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SUPPLEMENTARY INFORMATION
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Thivierge a,b,c, Roger C. Levesquea,e, Steve J. Charettea,b,c#
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a. Institut de biologie intégrative et des systèmes, Pavillon Charles-Eugène-Marchand, Université
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Laval, 1030 avenue de la Médecine, Quebec City, QC, Canada, G1V 0A6
Increasing genomic diversity and evidence of constrained lifestyle evolution due to insertion
sequences in Aeromonas salmonicida
Antony T. Vincenta,b,c, Mélanie V. Trudela,b,c, Luca Freschia,e, Vandan Nagard, Cynthia Gagné-
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b. Centre de recherche de l’Institut universitaire de cardiologie et de pneumologie de Québec
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(Hôpital Laval), 2725 Chemin Sainte-Foy, Quebec City, QC, Canada, G1V 4G5
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c. Département de biochimie, de microbiologie et de bio-informatique, Faculté des sciences et de
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génie, Université Laval, 1045 avenue de la Médecine, Quebec City, QC, Canada G1V 0A6
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d. Food Technology Division, Bhabha Atomic Research Centre, Mumbai, 400085, India
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e. Département de microbiologie-infectiologie et immunologie, Faculté de médecine, Université
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Laval, Quebec City, QC, Canada
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#
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Pavillon Charles-Eugène-Marchand, 1030 avenue de la Médecine, Université Laval, Quebec
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City, QC, Canada G1V 0A6
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Telephone: 418-656-2131, ext. 6914, Fax: 418-656-7176
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steve.charette@bcm.ulaval.ca
Corresponding author: Steve J. Charette, Institut de Biologie Intégrative et des Systèmes (IBIS),
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Supplementary Experimental Procedures
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Phylogenetic analyses
28
To perform a robust core genome phylogeny, we wrote an in-house Perl script called
29
CoreFinder.pl that relies on BioPerl modules [1] to find the genes involved in the core genome.
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The script uses coding sequences extracted from a GenBank file and sequentially performs
31
tblastn [2] searches in fasta or multi-fasta (for draft genomes) files (Figure S1). We used A.
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hydrophila ATCC 7966T [3], which is the A. hydrophila type strain, as a reference. The genome
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of this strain has been well studied and has a high-quality annotation. The others aeromonads
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used in the present study are listed in Table S1. The parameter used to search the CDSs was at
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least a 85% query cover for various similarity percent (25% to 100%, with 5% steps). The
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graphical interpretation of the results revealed three linear sections and two breakpoints estimated
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at 40% and 80% similarity (Figure S2). To verify the importance of this parameter (e.g., the
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similarity percent) with respect to the final phylogeny, we performed all subsequent analyses (as
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indicated in the main manuscript) at 40% and 80% similarity.
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To choose the most appropriate phylogenetic model, the Akaike Information Criterion (AIC) and
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the Bayesian Information Criterion (BIC) were computed using jModelTest version 2.1.7 [4] for
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both matrixes. In both cases, while the best-fit model was GTR+Γ closely followed by GTR+I+Γ
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(Table S2), there was no significant difference between the two. However, as discussed and
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reviewed elsewhere [5], the consideration of a rate class with a rate zero caused by invariable
46
sites is meaningless since the α parameter, which governs the shape of the gamma distribution,
47
already allows low-rate sites through an L-shaped gamma distribution caused by an α < 1.
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Moreover, the use of the mixture model +I+Γ might result in an over-parameterization since it
2
49
would be difficult to optimize both parameters. We thus used the GTR+Γ model for both
50
matrixes at 40% and 80% similarity.
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Species relatedness was inferred by average nucleotide identity (ANI) analyses for key taxa using
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JSpecies version 1.2.1 [6]. MUMmer version 3.23 [7] was used to perform the analyses since it
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provides more robust results for genomes sharing a high level of similarity (ANI > 90%) than
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blast searches [6]. Two taxa were considered to be members of the same species if they shared an
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ANI ≥ 96%, a value that is well adapted to the aeromonads [8].
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Bacterial growth at 7°C
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The Indian isolates (Y577, Y567 and Y47) as well as A. salmonicida subsp. pectinolytica
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(34melT), A. salmonicida subsp. smithia (JF4097), A. salmonicida subsp. masoucida (NBRC
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13784T), and A. salmonicida subsp. salmonicida (01-B526) were inoculated on furunculosis agar
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or on tryptic soy agar (TSA) from frozen stocks and were grown at 18°C for 24 to 48 h. The
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isolates were then inoculated in 3 ml of lysogeny broth (LB) and were incubated at 7°C overnight
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with shaking at 200 rpm. The turbidity was adjusted to an optical density of 0.1 at 595 nm
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(OD595), and the cultures were incubated at 7°C with shaking at 200 rpm. The ODs were read
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systematically every hour for 8 h. The experiment was performed in triplicate.
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PCR assays
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We performed PCR assays using previously published conditions [9] to verify whether the pAsa5
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plasmid of strain RS 534 had lost its type three secretion system (TTSS) by the recombination of
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ISAS11B and ISAS11C [10].
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Plasmid characterization
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The contigs for the strains sequenced in the present study were locally mapped on the
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chromosome sequence of the A. salmonicida reference strain A449 [11], the only A. salmonicida
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strain with a fully assembled chromosome, using CONTIGuator version 2.7.4 [12]. Identity
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searches of the unmapped contig sequences were performed by blast searches against the NCBI
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nr/nt database. Sequence manipulations were performed using the bioinformatics package
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EMBOSS version 6.6.0.0 [13].
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The plasmid sequences that were discovered were automatically annotated by the RAST
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webserver [14]. All the putative CDSs were manually curated by performing blastp searches
83
against the NCBI nr/nt database. Putative toxin-antitoxin systems were found by TAfinder [15].
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The average copy number of each new plasmid for each chromosome was calculated using the
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sequencing depth, a procedure that has been successfully used in the past [16]. We filtered the
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sequencing reads using Trimmomatic version 0.32 [17] with the parameters suggested in the
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manual. The resulting filtered sequencing reads were mapped on the gyrB gene (single copy per
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chromosome) with CUSHAW3 version 3.0.3 [18] without allowing any mismatches in order to
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avoid cross-mapping from reads related to other genes. The reads were also mapped on the
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plasmid sequences. The average coverages were calculated using Qualimap version 2.1.1-dev
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[19].
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Biochemical tests
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Three Indian A. salmonicida isolates (Y47, Y567 and Y577) were further phenotypically
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characterized using a set of biochemical tests as described by Pavan et al. [20] and Abbott et al.
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[21]. All tests were performed in triplicate and according to conventional protocols with suitable
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positive and negative controls and incubated at 35°C for 48 – 72 h (unless mentioned). The tests
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for carbohydrate fermentation and extracellular enzymes were read daily for 7 days; whereas,
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tests for Voges-Proskauer, polypectate degradation and production of brown diffusible pigment
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on tryptic soya agar (TSA) were incubated at 25°C for 2 to 4 days.
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Supplementary results
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Sequencing results
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Despite the stable average coverage of the assemblies, the N50 values, which are an indicator of
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contigs length, varied considerably (Table S3). For example, the Y567 Indian strain had a N50
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value two times higher than the other strains while JF4097 (smithia) had a lower N50 value and
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the smallest large contig. Large repeated elements such as ISs, duplicated genes, and ribosomal
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RNA clusters cause contig breaks during de novo assembly [22], which suggests that the A.
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salmonicida subsp. smithia genome contained numerous large repeated elements.
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Molecular phylogeny optimization
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At 40% similarity (of the translated sequences), the core genome was estimated at 1645 genes
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compared to 1190 genes at 80%. The functional categories of the genes (at 40 and 80%) were
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found using an in-house Perl script as explained in the main manuscript to verify whether there
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was an enrichment of one or more categories. There were major differences in the relative
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abundance of the functional categories at 40% and 80% similarity in only two categories (J:
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translational, ribosomal structure and biogenesis and K: transcription), indicating that the
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gains/losses were uniform in the other categories (Figure S3). The relative importance of the J
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category at 80% is higher than at 40%, which is consistent with this conserved process. The high
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relative importance for the K category at 40% is in accordance with the capacity of various
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aeromonads to react to a wide diversity of stimuli.
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The basic features of the phylogenetic analyses are presented in the Table S4. The matrix at 80%
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similarity had 35% fewer sites than the matrix at 40% similarity. In both cases, the values of the
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alignment patterns, which are the numbers of different patterns in the matrixes, corresponded to
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approximately 60% of the total number of sites. There was no significant difference between the
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α parameters of the two phylogenetic analyses as estimated by RAxML, meaning that the 35%
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more sites at 40% similarity shared the same rate as the other sites.
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There were differences between the resulting trees in terms of bootstrap values and topology
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(Figures S4 and S5). In fact, the phylogenetic analysis at 80% similarity had the weakest
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bootstrap values (Figure S4), indicating that the 35% more sites obtained at 40% similarity are
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important for obtaining a more robust tree (Figure S5). The topology diverged for the clade
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containing Aeromonas veronii. This observation was understandable since this clade had a weak
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bootstrap value, even with the matrix at 40% similarity (Figure S5). Based on the bootstrap
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values, we thus believe that the tree based on the core genome found at 40% similarity more
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accurately represents the true evolution links between the taxa, which is why we used this
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phylogenetic tree for the remainder of the study.
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Phylogenetic position of A. salmonicida
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As mentioned in the main manuscript, the molecular phylogeny of the present paper revealed that
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A. salmonicida CBA100, a recently deposited Chilean strain [23], is phylogenetically closer to A.
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bestiarum than to A. salmonicida (Figure S5). To verify the relatedness of the CBA100 strain and
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A. bestiarum, the average nucleotide identity (ANI) values were computed for some key taxa
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(Figure S6). The fact that the ANI value between CBA100 and A. bestiarum is above 96%
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reinforce the close evolutionary link between both taxa and let believe at a miss-classification of
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CBA100.
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Strain Y577 shared a clade with A. salmonicida subsp. pectinolytica. Based strictly on the
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molecular phylogeny and the ANI values, we cannot rule out the possibility that Y577 is in fact
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A. salmonicida subsp. pectinolytica. As previously published, the pectinolytica subspecies is the
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only known aeromonad with pectinase activity [20]. Interestingly, all the genes in the
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pectinolytica subspecies that are needed to degrade and use pectin as a carbon source [24] were
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found in the genome of Y577. Given this, the pectinase activity of Y577 was verified and was
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confirmed experimentally (Table S5). It is tempting to suggest that Y577 is a member of the
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pectinolytica subspecies or a new subspecies sharing a near common ancestor. However, the
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overall chromosomal organizations of Y577 and the pectinolytica subspecies strains appeared
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divergent (Figure 1, main manuscript), which is unusual for such closely related strains given the
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chromosomal uniformity of salmonicida subspecies strains. The results of some other
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biochemical tests also diverged (Table S5), suggesting that strains Y577 and A. salmonicida
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subsp. pectinolytica 34melT may not belong to the same subspecies.
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Strains Y47 and Y567 formed a basal clade to the masoucida and salmonicida subspecies.
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However, like the relation between Y577 and the pectinolytica 34melT strain, we cannot infer that
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Y47 and Y567 belong to the same subspecies based solely on the molecular phylogeny and the
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ANI values, especially since there were also macro-chromosomal differences between the two
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strains. If they belong to the same subspecies, this would indicate that they display significant
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genomic plasticity. There are also differences between many of the biochemical test results
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(Table S5), which also points to a potential taxonomic difference. Surprisingly, Y47 and Y567
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were also pectinolytic. While 34melT and Y577 bore genes coding for three lyases involved in the
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first step of pectin degradation, Y567 and Y47 did not. The genomes of strains Y567, Y47, and
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Y577 (as a positive control) were annotated using the RAST webserver [25] to verify whether
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they possessed a subsystem related to pectin degradation. The annotation of Y577 contained the
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three lyases (EC 4.2.2.2, EC 4.2.2.6 and EC 4.2.2.9) in the “D-galacturonate and D-glucuronate
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utilization” subsystem while the annotations of Y47 and Y567 did not contain any enzymes
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involved in pectin degradation. The pectinase activities of Y47 and Y567 likely involved an
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unknown pathway and are potentially the result of convergent evolution (i.e., when compared to
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the strains pectinolytica 34melT and Y577). This result is interesting since it evokes that
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pectinolytic activity could be important for mesophilic A. salmonicida.
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Bacterial growth at 7°C
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The capacity of various A. salmonicida isolates to grow at 7°C was tested in addition to 18°C and
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37°C (main manuscript). The same trend as at 18°C was observed, with the mesophilic strains
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growing more efficiently than the psychrophilic ones (Figure S7). The isolate JF4097 of the
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subspecies smithia was not able to grow at this temperature. This was expected knowing that this
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isolate had a weak growth capacity at 18°C (main manuscript).
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Investigation of the plasmidome
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The putative chromosomal contigs were removed and the remaining contigs were analyzed in
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order to investigate the plasmidome of the strains for which the DNA was sequenced in the
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present study. This resulted in the identification of three small cryptic plasmids in Indian strain
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Y47 (Figure S8). To our knowledge, this was the first time that these plasmids had been found.
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We named them pY47-1, pY47-2, and pY47-3 and deposited their sequences in GenBank under
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accession numbers KT334396, KT334397, and KT334398, respectively. There were no clear
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known functions associated with these plasmids. All bore a putative type II toxin-antitoxin
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maintenance system and/or a phage resistance mechanism [26]. The plasmids pY47-2 and pY47-
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3 are ColE2-type replicon plasmids with a short RNA (RNA I) replication regulator [27].
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Interestingly, a blastn (word size of 11) search revealed sequence identity and structural
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similarity between pY47-3 and the ColE2-type replicon plasmids pAQ2-1 and pAQ2-2 in
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Aeromonas sobria and Aeromonas hydrophila, respectively [28]. However, unlike these
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plasmids, pY47-3 did not bear the qnrS2 quinolone resistance gene.
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The high sequencing depth provided by the Illumina technology was used to infer the average
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copy number per chromosome of each plasmid. As it has been reported elsewhere [29], high copy
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numbers of ColE2-type replicon plasmids are maintained per cell ( 24 copies for pY47-2 and
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13 copies for pY47-3). The plasmid pY47-1, for which the incompatibility group is unknown,
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also had a high copy number (22 copies). It is important to mention that inferring relative
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plasmid copy numbers has an inherent bias since it is assumed that there is a single copy of the
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chromosome in each cell, which is not true after the replication. However, the results showed that
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these plasmids were maintained at much higher copy numbers than the bacterial chromosome. No
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plasmids were found in strain Y567 while Y577 harbored a pY47-3 plasmid that shared more
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than 99% identity with the one in Y47 (7 point mutations).
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A plasmid maintained at a high copy number (~40 copies/cell) was also found in A. salmonicida
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subsp. smithia JF4097 and was subsequently named pJF4097 and its sequence deposited in
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GenBank under the accession number: KT334395 (Figure S9). pJF4097 bears the mobABCD
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genes, which are related to mobilization, an ISAS11, and a gene encoding an ExoY-like protein,
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which is a type-three secretion system (TTSS) effector in the human pathogen Pseudomonas
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aeruginosa [30].
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The A. salmonicida subsp. salmonicida RS 534 strain harbors the same five plasmids as the A449
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reference strain [11], including the large plasmid pAsa4, which encodes many drug resistance
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genes; pAsa5, which normally bears the type-three secretion system, and the pAsa1, pAsa2, and
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pAsa3 cryptic plasmids [31]. Basic bioinformatics analyses showed that the pAsa5 plasmid of the
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RS 534 strain has lost its TTSS. It is known that this region is bordered by two ISAS11s (B and
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C) and that growth above 25°C may result in the recombination of the two ISAS11s and the loss
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of the TTSS [9,10]. We confirmed by PCR that the TTSS was lost by a recombination of
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ISAS11B and C (Figure S10).
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The pan-genome analyze
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We used an in-house Perl script as indicated in the main manuscript to find the pan-genome of A.
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salmonicida. The resulting binary matrix (i.e., presence/absence) was used to map the characters
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(i.e., the genes) on a phylogenetic tree based on the core genome (Figure S11A). This analysis
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made it possible to determine which genes were acquired or lost during evolution and,
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consequently, may have played a role in the adaption of a given strain. As indicated in the main
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manuscript, three functional categories (K, N and X) at branch 1 experienced many events (i.e.,
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gains and losses) (Figure S11B). The L, R, T and U categories have also acquired and lost many
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genes, but this can more likely be attributed to general rather than mesophilic-to-psychrophilic
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evolution. In the case of branch 2 (Figure S11C), the three functional categories exhibiting most
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important changes are energy production and conversion (C) (only losses for this category),
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carbohydrate transport and metabolism (G), replication, recombination, and repair (L).
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Interestingly only gains have been detected for the category related to the mobilome (X).
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Unfortunately, it was impossible to assign a cluster of orthologous groups (COGs) at 45,4 and
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59,1% of the genes for the branches 1 and 2, respectively and, consequently, to infer their
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functional categories. This highlights a drawback of bioinformatics analyses and their
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dependence on incomplete and poorly curated databases.
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Functional categories of the genes under positive selection in the mesophilic lineages
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A total of 322 genes in the A. salmonicida lineages appear to be under positive selection for
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various lineages among the salmonicida species, including 241 that were specific to at least one
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mesophilic lineage. We used a COG assignment of these 241 genes to find their relative
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functional categories (Figure S12). Many categories in the mesophilic lineages were under
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positive selection, indicating that these lineages may have a high evolutionary potential.
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Table S1. Aeromonads used in the study.
Species
Strain
Accession no.
Reference
T
A. allosaccharophila
CECT 4199
CDBR00000000 [8]
A. allosaccharophila
BVH88
CDCB00000000 [8]
A. australiensis
CECT 8023T
CDDH00000000 [8]
T
A. bestiarum
CECT 4227
CDDA00000000 [8]
A. bivalvium
CECT 7113T
CDBT00000000 [8]
A. caviae
CECT 838T
CDBK00000000 [8]
A. dhakensis
CIP 107500
CDBH00000000 [8]
A. diversa
CECT 4254T
CDCE00000000 [8]
A. encheleia
CECT 4342T
CDDI00000000
[8]
A. enteropelogenes
CECT 4487T
CDCG00000000 [8]
A. eucrenophila
CECT 4224T
CDDF00000000 [8]
A. fluvialis
LMG 24681T CDBO00000000 [8]
A. hydrophilaa
ATCC 7966T CP000462
[3]
T
A. jandaei
CECT 4228
CDBV00000000 [8]
A. media
CECT 4232T
CDBZ00000000 [8]
A. molluscorum
848T
AQGQ00000000 [32]
T
A. piscicola
LMG 24783
CDBL00000000 [8]
A. popoffii
CIP 105493T
CDBI00000000
[8]
A. rivuli
DSM 22539T CDBJ00000000
[8]
A. salmonicida subsp. salmonicida
A449
CP000644
[11]
A. salmonicida subsp. salmonicida
01-B526
AGVO01000000 [33]
A. salmonicida subsp. salmonicida
RS534
JYFF00000000
This study
A. salmonicida subsp. salmonicida
JF3224
JXTA00000000
[9]
A. salmonicida subsp. salmonicida
CIP 103209
CDDW00000000 [8]
A. salmonicida subsp. salmonicida
2009-144K3
JRYV00000000
[34]
A. salmonicida subsp. salmonicida
2004-05MF26 JRYW00000000 [34]
A. salmonicida
CBA100
JPWL00000000
[23]
A. salmonicida subsp. achromogenes
AS03
AMQG00000000 [35]
A. salmonicida subsp. smithia
JF4097
JZTI00000000
This study
T
A. salmonicida subsp. pectinolytica
34mel
ARYZ00000000 [36]
A. salmonicida subsp. masoucida
NBRC 13784T BAWQ00000000 N/Ab
A. salmonicida
Y47
JZTF00000000
This study
A. salmonicida
Y567
JZTG00000000
This study
A. salmonicida
Y577
JZTH00000000
This study
A. sanarellii
LMG 24682T CDBN00000000 [8]
A. schubertii
CECT 4240T
CDDB00000000 [8]
T
A. simiae
CIP 107798
CDBY00000000 [8]
A. sobria
CECT 4245T
CDBW00000000 [8]
A. species
AH4
ERX552948c
[8]
A. species
AMC34
AGWU00000000 N/A
A. taiwanensis
LMG 24683T CDDD00000000 [8]
A. tecta
CECT 7082T
CDCA00000000 [8]
T
A. veronii
CECT 4257
CDDK00000000 [8]
a: This strain was used as a model to find the genes involved in the core genome.
b: N/A means that no publication is associated with the sequence.
c: Only the sequencing reads were available via the SRA database for A. species AH4. The reads were de
novo assembled as indicated in the “Methods of the main manuscript” section.
13
262
Table S2. The five best models and their –InL, AIC, and BIC values.
Similarity
40%
Model
GTR+Γ
GTR+I+Γ
HKY+Γ
HKY+I+Γ
SYM+Γ
-lnL
12827871
12827940
12832779
12832849
13005302
AIC
25655928
25656069
25665737
25665878
26010784
80%
BIC
25656993
25657146
25666756
25666909
26011815
-lnL
7941102
7941148
7944260
7944305
8071711
AIC
15882391
15882484
15888698
15888791
16143602
BIC
15883417
15883521
15889679
15889783
16144595
263
264
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265
Table S3. Assembly results.
Strains
Contigs
Largest contigs (kbp)
N50 (kbp)
Average coverage
Assembly size (Mbp)
A449 fraction (%)a
266
267
268
Y47
Y567
Y577
JF4097
RS 534
118
395.27
117.77
66.92
4,710233
85.077
47
448.10
217.34
68.03
4,554847
85.607
104
383.04
101.76
78.53
4,736410
83.731
344
109.61
28.95
88.72
4,307768
84.059
123
382.43
119.02
62.59
4,889640
97.599
a: The chromosome sequence of the strain A449 (A. salmonicida subsp. salmonicida) [11] was
used. This feature was found using QUAST version 3.1 [37].
15
269
Table S4. Phylogenetic features.
Similarity percent
40%
80%
1645
1190
Genes
696,249 454,574
Sites
Alignment patterns 420,006 271,519
GTR+Γ
GTR+Γ
Best model
1.703415 1.686099
α parameter
270
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271
Table S5. Biochemical tests used for the mesophilic A. salmonicida strains.
Biochemical tests
Strains
Y577 Y47 Y567
Indole (35°C)
+
+
+
+
ONPG
+
+
+
+
VP (25°C)
+
+
+
+
Simmons citrate
+
+
+
+
Esculin hydrolysis
+
+
Polypectate degradation (25°C)
+
+
+
+
Motility (35°C)
+
+
+
Brown pigment (25°C)
+
Growth (37°C)
+
+
+
+
Dnase
+
+
+
+
Lipase
+
+
+
+
Gelatinase
+
+
+
+
H2S
VP (35°C)
ODC
LDC
+
+
+
ADH
+
+
+
Urease
Cellobiose
+
+
+
Salicin
+
+
Sorbitol
+
+
+
+
Rhamnose
Mannitol
+
+
+
+
Sucrose
+
+
+
+
Glucose (gas)
+
+
+
+
L-Arabinose
+
+
+
+
Lactose
+
+
+
+
Glycose
+
+
+
+
Inositol
Melibiose
Glu
+
+
+
+
Amygdalin
+
+
Hemolysin (sheep, horse)a
+
+
+
+
a: These results are from [20]. We have used horse blood agar for assessing hemolysis; whereas,
Pavan et al. (2000) [20] have used sheep blood agar plates.
34melT a
272
273
274
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Initial GenBank
file (Genome 1)
CDS extraction
tblastn
List of CDSs
276
277
278
279
Sequence(s)
(Genome 2)
...
Sequence(s)
(Genome n)
Sequence(s)
(Genome n-1)
tblastn
List of CDSs
List of CDSs
Core genome
Translated
sequences
List of CDSs
with known
biological function
Figure S1. Conceptual schematization of the in-house CoreFinder.pl Perl script.
18
Core genome(genes)
1500
1000
500
0
100
280
281
282
283
284
285
286
85
70
55
40
25
similarity (%)
Figure S2. Number of genes involved in the core genome based on the similarity percent used
with the CoreFinder.pl script. The blue dots at 40% and 80% indicate the similarity percent used
to perform the optimization analyses.
19
287
288
289
290
291
Figure S3. Relative abundance of 26 functional categories for genes used to construct the
phylogenetic matrixes at 40 and 80% similarity.
20
292
293
294
295
296
297
298
299
300
301
Figure S4. (A) Molecular core genome phylogeny of 43 aeromonads inferred from the sequences
of 1190 genes (determined using the 80% similarity) by maximum-likelihood using the GTR+Γ
model and a 1000 rapid bootstrap analysis. Only bootstrap values under 100 are shown. For
clarity, the bootstrap values have been removed for the taxa of the salmonicida species. The
mesophilic strains are in red while the psychrophilic strains are in blue. (B) Zoom of salmonicida
species with equal branch lengths. Only bootstrap values under 100 are shown. The mesophilic,
intermediate, and psychrophilic strains are shown in red, purple, and blue, respectively.
21
.p
e
56
A.
s
-Y
A.
ila
sis
i
7
sY5
77
ss
ub
sp
o ff ii
A.
A. p
op
A . pi sc ic
ol a
A. s - CBA100
m
ph
en
s
ae
dro
ne
nd
ak
ge
ja
sis
A
.s
47
-Y
u
ss
A.
s
A. ve
ron
A. sa cc ha ro
ph
lo
ien
dh
pe
A.
ro
iali
ial
hy
te
fluv
A.
str
H4
s-A
A.
en
A.
au
i ar u
A. be s t
ie
pec
A. s
A.
A.
ctin
oly
tica
302
bs
s
ma
p.
ub
ss
A.
ila - BV H8 8
sp.
subs
A. s
96
ii
ou
sm
A. s
sub
sp
34
ch
el
a
ei
A.
s
su
su
p.
bs
sa
si
m
iu m
ia
e
A.
omo
gene
s
52 6
id a - 01 -B
lm
p.
on
bs
sa
ic
p.
lm
id
a
sa
on
-J
s a lm o n
. sa
lm
lm
ici
F3
on
da
22
o n ic
icid
ic id a 2 0 0 9 -1
id a
aRS
-C
IP
10
32
- A4
4
53
44K3
9
4
09
4
div
sa
sch
er
A.
ube
rtii
um
A. mol l uscor
A. r i v u l i
rel
an
na
sa
A.
iw
ta
A.
bs
s
ss
u
A.
a lv
A . b iv
0.08
A. me
d ia
lii
A
n
.e
A.
A. c
avi
ae
en
op
la
hi
en
cr
A.
ta
s
tec
si
A.
eu
A.
304
305
306
307
308
309
310
a
chr
p. a
A. s su
bsp.
b ri a
A. so
303
ithi
A. s subsp. salmonic ida - 2004-05M F26
T 4199
A. sacc haro phila - CEC
C
- AM
ie s
pec
A. s
a
ic
p. sa lm on
A. s su bs
Aeromonads
96
cid
Figure S5. Molecular phylogeny of 43 aeromonads inferred from 1645 core genes by maximumlikelihood using the GTR+Γ model. Only bootstrap values under 100 are shown in this figure.
All the bootstrap values for the salmonicida subspecies are given on Figure 1 (main article) for
clarity. The red branches correspond to mesophilic taxa, the purple branch corresponds to
intermediate taxon and the blue branch corresponds to psychrophilic taxa. The strain numbers are
shown only when there are two taxa from the same species or subspecies.
22
312
313
314
315
316
---
A. s CBA100
97.49
---
A. s subsp.
pectinolytica
90.54
90.62
---
A. s Y577
90.51
90.63
97.52
---
A. s Y567
90.69
90.83
97.14
97.11
---
A. s Y47
90.63
90.78
97.11
97.13
97.64
---
A. s subsp.
masoucida
A. s subsp.
smithia
A. s subsp.
achromogenes
A. s subsp.
salmonicida
90.52
90.59
97.08
97.08
97.59
97.47
---
90.56
90.71
97.08
97.09
97.58
97.47
99.70
---
90.54
90.75
97.11
97.11
97.59
97.50
99.68
99.62
---
90.52
90.75
97.06
97.06
97.55
97.46
99.75
99.69
99.64
---
A. bestiarum
A. s CBA100
A. s subsp.
pectinolytica
A. s Y577
A. s Y567
A. s Y47
A. s subsp.
masoucida
A. s subsp.
smithia
A. s subsp.
achromogenes
A. s subsp.
salmonicida
311
A. bestiarum
Figure S6. Average nucleotide identity (ANI) analyses for some A. salmonicida subspecies
included in this study. A. bestiarum is also included for comparative purposes with A.
salmonicida CBA100. Two taxa were considered as belonging to the same subspecies if they
shared an ANI value ≥ 96 (yellow and green).
23
0.6
A. salmonicida
O.D (595 nm)
pectinolytica
Y577
Y567
0.4
Y47
masoucida
01-B526
0.2
0.0
0
317
2
4
6
8
Time (h)
318
Figure S7. Growth curves at 7°C for selected A. salmonicida subspecies. The growth curves
319
were determined three times in independent experiments. The means of three replicates with
320
standard error of the mean are shown for each subspecies.
321
24
repB
relE
repA
relB
RNA I
mob
chemotaxis
protein
Origin of
replication
repB
pY47-1
pY47-2
12 495 bp
6 042 bp
repB
parB
chemotaxis
protein
parA
mob
acyltransferase
parD
parE
repA
GTP-binding
protein
RNA I
pY47-3
ccdB
Origin of
replication
5 104 bp
ccdA
mobC
mobD
mobB
mobA
322
323
324
325
326
327
Figure S8. The three high-copy plasmids found in the Indian strain Y47. The pY47-3 plasmid
was also found in the Indian strain Y577. The blue arrows represent genes with a known
function, the green arrows represent genes encoding hypothetical proteins, and the black arrow
represents the putative RNA regulator.
25
mobD
mobA
mobB
tnp
ISAS11
pJF4097
6 231 bp
mobC
RNA I
exoY-like
RNA II
328
329
330
331
332
333
Figure S9. The high-copy plasmid pJF4097 found in A. salmonicida subsp. smithia. The blue
arrows represent genes with a known function, the green arrows represent genes encoding
hypothetical proteins, the black arrows represent the putative RNAs regulator, and the grey
rectangle represents the ISAS11.
26
1
2 3 4
334
335
336
337
338
339
Figure S10. Result of the PCR assay confirming that the RS 534 strain lost its TTSS by the
recombination of two ISAS11s (B-C rearrangement [10]). The wells are as follows: (1) 2-log
DNA ladder (New England Biolabs), (2) RS 534, (3) JF3224 (positive control), and (4) 01-B526
(negative control).
27
1700
1600
1500
148/547
pectinolytica
salmonicida
achromogenes
masoucida
smithia
A
370/348
1400
1300
1200
572/279
489/412
1100
514/335
Y47
Y567
Y577
1000
900
25/326
800
700
212/149
70/45
117/156
217/235
1
300
100
66/34
0
popoffii (outgroup)
B
Gain
0.09
Relative importance
500
200
46/119
Loss
1
0.06
0.03
0.00
A B C D E F G H I
J K L M N O P Q R S T U V W X Y Z
Functional category
2
Gain
Loss
0.10
Relative Importance
600
400
201/235
44/262
C
2
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
Q
R
S
T
U
V
W
X
Y
Z
RNA processing and modification
Chromatin structure and dynamics
Energy production and conversion
Cell cycle control, cell division, chromosome partitioning
Amino acid transport and metabolism
Nucleotide transport and metabolism
Carbohydrate transport and metabolism
Coenzyme transport and metabolism
Lipid transport and metabolism
Translation, ribosomal structure and biogenesis
Transcription
Replication, recombination and repair
Cell wall/membrane/envelope biogenesis
Cell motility
Posttranslational modification, protein turnover, chaperones
Inorganic ion transport and metabolism
Secondary metabolites biosynthesis, transport and catabolism
General function prediction only
Function unknown
Signal transduction mechanisms
Intracellular trafficking, secretion, and vesicular transport
Defense mechanisms
Extracellular structures
Mobilome: prophages, transposons
Nuclear structure
Cytoskeleton
0.05
0.00
340
A B C D E F G H I
J K L M N O P Q R S T U V W X Y Z
Functional category
28
341
342
343
344
345
346
347
Figure S11. Pan-genome analysis of selected A. salmonicida subspecies, including A. popoffii as
an outgroup. (A) Distribution of the pan-genome on a phylogenetic tree for some key taxa. The
phylogenetic tree was based on the tree found using the core genome. The green and black values
indicate the number of genes acquired and lost, respectively, for the specific branch using the
parsimonious Dollo model. The branch lengths represent the number of genes acquired or lost.
For A. salmonicida subsp. salmonicida the strain used was 01-B526. Relative importance of 26
functional categories for the genes implicated in branches 1 (B) and 2 (C).
29
348
Number of genes
30
20
10
0
A B C D E F G H
349
350
351
352
I
J
K
L M N O P Q R S T U V W X Y Z
Functional category
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
Q
R
S
T
U
V
W
X
Y
Z
RNA processing and modification
Chromatin structure and dynamics
Energy production and conversion
Cell cycle control, cell division, chromosome partitioning
Amino acid transport and metabolism
Nucleotide transport and metabolism
Carbohydrate transport and metabolism
Coenzyme transport and metabolism
Lipid transport and metabolism
Translation, ribosomal structure and biogenesis
Transcription
Replication, recombination and repair
Cell wall/membrane/envelope biogenesis
Cell motility
Posttranslational modification, protein turnover, chaperones
Inorganic ion transport and metabolism
Secondary metabolites biosynthesis, transport and catabolism
General function prediction only
Function unknown
Signal transduction mechanisms
Intracellular trafficking, secretion, and vesicular transport
Defense mechanisms
Extracellular structures
Mobilome: prophages, transposons
Nuclear structure
Cytoskeleton
Figure S12. Functional categories of the genes under positive selection in the A. salmonicida
mesophilic lineages.
30
353
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