In this investigation, we have predicted putative recombination signals in mature tmRNA of species belong to Enterobacteriaceae. A total of 53 species from 16 genera were taken for analysis. Supplementary Table 1 summarizes the name of species taken for analysis and information about the total length of tmRNA and sequence of peptide tag. The length of tmRNA among the species did not show significant variation except a few. The size of the smallest tmRNA is 312 nts, which belongs to Enterobacter aerogenes whereas the length of the largest tmRNA is 408 nts and it belongs to Erwinia pyrifoliae (Supplementary Table 1). Variations in the length of mature tmRNA largely arise due to the sequence difference in the mRNA region, pseudoknot 2 (PK2) (Fig. 1b), however differences in pseudoknot 3 (PK3) were observed in Blochmania spp. The nucleotide coordinates of the HGT events were summarized in Table 1 and Fig. 1. Putative recombination signals were analyzed in the following ways. Primarily the recombination signals were analyzed in the context of tmRNA domains (i.e. the regions which are involved in HGT and the regions which are not). Secondly, recombination signals were analyzed as individual events with their donor and recipient status. Thirdly, phylogenetic analysis to access the closeness of minor and major parents towards the recombinant in a segmented alignment files which are specific to the seven recombination events.
Table 1
Recombination events predicted by RDP5 and their details.
Recombination event
|
Nucleotide co-ordinates
|
Potential Recombinant
|
Minor parent
|
Major parent
|
Programs predicted and their E-values
|
Domains of tmRNA in the co-ordinates
|
Species having sequence evidence of the same Recombination signature
|
Event 1
|
101–153
|
D. dadantii
|
B. pennsylvanicus
|
D. zeae
|
GENECONV (1.94 X 10− 9),
Maxchi (4.88 X 10− 6),
SiSscan (8.37 X 10− 27),
3Seq (1.65 X 10− 4)
|
Partial region of ORF and partial region of PK2
|
E. amylovora,
E. tasmaniensis,
D. chrysanthemi,
Y. intermedia,
Y. bercovieri,
Y. mollaretti,
Y. rohdei, Y. enterocolitica
|
Event 2
|
81–131
|
P. vagans
|
S. boydii
|
-
|
SiSscan (2.07 X 10− 9),
3Seq (4.12 X 10− 3)
|
ORF
|
P. stewartii,
P. atrosepticum,
P. wasabiae
|
Event 3
|
60–84
|
B. floridanus
|
D. zeae
|
-
|
GENECONV (1.59 X 10− 5)
|
Partial region of PK1
|
-
|
Event4
|
252–345
|
E. amylovora
|
Y. intermedia
|
P. stewartii
|
Maxchi (4.66 X 10− 3),
Chimaera (6.08 X 10− 4),
SiSscan (1.54 X 10− 5),
3Seq (4.20 X 10− 5)
|
Partial region of PK4 and T-arm
|
E. tasmaniensis
|
Event 5
|
89–179
|
P. ananatis
|
Y. frederiksenii
|
-
|
Maxchi (1.41 X 10− 3),
Chimaera (4.89 X 10− 2),
SiSscan (5.97 X 10− 5),
3Seq (7.89 X 10− 4)
|
ORF and Partial region of PK2
|
E. billingiae, P. stewartii
|
Event 6
|
246–359
|
Y. intermedia
|
P. vagans
|
P. rettgeri
|
Maxchi (4.55 X 10− 2),
SiSscan (9.67 X 10− 4),
3Seq (3.04 X 10− 2)
|
PK4 and T-arm
|
Y. bercovieri,
Y. mollaretti,
Y. enterocolitica
|
Event 7
|
167–211
|
E. amylovora
|
Y. frederiksenii
|
-
|
SiSscan (5.18 X 10− 2),
3Seq (9.32 X 10− 3)
|
Partial region of PK2 and partial region of PK3
|
E. tasmaniensis
|
3.1 Domain-specific gene traffic in mature tmRNA
Out of 7 recombination events predicted, putative recombination signals were identified in domains of tmRNA like PK1, mRNA domain, PK2, PK3, PK4, and T arm except D-arm (refer Fig. 1c). The gene traffic tends to cluster near mRNA domain (Events 1, 2, and 5). Other domains showed one (PK1 in Event3 and PK3 in Event 7) or two (PK2 in Event 5 and 7; PK4 and T-arm in Event 4 and 6) events where as the D arm is free from the genetic traffic. This shows domain-specific nature of tmRNA genetic traffic.
3.2 Phylogenetic and Neighbor net analysis
For each event, the sequence alignment was split into two in such a way that one has the recombined region (derived from the minor parent) and the other has the rest of the sequence (sequence in recombinant donated by the major parent). All the files were subjected to the phylogenetic tree construction. The recombinant, major and minor parent sequences were highlighted with different color (Supplementary Fig. S1-S7). Phylogenetic trees constructed for the events 1, 6 and 7 showed the predicted major parent and minor parent sequences shared the nearer clad with the recombinant sequence. This also adds additional weightage for our claim. The middle portion of the ssrA is more prone to recombination than the flanks.
The neighbor net analysis was performed to analyze the genetic relationship among species. Reticulation reflects the evidence of recombination. The PhiTest gave significance signals for recombination with a p-value of 0.006016. The recombinants are highlighted in red (Fig. 3). Except for B. floridanus, all the recombinants are found in the main group. Species belong to Yersinia form two different groups which are incomparable with recombination event 1 and 6. Specieslike B. floridanus, B. pennsylvanicus, P. rettgerii, P. vulgaris, P. mirablis clustered together as individual groups and well separated from the rest of the strains. These suggest a divergent evolution in the tmRNA from the rest of the population.
3.3 Recombination events predicted a donor-recipient relationship
tmRNA of D. dadatii (refer Fig. 2a), P. vagans (refer Fig. 2b), B. floridanus (Fig. 2c),
E. amylovora (refer Fig. 2d), P. ananalis (refer Fig. 2e) and Y. intermedia (refer Fig. 2f) were found to be recombinants (i.e. act as recipients) (refer Table 1). Species like B. pennsylvanicus, S. boydii, D. zeae, Y. intermedia, Y. frederiksenii acts as donors in various recombination events. The details of each of the recombinant signals were discussed below in detail. Interestingly
Y. frederiksenii was found to donate different regions corresponding to (89 – 179 and 167 - 211) to P. ananatis and E. amylovora. Two recombinant signals were found in different domains of
E. amylovora tmRNA (corresponds to Event 4 and Event 7) in which Y. intermedia and
Y. frederiksenii act as donors, respectively. Species like P. vagans and Y. intermedia acts both as donor and recipient in different recombination events (refer Table 1). Sequences of recombinant tmRNA belong to Y. intermedia and E. amylovora also showed evidence for recombination
event 1.
Event 1
In Recombination event 1, the recombination breakpoints were between 101 - 154 nts. Putative recombinant tmRNA was found in D. dadantii, (refer Fig. 2a) which is a plant pathogen formerly called Erwinia Chrysanthemi. It infects tubers, bulbs, and ornamental crops [9].
B. pennsylvanicuss acts as a donor in this event which lives as a symbiont in insects [10]. Surprisingly evidence of this recombination event was also found in E. amylovora, E. tasmaniensis, D. chrysanthemi, Y. intermedia, Y. bercovieri, Y. rhodei and Y. enterocolitica.
Event 2
In Recombination event 2, the recombination breakpoints were between 81 - 131 nts. Putative recombinant tmRNA was found in P. vagans (refer Fig. 2b) which is an epiphyte [11]. Human pathogen S. boydii [12] acts as a donor in this event. Evidence for similar recombination event was found in P. stewartii, P. atrosepticum and P. Wasabiae.
Event 3
In Recombination event 3, the recombination breakpoints were between 60 – 84 nts. Putative recombinant tmRNA was found in B. floridanus (refer Fig. 2c) which is an endosymbiont of carpenter ant [13]. Plant pathogen D. zeae acts as a donor in this event.
Event 4
In Recombination event 4, the recombination breakpoints were between 252 -315 nts. Putative recombinant tmRNA was found in E. amylovora (refer Fig. 2d) which is a plant pathogen [14]. Donor for this event is Y. intermedia which is a Human pathogen [15]. Evidence for similar recombination event was found in tmRNA of E. pyrifoliae and E. tasmaniensis
Event 5
In Recombination event 5, the recombination breakpoints were between 89 -179 nts. Putative recombinant tmRNA was found in P. ananatis (refer Fig. 2e) which is both human and plant pathogen [16]. Insect pathogen, Y. frederiksenii [17] acts as a donor. Evidence for similar recombination event was found in E. billingiae and P. stuartii.
Event 6
In Recombination event 6, the recombination breakpoints were between 246 -359 nts. Putative recombinant tmRNA was found in human pathogen Y. intermedia (refer Fig. 2f). P. vagans acts as a donor. Evidence for similar recombination event were found in species Y. intermedia, Y. bercovieri, Y. mollaretii, Y. rohdei, Y. enterocolitica and Y. frederiksenii
Event 7
In Recombination event 7, the recombination breakpoints were between 167 -211 nts. Putative recombinant tmRNA was found in E. amylovora (refer Fig. 2d). None of the tested tmRNA have similarity in this region. Evidence for similar recombination event was found in E. amylovora, E. pyrifoliae and E. tasmaniensis.
The recombination events occurred between a functionally diverse set of species. tmRNA of many species were reported to have evidence for recombination event. These results depicted a diverse nature of gene traffic in tmRNA of Enterobacteriaceae.