosymbiont present in many aphids (Hemiptera: Aphididae), displays various characteristics that make it a good model organism for studying genome reduction. While some strains are of facultative nature, others have established co-obligate associations with their respective aphid host and its primary endosymbiont (Buchnera). Furthermore, the different strains hold genomes of contrasting sizes and features, and have strikingly disparate cell shapes, sizes, and tissue tropism. Finally, genomes from closely related free-living Serratia marcescens are also available. In this study, we describe in detail the genome reduction process (from free-living to reduced obligate endosymbiont) undergone by S. symbiotica, and relate it to the stages of integration to the symbiotic system the different strains find themselves in. We establish that the genome reduction patterns observed in S. symbiotica follow those from other dwindling genomes, thus proving to be a good model for the study of the genome reduction process within a single bacterial taxon evolving in a similar biological niche (aphid-Buchnera).

Obligate microbial symbionts (whether primary, secondary, tertiary, or other) are present in a variety of eukaryotic organisms, such as leeches (Annelida: Hirudinida)1, gutless oligochaetes (Annelida: Oligochaeta)2, and insects (Arthropoda: Insecta)3 (see ref. 4). These have the capacity to produce essential nutrients their hosts cannot synthesise nor obtain from their diet5,6,7,8, making them essential for the correct development and survival of their partners. On the other hand, facultative symbionts are dispensable, although under certain environmental challenges/niches, they can endow the host with desirable traits, ranging from defence against parasitoids or fungal parasites to survival after heat stress (reviewed in refs 9 and 10). Moreover, these facultative endosymbionts can even affect the performance of its host on a certain food source (e.g. a plant)11,12,13.

Whichever the symbiont’s function, taxonomic position, or status (facultative or obligate), a common feature from maternally inherited endosymbiotic organisms is the possession of a reduced genome, when compared to their free-living counterparts8,14,15,16,17,18,19,20,21,22. The sequencing of these genomes has undoubtedly provided important clues into the distinct features these display along the erosion process. While mildly-reduced genomes such as the one from Sodalis glossinidius, (facultative endosymbiont of the tsetse fly Glossina morsitans morsitans), Sodalis pierantonius (primary obligate endosymbiont from the rice weevil Sitohpilus oryzae), and Hamiltonella defensa (facultative endosymbiont from the aphid Acyrthosiphon pisum) show intermediate guanine-cytosine (hereafter GC) contents and a massive presence of both mobile elements (hereafter MEs) and pseudogenes23,24,25, highly reduced genomes such as the ones from Buchnera (primary obligate endosymbiont of aphids) and Blochmannia (primary obligate endosymbiont from carpenter ants) are highly compact with few pseudogenes and show no traces of MEs15,26. However valuable the study of these genomes is, most interesting is the study of different bacterial strains, belonging to a single genus, holding differentially-reduced genomes. Examples of these include Arsenohponus symbionts of a parasitic wasp (Nasonia vitripennis) (INSDC:AUCC00000000.1), the brown planthopper (Nilaparvata lugens)27, and louse flies (Diptera: Hippoboscidae)28 (INSDC:CP013920.1); Coxiella symbionts of ticks21,22; Tremblaya symbionts of mealybugs29,30,31; and Sodalis symbionts from the tsetse fly23 and rice weevil24.

Serratia symbiotica, a secondary endosymbiont harboured by many aphids32,33,34,35, is particular among currently sequenced bacterial symbionts. While strains harboured by Aphis fabae and Acyrthosiphon pisum (Aphidinae subfamily) are of facultative nature32,36,37, strains from the aphids Cinara tujafilina, Cinara cedri, and Tuberolachnus salignus (Lachninae subfamily) have established co-obligate associations with both the aphids and its primary obligate endosymbiont, Buchnera38,39,40. This co-obligate association was putatively triggered by a loss of the riboflavin biosynthetic genes in Buchnera from the Lachninae last common ancestor40. In addition, depending on the strain, its cell shape is either rod-like (strain CWBI-2.3 from Ap. fabae41, hereafter SAf), filamentous (strain Tucson from Ac. pisum42, hereafter SAp; and strain SCt-VLC from C. tujafilina38, hereafter SCt), or spherical (strain SCc from Cinara cedri39, hereafter SCc; and strain STs-Pazieg from T. salignus40, hereafter STs). Not surprisingly, these strains hold genomes of contrasting sizes, ranging from 3.58 to 0.65 mega base pairs (hereafter Mbp)16,20,38,39,40. Furthermore, although most strains have not yet (to our knowledge) been cultured, similarly to many insect obligate endosymbionts (reviewed in ref. 43), SAf is able to grow freely in anaerobic conditions on a rich medium41. Finally, while the phylogenetic relations of S. symbiotica are not fully resolved40, they show a clear sister relationship to Serratia marcescens, a species comprised of various free-living bacterial strains for which complete genomes are available.

In the current study, we analysed the genomes of currently-available S. symbiotica strains and compared them to the free-living insect pathogen S. marcescens strain Db11 (hereafter Db11)44. Through comparative genomics we investigated genome rearrangement, the enrichment, and loss, of MEs, and the erosion undergone by RNA features and the informational machinery in S. symbiotica. Additionally, we describe the diminution of certain genes and the possible functional consequences of these reductions. Finally, we relate all these features to different stages of the symbionts’ integration to the aphid-Buchnera symbiotic consortia and discuss the features which are convergent with other dwindling endosymbiotic genomes.

Results and Discussion

S. symbiotica strains and their shrinking genomes

Generally, “ancient” obligate endosymbionts hold highly reduced genomes, as small as 112 kilo base pairs45 (hereafter kbp). Conversely, more “recently” derived endosymbionts (including facultative ones) tend to display larger genomes, all the way up to the 4.5 Mbp genome of S. glossinidius (reviewed in refs 46 and 47). Accordingly, the different genomes of S. symbiotica strains land within and along this spectrum, from the large 3.58 Mbp genome of the facultative SAf to the small 0.65 Mbp genome of the co-obligate STs (Fig. 1). Similarly to the other large endosymbiotic genomes23,24,25,48,49, SAf, SAp, and SCt’s display a large enrichment of MEs, both in terms of diversity and number of them (Fig. 1; Supplementary Table S1 and Dataset S1). While Db11 holds nine insertion sequence (hereafter IS) elements and one TnTIR transposon; SAf, SAp, and SCt all hold over one hundred IS elements, show an enrichment of TnTIR transposons, and have gained group II intron mobile elements. Interestingly, the composition of the IS families (the most common type of MEs found within these genomes) seems to be lineage-specific. While IS3 and IS256 are the most prevalent in SAf and SAp (both facultative endosymbionts from Aphidinae aphids), IS481 and IS5 are the most common in SCt (co-obligate endosymbiont from C. tujafilina [Lachninae]). Conversely, the smaller genomes of SCc and STs lack any traces of MEs, congruent with similar-sized endosymbiotic genomes (see ref. 3). It is important to note that, given the highly fragmented assembly of SAp, the absolute counts of long repetitive mobile elements such as Tn3, IS3, and group II intron, might be underestimated. Following the trend of many other dwindling genomes3, all S. symbiotica have a GC content lower than that of their free-living counterpart, Db11. While this GC content is very similar among SAf, SAp, and SCt (52.1%), there is a marked decrease in SCc (29.2%) and even more so in STs (20.9%). Additionally, while there is a great enrichment of pseudogenes in SAf (126), SAp (550), SCt (916), and SCc (110), the small STs is almost deprived of these gene remnants. This genetic erosion comes along with a decrease in coding density. Accordingly, while SAf shows only a small decrease when compared to Db11 (87.9% to 78.2%), SAp, SCt, and SCc exhibit a marked drop down (56.8%, 53.4%, and 39.0%, respectively), mainly due to the increased pseudogenisation and “junk” DNA. On the other hand, the highly-reduced STs shows a high coding density (77.5%). This difference between SCc and STs is mainly due to high-amount “junk” DNA that is present in SCc’s genome, amounting to almost half of it39. Finally, we also found a gradual loss (from free-living Db11 to co-obligate intracellular STs) of RNA features (rRNAs, tRNAs, and other non-coding RNAs [hereafter ncRNAs]), revealing their different levels of genomic erosion. As has been previously observed in other endosymbionts, genome erosion comes with a “disturbance” of the functional profile of the organism, when compared to their free-living relatives14,18. Accordingly, prior analyses have described that while the functional profiles of free-living Serratia strains were very stable, a displacement of it was evident in SCc and SAp50. Through a similar analysis using all five currently available S. symbiotica strains, we have determined that while the recently-derived SAf, SAp, and SCt strains cluster together and are most similar to DB11, the highly-reduced SCc and STs form a divergent cluster from the rest of Serratia strains (Fig. 2A). These two S. symbiotica clusters differ mainly in the relative presence of MEs (category X) and translation-related genes (category J). While the former reflects the enrichment of SAf, SAp, and SCt’s in MEs, the latter evidences the common trend in highly reduced endosymbionts to retain housekeeping genes (e.g. category J includes all ribosomal proteins) (see ref. 3).

Figure 1. Genome reduction in S. symbiotica.

Figure 1

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Serratia genomes are depicted as circular plots and are arranged from largest (leftmost) to smallest (rightmost). From outermost to innermost, the rings within the genome plots display features on the direct strand, the reverse one, and RNA genes. Inside the circles, coloured lines connect the same-family IS elements scattered throughout the genome, following the colour code at the very bottom of the image. The grey bars on top of the genome plots describe the lifestyle and genome reduction stage. Underneath the genome plots, the strain alias and the host, between parenthesis, are shown. Below, a table showing the genomic features of each strain and pie charts displaying the relative abundance of IS-family elements, with the two most abundant highlighted by name. Underneath, the colour code for the different IS elements.

Figure 2. S. symbiotica functional profile displacement and genome rearrangement.

Figure 2

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(A) Heat map showing the two-way clustering of the S. symbiotica COG profile’s differences, relative to the free-living Db11 On the right, one-letter code for the COG categories. (B) Graphic linear representation of the rearrangements undergone in S. symbiotica. Orange and red vertical bars mark the position of single-copy conserved genes. Contigs that do not have single-copy conserved genes are not displayed. (C) Unrooted tree as calculated by MGR for the minimum number of rearrangements (reversals) undergone by S. symbiotica.

In the early stages of an endosymbiont’s genomic reduction, the genome’s enrichment in MEs can lead to rearrangement18,38. Generally, these rearrangements get fixed in the endosymbiotic lineage once the MEs have been lost, as is observed by the general genome-wide synteny displayed in Buchnera15,51, Blochmannia26, or Blattabacterium52. Nonetheless, some endosymbionts such as Portiera have been found to present lineage-specific genome rearrangements, putatively mediated by large repetitive intergenic regions53. Free-living Serratia strains display general genome-wide synteny50, on the contrary, S. symbiotica genomes display various rearrangements when compared to free-living Db11’s, and even among each other’s (Fig. 2B,C). Interestingly, while the less-reduced genome of SAf displays the most similarity (in terms of rearrangements) to Db11, the drastically-reduced genome of STs has accumulated the highest number of rearrangements. Also, SCc and STs’ genomes, which both lack MEs, display no synteny between them. These observations suggest that all S. symbiotica lineages have diverged before the loss of MEs, allowing a great number of lineage-specific reorganisation.

Erosion of essential amino acid biosynthetic routes

A general feature of endosymbiotic genomes is the loss of non-essential genes, leading to highly reduced genomes with a genetic repertoire specialised in the symbiotic function (reviewed in ref. 3). In aphids, Buchnera, the primary obligate endosymibont, is mainly in charge of producing essential amino acids (hereafter EAAs) for its host. Therefore, it is expected that co-existing symbionts show degraded biosynthetic routes involved in the production of these compounds. By analysing these routes in S. symbiotica, the gradual degradation of genes and operon attenuators implicated in the synthesis of EAAs becomes immediately evident (Fig. 3). The recently-derived SAf shows intact routes for most EAAs, with the notable expections of lysine and methionine. As already described in a previous study, there is a marked difference in the retention of leucine, arginine, and histidine biosynthetic-related genes even between the closely related facultative SAp and co-obligate SCt38. Finally, by comparing SCc and STs against the other S. symbiotica strains and each other, it becomes evident that both have become highly dependent on Buchnera for the supply of EAAs, with the main difference between SCc and STs being the purging of the remaining pseudogenes in the latter.

Figure 3. Erosion of essential amino acid biosynthetic genes in S. symbiotica.

Figure 3

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Inactivation tables showing the genes and leader sequences involved in the essential amino acid biosynthetic routes in S. symbiotica genomes compared to free-living Db11. At the top and left of the table, gene names for each enzymatic step and abbreviation for each Serratia strain, respectively. At the bottom of the table, black lines encompass the enzymatic steps required for the biosynthesis of each compound. Amino acid names are displayed using standard three-letter abbreviations. At the bottom-right, colour code for squares. Half-coloured boxes mean the genes catalysing the enzymatic step are present in different states.

Decay of RNA features and the loss of regulation

Typically, highly-reduced endosymbionts retain only a small number of ncRNAs and other RNA features54 (see Supplementary Figs S1 and S2). Through an annotation of these in the genomes of S. symbiotica and Db11, we have explored the erosion of RNA features (Fig. 4: top panel). We found that in the recently-derived endosymbionts SAf, SAp, and SCt, many of these features are still retained, although differentially. This points towards drift acting behind the loss of these features at the early stages of genome reduction. As expected, these three genomes show the acquisition of ME related ncRNAs, which all belong to the large class of self-catalytic group II introns (RF00029, RF01999, RF02001, RF02003, RF02005, RF02012). In the intermediate and late stages of drastic genome reduction SCc and STs find themselves in, respectively, most of the RNA features have been lost. Conserved features across S. symbiotica are the the 4.5S RNA component of the signal recognition particle (SRP) (ffs), the RNase P M1 RNA component (rnpB), the tmRNA (ssrA), the tpke11 small RNA (of unknown function), the leader sequence from the rnc-era transcription unit (coding for the ribonuclease 3 and the GTPase Era), and the alpha operon leader (coding for the 30S ribosomal subunits S13, S11, and S4; the 50S ribosomal subunit L17; and the DNA-directed RNA polymerase subunit alpha). The first three are interestingly also retained in other small genomes, but unidentifiable in some tiny genomes (Supplementary Fig. S1), hinting at these being essential functions retained until the last stages of genome reduction. Since most of these RNA features are related to the regulation of gene expression (small antisense RNAs, riboswitches, and leader sequences [including amino acid operon attenuators]), these losses would reflect a general trend of gene-regulation-loss in endosymbiotic genomes through the erosion of RNA features.

Figure 4. Decay of tRNA and other RNA features in Serratia genomes.

Figure 4

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(Top) Colour-coded diagram showing the decay of RNA features in different S. symbiotica genomes. On top of the matrix, gene names (for the first four columns) and RNA categories (for the rest) are indicated. On the bottom of the matrix, feature names are indicated for those features retained in SCc and STs. (Bottom) Colour-coded diagram showing the decay of tRNA features in different S. symbiotica genomes. On the top of the matrix, aminoacyl charging potential for each tRNA species (as inferred by TFAM). Each column represents a different anticodon. Asterisks indicate putative codon reassignments as judged by TFAM. fMet = N-Formylmethionine, kIle = lysylated isoleucine.

Regarding tRNAs, we observed a drastic reduction in tRNA-gene number, particularly marked in SCc and STs (Fig. 4: bottom panel). These losses, as in other reduced endosymbionts (see Supplementary Fig. S2), mainly affect redundancy rather than variety. Contrasting the other S. symbiotica genomes, we were unable to detect a tRNA with aminoacyl charging potential for glutamate in SCc. This is similar to what is observed in other tiny genomes, where some tRNAs with certain aminoacyl charging potential are absent (Supplementary Fig. S2). However, the presence of a tRNAGlu in a yet-unidentified plasmid cannot be discarded. Also, a loss of the selenocysteine tRNA is already present in the early co-obligate SCt, consistent with the loss of other selenocysteine-related genes, and completely absent in the smaller SCc and STs. It is important to remark that, in SAp, one of the tRNAMet copies has undergone a mutation in its anticodon (CAT → AAT), which could theoretically lead to the ATT codon to be recognised as coding for methionine. Finally, the tRNAs for formyl-methionine (tRNAfMet), in charge of aminoacylation of the starting methionine, and lysylated isoleucine (tRNAkIle) are conserved even in the two smallest S. symbiotica. This follows the trend observed in other reduced genomes (Supplementary Fig. S2) and points towards the essential nature of these tRNAs.

Informational machinery

By analysing and comparing the informational machinery (ribosome-, transcription-, translation-, and DNA replication/repair-related genes) in S. symbiotica strains, both high preservation as well as gradual patterns of deterioration become evident in different categories. The ribosome, as well as the tRNA aminoacylation genes are mostly perfectly preserved (Fig. 5: top). Marked differences include the presence of multiple copies of the three rRNA genes in SAf, SAp, and SCt, and the absence of two ribosomal proteins (rpsI and rplM) as well as the prolyl–tRNA synthetase gene (proS) in STs. While the retention of only one copy of the rRNA genes reflects the tendency of endosymbiotic, and other reduced genomes, to eliminate redundancy55, the loss of the rpsI gene (coding for the 30S ribosomal subunit S9) reflects the loss of a non-essential gene. In Escherichia coli, it has been experimentally proven that a null mutant of the rplI gene is able to grow, albeit showing a slow growth phenotype56,57. Most intriguing are the losses of the rplM (coding for the 50S ribosomal subunit L13) and proS genes. The former has been described as essential in E. coli57, and its loss could be related to the loss of rpsI, that together with rplM forms an operon. The latter, could reflect a putative functional replacement of the ProS protein activity by another non-specific aminoacyl–tRNA synthetase. This phenomenon has been observed for the prolyl–tRNA synthetase of Deinococcus radiodurans, which has the ability to charge cysteine to tRNACys58. This non-specific aminoacyl–tRNA synthetases have also been observed in archeal organisms (reviewed in ref. 59), suggesting this to be a common mechanism to cope with the lack of a specific aminoacyl–tRNA synthetase.

Figure 5. Informational machinery in S. symbiotica genomes.

Figure 5

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Circular plots displaying the different genes involved in functional categories (top left, ribosome; top right, tRNA aminoacylation; middle left, rRNA modifications; middle right, tRNA modification; bottom left, DNA replication and repair; bottom right, transcription and translation) in the informational machinery of S. symbiotica strains and Db11. Outer lines in each circular plot delimit the subcategory. Form outer to inner, the rings in the plot stand for the gene name, the colour-coded lines delimiting categories/complexes, and boxes standing for the presence or absence of the genes in Db11, SAf, SAp, SCt, SCc and STs.

Both rRNAs and tRNAs undergo a series of modifications that are required to produce the mature version of these ncRNAs (reviewed in refs 60 and 61). By analysing the genes involved in both rRNA and tRNA modifications, we observed that while the recently-derived SAf, SAp, and SCt hold a rather complete set (with particularly marked losses of 23S rRNA methyltransferases), the highly-reduced SCc and STs retain only a small fraction of these genes (Fig. 5: middle). With the notable exceptions of the fmt, tilS, trmD, tsaB, tsaC, tsaD, and tadA genes (all retained in the small SCc and STs), individual knockout mutants all of the rRNA and tRNA modification-related genes in E. coli (except miaE, which is not present in this organism) have dimmed them non-essential62,63,64,65. The fmt and tilS genes code for the proteins responsible for the attachment of a formyl group to the free amino group of methionyl-tRNAfMet (for initiator methionine)66 and the modification of the wobble base of the CAU anticodon of the tRNAkIle 67,68, respectively. The retention of these two genes thereby insure both the correct charging of the initiator methionine in proteins (which is posttranslationally-removed) and the accurate recognition of the AUA codon as coding for Isoleucine. On the other hand, the genes tsaB, tsaC, and tsaD (along with the tsaE gene) are responsible for the biosynthesis of the threonylcarbamoyladenosine (t6A) residue at position 37 of ANN-decoding tRNAs69. Interestignly, the tsaE gene (an ATPase), which has been found to be non-essential in E. coli under anaerobic conditions70, is missing from STs, thus the biosynthesis of t6A would be either putatively impaired or working in an unknown way. Finally, the tadA gene, which codes for a tRNA-specific adenosine deaminase that is essential for viability in E. coli71, is retained even in the small STs.

In regards to DNA replication and repair, the gene losses are particularly marked in the most genomically reduced symbionts, SCc and STS, affecting mostly DNA repair-related genes (Fig. 5: bottom left). This is also observed in other reduced endosymbionts (see ref. 47), and is possibly related to the triggering of more drastic genome erosion (reviewed in ref. 3). DNA replication-related losses affect the non-essential holE gene of the DNA polymerase, the priA-dependent primosome (retaining an elementary DNA-dependent one [missing the auxiliary Hup proteins]), and the gyrA subunit of the DNA gyrase. These latter, although identified as essential in E. coli63, has also been found to be missing from tiny genomes46,47, thereby suggesting its function could be taken over by an alternative enzyme or it actually being non-essential in some endosymbiotic organisms.

In terms of transcription- and translation-related genes, a high degree of retention in all S. symbiotica genomes can be observed (Fig. 5: bottom right). Gene losses mainly affect the sigma factors, with STs retaining only the rpoD and rpoH genes, coding for σ70 and σ32, respectively. While the former is generally preserved in endosymbionts47, the latter is missing from endosymbionts such as Blattabacterium and Nasuia. σ32 is required for the normal expression of heat shock genes and for the heat shock response through the regulation of the synthesis of heat shock proteins72, and thus its retention/loss could be specific of certain endosymbiotic systems.

Dwindling genes: stripping proteins down to the bones

Through the manual curation of the annotation of SCt, SCc, and STs endosymbionts38,40, we noted that some genes (atpC, cysJ, deaD, dnaX, ftsN, hscA, metG, pcnB, rnr, and tolC) seemed to be shorter in STs, and sometimes consistently shrunken across S. symbiotica, compared to those of free-living E. coli and even Db11. However, while these genes showed truncated or missing domains, they displayed a high degree of sequence conservation when compared to Db11. Thorough examination of these shrunken genes, revealed that experimental evidence, mainly from E. coli, have proven that truncated versions of these proteins were able to function with few to none obvious phenotypic consequences (details recorded in the annotation files available from the INSDC). Particularly evident is the loss of non-essential domains in six proteins: AceF, DnaX, FtsK, FtsN, and Rnr (Fig. 6). The AceF protein (E2 component of pyruvate dehydrogenase complex) has undergone the loss of one or two biotin/lypoyl domains (PF00364) in all S. symbiotica, namely STs retains only one. In E. coli, it has been shown, through the in vitro deletion of biotin/lipoyl domains, that one single domain suffices with respect to enzyme activity and protein function73. The tau subunit of the DNA polymerase III is coded by the dnaX gene, however an alternative isoform, denominated gamma subunit, is produced due to a programmed ribosomal frameshifting, which leads to a premature stop codon in the −1 frame at codon 43074. in vitro experiments with the shorter isoform, which lacks the tau 4 and 5 domains (PF12168 and PF12170), indicate that gamma is sufficient for replication75. The most drastic gene diminutions are observed in the ftsK and ftsN genes (whose products are involved in cell division), where SCc and STs preserve only a very small portion of the original gene. Independent in vivo experiments in E. coli mutants coding only for truncated FtsK (amino acids 1–200)76 or FtsN (amino acids 1–119)77 proteins, have corroborated that these tiny versions are sufficient for cell division, although short to long filamentous cells were observed to occur. Regarding MetG (Methionyl tRNA–synthetase), both SCc and STs are lacking the C-terminal putative tRNA binding domain (PF01588). Genetic complementation studies and characterization of C-terminally truncated enzymes in E. coli, established that MetG can be reduced to 547 residues without significant effect on either the activity or stability of the enzyme78. Finally, a deletion of the C-terminal basic domain of the Rnr protein (ribonuclease R) can be observed only in STs. This could lead to an increase in activity of this enzyme, since assays using purified truncated Rnr proteins from mutant E. coli, lacking the 83 residues from the C-terminus, were shown to display higher affinity and circa 2-fold higher activity than full length wild-type Rnr (on poly[A], A[17] and A[4] substrates)79. Through the alignment of the aforementioned putatively-functional proteins against other small and tiny genomes, we corroborated most of these gene diminutions are common among these organisms (Supplementary Dataset S1). This suggests that selection might favour gene diminution (the retention of only essential domains of the coded protein), relaxing selective constraints in non-essential gene regions, thus further contributing to genome reduction.

Figure 6. Diminution of genes in S. symbiotica.

Figure 6

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Graphic representation of the proteins that show an evident loss of non-essential domains (as judged by experimental evidence in E. coli) in S. symbiotica. Domains in each frame are represented by coloured boxes, with similar colours used for repeated domains in each protein. On top of each box, the domain’s is provided. Above each frame, the protein’s name is stated. On the bottom-right of every frame, a scale bar is provided.

Conclusion

S. symbiotica strains analysed here have all been evolving under a similar environment, the aphid-Buchnera symbiotic system. We have established that S. symbiotica strains can be considered to be along the genome reduction spectrum from a free-living bacterium to a drastically-reduced endosymbiont, thus providing “snapshots” of the genome reduction process. SAf would thus represent the very first stages of genome reduction, having not yet lost its ability to be grown in axenic culture and having undergone a mild genome shrinkage and few rearrangements, when compared to the free-living Db11. SAp and SCt would be a stage further down the path, having a more reduced genome than SAf and showing a massive enrichment in both pseudogenes and MEs. However, SCt has already done the transition to becoming a co-obligate endosymbiont, and thus shows more drastic gene losses in the EAAs’ biosynthetic pathways. SCc and STs find themselves in more advanced stages of genome reduction and integration to their symbiotic systems, having established a series of metabolic dependencies and complementation with Buchnera for the synthesis of several essential compounds. Nonetheless, SCc differs greatly from STs in genome size, which is explained by the former being in a recent stage of an advanced genome erosion, thus retaining several pseudogenes and “junk” DNA. Both SCc and STs display a drastic genome-wide gene loss, and particularly in their ncRNA repertoire and informational machinery. Through the comparison of these S. symbiotica strains, we were able to hint at essential retained functions, which not surprisingly are shared with other highly-reduced endosymbionts. The detailed study of protein diminution in S. symbiotica revealed a common tendency of endosymbionts to loose non-essential protein domains, and thus constituting an additional route towards genome reduction. We expect the further study of this particular endosymbiont of aphids will continue to provide important clues into the intriguing process of genome reduction.

Methods

Annotation of protein-coding genes

All protein-coding genes that were not found in their respective S. symbiotica genomes were searched for using the online version of tblastn80 with S. marcescens’ or E. coli’s protein as query. All positive hits with an e-value ≤10−3 were then manually curated. Domains within a protein were annotated using the InterProScan81 webserver and through alignments against E. coli’s proteins using MAFFT v7.22082. Circular representations of presence/absence of genes were done using circos v0.6783 and edited in Inkscape v0.91. COG categories for proteins were assigned using blastx and ad hoc perl scripts to select the best non-overlapping hits with an e-value threshold of ≤10−3. Then, COG categories absolute counts were converted to relative ones per organism. Finally, in order to analyse the disturbance of S. symbiotica’s functional profiles from that of free-living Db11, we subtracted Db11’s relative frequency per COG from each value within the same row. Visual display of COG categories was done using R and the gplots library, followed by manual editing in Inkscape.

Annotation of RNA features

tRNA features were annotated using tRNAscan-SE v1.3.184 (-B option for the bacterial model) and TFAM v1.4, followed by manual curation. All other RNA features were searched for using Infernal v1.1.185 (–cut_tc –mid) against the Rfam v12.0 database86. All hits with an e-value ≤10−3 were considered and manually curated. Visual displays were done using R and the gplots library, followed by manual editing in Inkscape. Plain-text source files used for the plotting of RNA features can be found in https://dx.doi.org/10.6084/m9.figshare.3413932.v1.

Table 1. Accession codes.

Species Strain Genome size (Mbp) Accession      
Serratia marcescens Db11 5.11 HG326223      
Serratia symbiotica CWBI-2.3 3.58 GCA_000821185.1      
Serratia symbiotica Tucson 2.57 GCA_000186485.2      
Serratia symbiotica SCt-VLC 2.49 GCA_900002265.1      
Serratia symbiotica SCc 1.76 CP002295      
Serratia symbiotica STs-Pazieg 0.65 LN890288      
Baumannia cicadellinicola BGSS 0.76 CP008985      
Blochmannia chromaiodes 640 0.79 CP003903      
Blattabacterium sp. Bge 0.64 CP001487      
Buchnera aphidicola BCc 0.42 CP000263      
Nasuia deltocephalinicola PUNC 0.11 CP013211      
Sulcia muelleri GWSS 0.25 CP000770      
Tremblaya phenacola PAVE 0.17 CP003982      
Wigglesworthia glossinidia brevipalpis 0.70 BA000021      
Zinderia insecticola CARI 0.21 CP002161      
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Accessions for all organisms used in this study.

Rearrangement analysis

Single copy shared proteins among S. symbiotica strains and Db11 were calculated as in ref. 40. Briefly, we used OrthoMCL v2.0.987 to build the orthologous groups of proteins, followed by manual curation aimed at joining rapidly-evolving proteins such as outer membrane proteins. This proteins were then used as rearrangement markers for calculating a minimal rearrangement phylogeny in MGR v2.0.388 (no heuristics). Scaffold for unfinished genomes (SAf, SAp, and SCt) were arranged as to minimise the distance against Db11. Tree visualisation was done in FigTree v1.4.1. Rearrangement graphic was done in R using the genoPlotR library89. All graphics were edited in Inkscape.

Additional Information

How to cite this article: Manzano-Marín, A. and Latorre, A. Snapshots of a shrinking partner: Genome reduction in Serratia symbiotica. Sci. Rep. 6, 32590; doi: 10.1038/srep32590 (2016).

Supplementary Material

Supplementary Information
srep32590-s1.pdf (215.3KB, pdf)
Supplementary Dataset 1
srep32590-s2.zip (22.7KB, zip)

Acknowledgments

This work has been funded by the Ministerio de Economía y Competitividad (Spain) co-financed by FEDER funds [BFU2015-64322-C2-1-R to A.L.]; the European Commission [Marie Curie FP7 PITN-GA-2010-264774-SYMBIOMICS to A.M.-M]; and the Consejo Nacional de Ciencia y Tecnología (Mexico) [Doctoral scholarship CONACYT 327211/381508 to A.M.-M.]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Footnotes

Author Contributions A.M.-M. and A.L. conceived and designed the study. A.M.-M. analysed the data. A.M.-M. and A.L. wrote, reviewed, and approved the final version of the manuscript.

References

  1. Perkins S. L., Budinoff R. B. & Siddall M. E. New Gammaproteobacteria associated with blood-feeding leeches and a broad phylogenetic analysis of leech endosymbionts. Appl. Environ. Microbiol. 71, 5219–5224 (2005). [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Giere O. & Langheld C. Structural organisation, transfer and biological fate of endosymbiotic bacteria in gutless oligochaetes. Mar. Biol. 93, 641–650 (1987). [Google Scholar]
  3. Moran N. A., McCutcheon J. P. & Nakabachi A. Genomics and Evolution of Heritable Bacterial Symbionts. Annu. Rev. Genet. 42, 165–190 (2008). [DOI] [PubMed] [Google Scholar]
  4. Buchner P. Endosymbiose der Tiere mit Pflanzlichen Mikroorganismen (Birkhäuser Basel, Basel, 1953). [Google Scholar]
  5. Nogge G. Significance of symbionts for the maintenance of an optimal nutritional state for successful reproduction in haematophagous arthropods. Parasitology 82, 101–104 (1981). [Google Scholar]
  6. Douglas A. E. Nutritional interactions in insect-microbial symbioses: aphids and their symbiotic bacteria Buchnera. Annu. Rev. Entomol. 43, 17–37 (1998). [DOI] [PubMed] [Google Scholar]
  7. Woyke T. et al. Symbiosis insights through metagenomic analysis of a microbial consortium. Nature 443, 950–5 (2006). [DOI] [PubMed] [Google Scholar]
  8. Manzano-Marín A., Oceguera-Figueroa A., Latorre A., Jiménez-García L. F. & Moya A. Solving a Bloody Mess: B-Vitamin Independent Metabolic Convergence among Gammaproteobacterial Obligate Endosymbionts from Blood-Feeding Arthropods and the Leech Haementeria officinalis. Genome Biol. Evol. 7, 2871–2884 (2015). [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Oliver K. M., Degnan P. H., Burke G. R. & Moran N. A. Facultative Symbionts in Aphids and the Horizontal Transfer of Ecologically Important Traits. Annu. Rev. Entomol. 55, 247–266 (2010). [DOI] [PubMed] [Google Scholar]
  10. Oliver K. M., Smith A. H. & Russell J. A. Defensive symbiosis in the real world - advancing ecological studies of heritable, protective bacteria in aphids and beyond. Funct. Ecol. 28, 341–355 (2014). [Google Scholar]
  11. Leonardo T. E. & Muiru G. T. Facultative symbionts are associated with host plant specialization in pea aphid populations. Proc. Biol. Sci. 270, S209–S212 (2003). [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Tsuchida T., Koga R. & Fukatsu T. Host Plant Specialization Governed by Facultative Symbiont. Science 303, 1989–1989 (2004). [DOI] [PubMed] [Google Scholar]
  13. Ferrari J., Scarborough C. L. & Godfray H. C. J. Genetic Variation in the Effect of a Facultative Symbiont on Host-Plant Use by Pea Aphids. Oecologia 153, 323–329 (2007). [DOI] [PubMed] [Google Scholar]
  14. Gil R. et al. The genome sequence of Blochmannia floridanus: Comparative analysis of reduced genomes. Proc. Natl. Acad. Sci. USA 100, 9388–9393 (2003). [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. van Ham R. C. H. J. et al. Reductive genome evolution in Buchnera aphidicola. Proc. Natl. Acad. Sci. USA 100, 581–586 (2003). [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Burke G. R. & Moran N. A. Massive Genomic Decay in Serratia symbiotica, a Recently Evolved Symbiont of Aphids. Genome Biol. Evol. 3, 195–208 (2011). [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Nikoh N., Hosokawa T., Oshima K., Hattori M. & Fukatsu T. Reductive Evolution of Bacterial Genome in Insect Gut Environment. Genome Biol. Evol. 3, 702–714 (2011). [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Clayton A. L. et al. A novel human-infection-derived bacterium provides insights into the evolutionary origins of mutualistic insect-bacterial symbioses. Plos Genet. 8, e1002990 (2012). [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Nakabachi A. et al. Defensive Bacteriome Symbiont with a Drastically Reduced Genome. Curr. Biol. 23, 1478–1484 (2013). [DOI] [PubMed] [Google Scholar]
  20. Foray V. et al. Whole-Genome Sequence of Serratia symbiotica Strain CWBI-2.3T, a Free-Living Symbiont of the Black Bean Aphid Aphis fabae. Genome Announc. 2, e00767–14 (2014). [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Gottlieb Y., Lalzar I. & Klasson L. Distinctive Genome Reduction Rates Revealed by Genomic Analyses of Two Coxiella-Like Endosymbionts in Ticks. Genome Biol. Evol. 7, 1779–1796 (2015). [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Smith T. A., Driscoll T., Gillespie J. J. & Raghavan R. A. Coxiella-Like Endosymbiont Is a Potential Vitamin Source for the Lone Star Tick. Genome Biol. Evol. 7, 831–838 (2015). [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Toh H. et al. Massive genome erosion and functional adaptations provide insights into the symbiotic lifestyle of Sodalis glossinidius in the tsetse host. Genome Res. 16, 149–156 (2005). [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Oakeson K. F. et al. Genome degeneration and adaptation in a nascent stage of symbiosis. Genome Biol. Evol. 6, 76–93 (2014). [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Degnan P. H., Yu Y., Sisneros N., Wing R. A. & Moran N. A. Hamiltonella defensa, genome evolution of protective bacterial endosymbiont from pathogenic ancestors. Proc. Natl. Acad. Sci. USA 106, 9063–9068 (2009). [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Williams L. E. & Wernegreen J. J. Genome evolution in an ancient bacteria-ant symbiosis: parallel gene loss among Blochmannia spanning the origin of the ant tribe Camponotini. PeerJ 3, e881 (2015). [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Xue J. et al. Genomes of the rice pest brown planthopper and its endosymbionts reveal complex complementary contributions for host adaptation. Genome Biol. 15, 521 (2014). [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Nováková E., Husník F., Šochová E. & Hypša V. Arsenophonus and Sodalis Symbionts in Louse Flies: an Analogy to the Wigglesworthia and Sodalis System in Tsetse Flies. Appl. Environ. Microbiol. 81, 6189–6199 (2015). [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. McCutcheon J. P. & von Dohlen C. D. An interdependent metabolic patchwork in the nested symbiosis of mealybugs. Curr. Biol. 21, 1366–1372 (2011). [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Husnik F. et al. Horizontal gene transfer from diverse bacteria to an insect genome enables a tripartite nested mealybug symbiosis. Cell 153, 1567–1578 (2013). [DOI] [PubMed] [Google Scholar]
  31. Husnik F. & McCutcheon J. P. Repeated replacement of an intrabacterial symbiont in the tripartite nested mealybug symbiosis. bioRxiv 042267 (2016). [DOI] [PMC free article] [PubMed]
  32. Chen D.-Q. & Purcell A. H. Occurrence and Transmission of Facultative Endosymbionts in Aphids. Curr. Microbiol. 34, 220–225 (1997). [DOI] [PubMed] [Google Scholar]
  33. Russell J. A., Latorre A., Sabater-Munoz B., Moya A. & Moran N. A. Side-stepping secondary symbionts: widespread horizontal transfer across and beyond the Aphidoidea. Mol. Ecol. 12, 1061–1075 (2003). [DOI] [PubMed] [Google Scholar]
  34. Lamelas A. et al. Evolution of the Secondary Symbiont “Candidatus Serratia symbiotica” in Aphid Species of the Subfamily Lachninae. Appl. Environ. Microbiol. 74, 4236–4240 (2008). [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Burke G. R., Normark B. B., Favret C. & Moran N. A. Evolution and diversity of facultative symbionts from the aphid subfamily Lachninae. Appl. Environ. Microbiol. 75, 5328–5335 (2009). [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Chandler S. M., Wilkinson T. L. & Douglas A. E. Impact of plant nutrients on the relationship between a herbivorous insect and its symbiotic bacteria. Proc. Biol. Sci. 275, 565–70 (2008). [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Chen D.-Q., Montllor C. B. & Purcell A. H. Fitness effects of two facultative endosymbiotic bacteria on the pea aphid, Acyrthosiphon pisum, and the blue alfalfa aphid, A. kondoi. Entomol. Exp. Appl. 95, 315–323 (2000). [Google Scholar]
  38. Manzano-Marín A. & Latorre A. Settling Down: The Genome of Serratia symbiotica from the Aphid Cinara tujafilina Zooms in on the Process of Accommodation to a Cooperative Intracellular Life. Genome Biol. Evol. 6, 1683–1698 (2014). [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Lamelas A. et al. Serratia symbiotica from the Aphid Cinara cedri: A Missing Link from Facultative to Obligate Insect Endosymbiont. Plos Genet. 7, e1002357 (2011). [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Manzano-Marín A., Simon J.-C. & Latorre A. Reinventing the wheel and making it round again: Evolutionary convergence in Buchnera-Serratia symbiotic consortia between the distantly related Lachninae aphids Tuberolachnus salignus and Cinara cedri. Genome Biol. Evol. evw085 (2016). [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Sabri A. et al. Isolation, pure culture and characterization of Serratia symbiotica sp. nov., the R-type of secondary endosymbiont of the black bean aphid Aphis fabae. Int. J. Syst. Evol. Microbiol. 61, 2081–2088 (2011). [DOI] [PubMed] [Google Scholar]
  42. Moran N. A., Russell J. A., Koga R. & Fukatsu T. Evolutionary Relationships of Three New Species of Enterobacteriaceae Living as Symbionts of Aphids and Other Insects. Appl. Environ. Microbiol. 71, 3302–3310 (2005). [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Kikuchi Y. Endosymbiotic Bacteria in Insects: Their Diversity and Culturability. Microbes Environ. 24, 195–204(2009). [DOI] [PubMed] [Google Scholar]
  44. Flyg C., Kenne K. & Boman H. G. Insect Pathogenic Properties of Serratia marcescens: Phage-resistant Mutants with a Decreased Resistance to Cecropia Immunity and a Decreased Virulence to Drosophila. Microbiology 120, 173–181 (1980). [DOI] [PubMed] [Google Scholar]
  45. Bennett G. M. & Moran N. A. Small, smaller, smallest: The origins and evolution of ancient dual symbioses in a phloem-feeding insect. Genome Biol. Evol. 5, 1675–1688 (2013). [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. McCutcheon J. P. & Moran N. A. Extreme genome reduction in symbiotic bacteria. Nat. Rev. Microbiol. 10, 13–26 (2012). [DOI] [PubMed] [Google Scholar]
  47. Moran N. A. & Bennett G. M. The Tiniest Tiny Genomes. Annu. Rev. Microbiol. 68, 195–215 (2014). [DOI] [PubMed] [Google Scholar]
  48. Degnan P. H. et al. Dynamics of genome evolution in facultative symbionts of aphids. Environ. Microbiol. 12, 2060–2069 (2009). [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Hansen A. K., Vorburger C. & Moran N. A. Genomic basis of endosymbiont-conferred protection against an insect parasitoid. Genome Res. 22, 106–114 (2012). [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Manzano-Marín A., Lamelas A., Moya A. & Latorre A. Comparative Genomics of Serratia spp. Two Paths towards Endosymbiotic Life. Plos One 7, e47274 (2012). [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Tamas I. et al. 50 Million Years of Genomic Stasis in Endosymbiotic Bacteria. Science 296, 2376–2379 (2002). [DOI] [PubMed] [Google Scholar]
  52. Patino-Navarrete R., Moya A., Latorre A. & Peretó J. Comparative Genomics of Blattabacterium cuenoti: The Frozen Legacy of an Ancient Endosymbiont Genome. Genome Biol. Evol. 5, 351–361 (2013). [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Sloan D. B. & Moran N. A. The evolution of genomic instability in the obligate endosymbionts of whiteflies. Genome Biol. Evol. 5, 783–793 (2013). [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Matelska D., Kurkowska M., Purta E., Bujnicki J. M. & Dunin-Horkawicz S. Loss of conserved noncoding RNAs in genomes of bacterial endosymbionts. Genome Biol. Evol. 8, 426–438 (2016). [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Mendonça A. G., Alves R. J. & Pereira-Leal J. B. Loss of Genetic Redundancy in Reductive Genome Evolution. Plos Comput. Biol. 7, e1001082 (2011). [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Bubunenko M., Baker T. & Court D. L. Essentiality of Ribosomal and Transcription Antitermination Proteins Analyzed by Systematic Gene Replacement in Escherichia coli. J. Bacteriol. 189, 2844–2853 (2007). [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Shoji S., Dambacher C. M., Shajani Z., Williamson J. R. & Schultz P. G. Systematic Chromosomal Deletion of Bacterial Ribosomal Protein Genes. J. Mol. Biol. 413, 751–761 (2011). [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Zhang C.-M. & Hou Y.-M. Synthesis of Cysteinyl-tRNACys by A Prolyl-tRNA Synthetase. RNA Biol. 1, 34–40 (2014). [PubMed] [Google Scholar]
  59. Jacquin-Becker C. et al. Cysteinyl-tRNA formation and prolyl-tRNA synthetase. FEBS Lett. 514, 34–36 (2002). [DOI] [PubMed] [Google Scholar]
  60. Decatur W. A. & Fournier M. J. rRNA modifications and ribosome function. Trends Biochem. Sci. 27, 344–351 (2002). [DOI] [PubMed] [Google Scholar]
  61. Hagervall T. G. & Björk G. R. Transfer RNA Modification. EcoSal Plus 1 (2013). [DOI] [PubMed] [Google Scholar]
  62. Gerdes S. Y. et al. Experimental Determination and System Level Analysis of Essential Genes in Escherichia coli MG1655. J. Bacteriol. 185, 5673–5684 (2003). [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Baba T. et al. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol. Syst. Biol. 2, 2006.0008 (2006). [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Joyce A. R. et al. Experimental and Computational Assessment of Conditionally Essential Genes in Escherichia coli. J. Bacteriol. 188, 8259–8271 (2006). [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Purta E., Kaminska K. H., Kasprzak J. M., Bujnicki J. M. & Douthwaite S. YbeA is the m3 methyltransferase RlmH that targets nucleotide 1915 in 23S rRNA. RNA 14, 2234–2244 (2008). [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Kahn D., Fromant M., Fayat G., Dessen P. & Blanquet S. Methionyl-Transfer-RNA Transformylase from Escherichia coli. Purification and Characterisation. Eur. J. Biochem. 105, 489–497 (1980). [DOI] [PubMed] [Google Scholar]
  67. Muramatsu T. et al. Codon and amino-acid specificities of a transfer RNA are both converted by a single post-transcriptional modification. Nature 336, 179–81 (1988). [DOI] [PubMed] [Google Scholar]
  68. Grosjean H. & Björk G. R. Enzymatic conversion of cytidine to lysidine in anticodon of bacterial tRNAIle – an alternative way of RNA editing. Trends Biochem. Sci. 29, 165–168 (2004). [DOI] [PubMed] [Google Scholar]
  69. Deutsch C., El Yacoubi B., de Crécy-Lagard V. & Iwata-Reuyl D. Biosynthesis of Threonylcarbamoyl Adenosine (t6A), a Universal tRNA Nucleoside. J. Biol. Chem. 287, 13666–13673 (2012). [DOI] [PMC free article] [PubMed] [Google Scholar]
  70. Mangat C. S. & Brown E. D. Known Bioactive Small Molecules Probe the Function of a Widely Conserved but Enigmatic Bacterial ATPase, YjeE. Chem. Biol. 15, 1287–1295 (2008). [DOI] [PubMed] [Google Scholar]
  71. Wolf J., Gerber A. P. & Keller W. tadA, an essential tRNA-specific adenosine deaminase from Escherichia coli. EMBO J. 21, 3841–51 (2002). [DOI] [PMC free article] [PubMed] [Google Scholar]
  72. Grossman A. D., Straus D. B., Walter W. A. & Gross C. A. Sigma 32 synthesis can regulate the synthesis of heat shock proteins in Escherichia coli. Genes Dev. 1, 179–184 (1987). [DOI] [PubMed] [Google Scholar]
  73. Guest J. R., Lewis H. M., Graham L. D., Packman L. C. & Perham R. N. Genetic reconstruction and functional analysis of the repeating lipoyl domains in the pyruvate dehydrogenase multienzyme complex of Escherichia coli. J. Mol. Biol. 185, 743–754 (1985). [DOI] [PubMed] [Google Scholar]
  74. Tsuchihashi Z. & Kornberg A. Translational frameshifting generates the gamma subunit of DNA polymerase III holoenzyme. Proc. Natl. Acad. Sci. USA 87, 2516–2520 (1990). [DOI] [PMC free article] [PubMed] [Google Scholar]
  75. Walker J. R. et al. Escherichia coli DNA polymerase III tau- and gamma-subunit conserved residues required for activity in vivo and in vitro. J. Bacteriol. 182, 6106–13 (2000). [DOI] [PMC free article] [PubMed] [Google Scholar]
  76. Draper G. C., McLennan N., Begg K., Masters M. & Donachie W. D. Only the N-terminal domain of FtsK functions in cell division. J. Bacteriol. 180, 4621–7 (1998). [DOI] [PMC free article] [PubMed] [Google Scholar]
  77. Gerding M. A. et al. Self-Enhanced Accumulation of FtsN at Division Sites and Roles for Other Proteins with a SPOR Domain (DamX, DedD, and RlpA) in Escherichia coli Cell Constriction. J. Bacteriol. 191, 7383–7401 (2009). [DOI] [PMC free article] [PubMed] [Google Scholar]
  78. Mellot P., Mechulam Y., Le Corre D., Blanquet S. & Fayat G. Identification of an amino acid region supporting specific methionyl-tRNA synthetase: tRNA recognition. J. Mol. Biol. 208, 429–443 (1989). [DOI] [PubMed] [Google Scholar]
  79. Vincent H. A. & Deutscher M. P. The Roles of Individual Domains of RNase R in Substrate Binding and Exoribonuclease Activity: The Nuclease Domain is Sufficinet for Digestion of Structured RNA. J. Biol. Chem. 284, 486–494 (2009). [DOI] [PMC free article] [PubMed] [Google Scholar]
  80. Johnson M. et al. NCBI BLAST: a better web interface. Nucleic Acids Res. 36, W5–W9 (2008). [DOI] [PMC free article] [PubMed] [Google Scholar]
  81. Jones P. et al. InterProScan 5: genome-scale protein function classification. Bioinformatics 30, 1236–1240 (2014). [DOI] [PMC free article] [PubMed] [Google Scholar]
  82. Katoh K. & Standley D. M. MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability. Mol. Biol. Evol. 30, 772–780 (2013). [DOI] [PMC free article] [PubMed] [Google Scholar]
  83. Krzywinski M. et al. Circos: an information aesthetic for comparative genomics. Genome Res. 19, 1639–45 (2009). [DOI] [PMC free article] [PubMed] [Google Scholar]
  84. Lowe T. M. & Eddy S. R. tRNAscan-SE: A Program for Improved Detection of Transfer RNA Genes in Genomic Sequence. Nucleic Acids Res. 25, 955–964 (1997). [DOI] [PMC free article] [PubMed] [Google Scholar]
  85. Nawrocki E. P. & Eddy S. R. Infernal 1.1: 100-fold faster RNA homology searches. Bioinformatics 29, 2933–2935 (2013). [DOI] [PMC free article] [PubMed] [Google Scholar]
  86. Nawrocki E. P. et al. Rfam 12.0: updates to the RNA families database. Nucleic Acids Res. 43, D130–D137 (2015). [DOI] [PMC free article] [PubMed] [Google Scholar]
  87. Li L. OrthoMCL: Identification of Ortholog Groups for Eukaryotic Genomes. Genome Res. 13, 2178–2189 (2003). [DOI] [PMC free article] [PubMed] [Google Scholar]
  88. Bourque G. & Pevzner P. A. Genome-scale evolution: Reconstructing gene orders in the ancestral species. Genome Res. 12, 26–36 (2002). [PMC free article] [PubMed] [Google Scholar]
  89. Guy L., Roat Kultima J. & Andersson S. G. E. genoPlotR: comparative gene and genome visualization in R. Bioinformatics 26, 2334–2335 (2010). [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Information
srep32590-s1.pdf (215.3KB, pdf)
Supplementary Dataset 1
srep32590-s2.zip (22.7KB, zip)

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