# Comparative Genomics Reveals Pathogenicity-Related Loci inShewanella algae

Authors: Jui-Hsing Wang; Guo-Cheng He; Yao-Ting Huang; Po-Yu Liu
Journal: Canadian Journal of Infectious Diseases and Medical Microbiology
 (2020)

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## Abstract

Shewanella algae is an emerging marine zoonotic pathogen and accounts for considerable mortality and morbidity in compromised hosts. However, there is scarce literature related to the understanding of the genetic background of virulence determinants in S. algae. In this study, we aim to determine the occurrence of common virulence genes in S. algae using whole-genome sequence and comparative genomic analysis. Comparative genomics reveals putative-virulence genes related to bile resistance, chemotaxis, hemolysis, and motility. We detected the existence of hlyA, hlyD, and hlyIII involved in hemolysis. We also found chemotaxis gene cluster cheYZA operon and cheW gene. The results provide insights into the genetic basis underlying pathogenicity in S. algae.


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## Body

## 1. Introduction

Shewanella algae is an emerging marine zoonotic pathogen. The organism was first classified in 1990 by Simidu et al. [1], emended by Nozue et al. [2], and described as a Gram-negative, motile bacillus, with hydrogen sulfide production, exhibiting hemolysis on sheep blood agar. S. algae is found in marine environments throughout the world and has been linked with both human and marine animal infections [3, 4]. Currently, there are at least three other Shewanella species found in clinical specimens and S. algae accounts for the majority of isolates from humans [5, 6]. S. algae has also been reported to cause diseases in marine animal, both wild and cultured [7–9]. However, there is scarce literature related to the understanding of the genetic background of virulence determinants in S. algae.Marine ecosystem consists of a large variety of organisms that impact human health [10]. The advance of sequencing technology allows the identification of determinants in pathogenic microorganisms and has become an important approach to study the fundamental mechanisms of pathogenesis [11, 12]. Comparative genomics further enables the investigation of core elements of pathogenesis factors in great detail [13]. Recently, there have been attempts to use whole-genome sequencing in the study of marine pathogens [14]. Therefore, genomic comparison of the clinical S. algae isolates could provide clues for pathogenic or fitness determinants [15].The aims of the study were to determine the occurrence of common virulence genes found inS. algae isolates from clinical setting using whole-genome sequence and comparative genomic analysis and to explore the relationship among the tested genomes.


## 2. Materials and Methods

### 2.1. Bacterial Strains, Media, and Growth Conditions

S. algae strains ACCC, YHL, and CHL were obtained from various clinical sources (Table 1). Glycerol stock of stored isolates was grown in trypticase soy agar with 5% sheep blood (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) at 30°C for 24 hours. Single colonies were inoculated in tryptic soy broth (Becton, Dickinson and Company, Franklin Lakes, NJ). The isolates were preliminarily identified using 16S rRNA gene sequencing and matrix-assisted laser desorption ionization-time of flight mass spectrometry (bioMérieux, Marcy l’Etoile, France). A part of 16S rRNA gene was amplified using the primers of B27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and U1492R (5′-GGTTACCTTGTTACGACTT-3′) [9, 16]. The nucleotide sequences were aligned, and BLAST search was performed against the GenBank database of the National Center for Biotechnology Information (NCBI) [17].Table 1
Strains and genomic features ofS. algae strains in this study.
Strain
Isolation source
Geographic origin
Genome assembly status
Genome coverage
Genome size (bp)
GC content (%)
CDSs
Pseudogenes
rRNA operons
tRNAs
CHL
Bile
Taiwan
Scaffold
243.0x
4,888,589
52.96
4,281
122
6, 5, 2 (5S, 16S, 23S)
88
YHL
Wound
Taiwan
Scaffold
257.0x
4,850,439
53.00
4,212
71
6, 5, 2 (5S, 16S, 23S)
86
ACCC
Bile
Taiwan
Scaffold
186.0x
4,744,804
53.08
4,223
143
4, 4 (5S, 16S)
91
MARS 14
Lung
France
Scaffold
91.0x
5,005,849
52.90
4,347
90
6, 3, 3 (5S, 16S, 23S)
104


### 2.2. Genome Sequencing and Assembly

Nucleic acids were extracted from overnight culture using the QIAGEN Genomic-tip 100/G kit and the Genomic DNA Buffer Set (QIAGEN, Paisley, UK) according to the manufacturer’s protocol. The DNA concentrations were measured by Qubit dsDNA HS Assay kit using Qubit 2.0 fluorometer (Life Technologies, Carlsbad, CA, USA). The DNA sample was sheared, in a microTUBE using Covaris S2 (Covaris, Woburn, MA, USA), into the desired size fragment of the library. The indexed PCR-free library preparation was performed using multiplexed high-throughput sequencing TruSeq DNA Sample Preparation Kit (Illumina) with 2μg of DNA on the basis of the manufacturer’s introduction. Genome sequencing was performed using paired-end 250 bp sequencing on the Illumina MiSeq platform (Illumina, Inc., San Diego, CA). Raw sequence files were artifact-filtered and trimmed with DUK (http://duk.sourceforge.net/) and FASTX-toolkit fastx_trimmer (https://github.com/agordon/fastx_toolkit), respectively. Assembly was performed with a hybrid approach by ALLPATHS, version R46652 and Velvet version 1.2.07.


### 2.3. Public Genome Download

Genome sequence of human isolatedS. algae MARS 14 was retrieved from the NCBI Genome website (https://www.ncbi.nlm.nih.gov/assembly/GCF_000947195.1/).


### 2.4. Phylogenetic Analysis Based on Whole-Genome Sequences

Genome-based phylogenic analysis was performed using pairwise comparison of average nucleotide identity. The whole-genome average nucleotide identity (ANI) was calculated with the use of a modified algorithm [18]. Phylogenetic trees were visualized using MEGA7.


### 2.5. Annotation and Comparative Genomics

The annotation was performed using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) [19] and the DOE-JGI Microbial Genome Annotation Pipeline version 4.10.5 [20]. The prediction was done using Glimmer 3.02 [21]. The nontranslated genes were predicted by tRNAscan-SE [22], RNAmmer [23], and RFAM [24]. Functional classification of the predicted genes was carried out using RPSBLAST program v. 2.2.15 [25]. Analysis of the functional annotation was further performed using the Integrated Microbial Genomes & Microbiomes system v.5.0 [26] and the Pathosystems Resource Integration Center [27]. CDS count for these strains was derived. Comparative genome analysis was performed using EDGAR platform (http://edgar.computational.bio) [28]. The core genome and the singletons for the 4 related S. algae genomes were generated for Prokka-annotated genomes using EDGAR (http://edgar.computational.bio). We compared the S. algae genomes using the MUMmer software package [29] together with the Circos visualization engine [30].


## 2.1. Bacterial Strains, Media, and Growth Conditions

S. algae strains ACCC, YHL, and CHL were obtained from various clinical sources (Table 1). Glycerol stock of stored isolates was grown in trypticase soy agar with 5% sheep blood (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) at 30°C for 24 hours. Single colonies were inoculated in tryptic soy broth (Becton, Dickinson and Company, Franklin Lakes, NJ). The isolates were preliminarily identified using 16S rRNA gene sequencing and matrix-assisted laser desorption ionization-time of flight mass spectrometry (bioMérieux, Marcy l’Etoile, France). A part of 16S rRNA gene was amplified using the primers of B27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and U1492R (5′-GGTTACCTTGTTACGACTT-3′) [9, 16]. The nucleotide sequences were aligned, and BLAST search was performed against the GenBank database of the National Center for Biotechnology Information (NCBI) [17].Table 1
Strains and genomic features ofS. algae strains in this study.
Strain
Isolation source
Geographic origin
Genome assembly status
Genome coverage
Genome size (bp)
GC content (%)
CDSs
Pseudogenes
rRNA operons
tRNAs
CHL
Bile
Taiwan
Scaffold
243.0x
4,888,589
52.96
4,281
122
6, 5, 2 (5S, 16S, 23S)
88
YHL
Wound
Taiwan
Scaffold
257.0x
4,850,439
53.00
4,212
71
6, 5, 2 (5S, 16S, 23S)
86
ACCC
Bile
Taiwan
Scaffold
186.0x
4,744,804
53.08
4,223
143
4, 4 (5S, 16S)
91
MARS 14
Lung
France
Scaffold
91.0x
5,005,849
52.90
4,347
90
6, 3, 3 (5S, 16S, 23S)
104


## 2.2. Genome Sequencing and Assembly

Nucleic acids were extracted from overnight culture using the QIAGEN Genomic-tip 100/G kit and the Genomic DNA Buffer Set (QIAGEN, Paisley, UK) according to the manufacturer’s protocol. The DNA concentrations were measured by Qubit dsDNA HS Assay kit using Qubit 2.0 fluorometer (Life Technologies, Carlsbad, CA, USA). The DNA sample was sheared, in a microTUBE using Covaris S2 (Covaris, Woburn, MA, USA), into the desired size fragment of the library. The indexed PCR-free library preparation was performed using multiplexed high-throughput sequencing TruSeq DNA Sample Preparation Kit (Illumina) with 2μg of DNA on the basis of the manufacturer’s introduction. Genome sequencing was performed using paired-end 250 bp sequencing on the Illumina MiSeq platform (Illumina, Inc., San Diego, CA). Raw sequence files were artifact-filtered and trimmed with DUK (http://duk.sourceforge.net/) and FASTX-toolkit fastx_trimmer (https://github.com/agordon/fastx_toolkit), respectively. Assembly was performed with a hybrid approach by ALLPATHS, version R46652 and Velvet version 1.2.07.


## 2.3. Public Genome Download

Genome sequence of human isolatedS. algae MARS 14 was retrieved from the NCBI Genome website (https://www.ncbi.nlm.nih.gov/assembly/GCF_000947195.1/).


## 2.4. Phylogenetic Analysis Based on Whole-Genome Sequences

Genome-based phylogenic analysis was performed using pairwise comparison of average nucleotide identity. The whole-genome average nucleotide identity (ANI) was calculated with the use of a modified algorithm [18]. Phylogenetic trees were visualized using MEGA7.


## 2.5. Annotation and Comparative Genomics

The annotation was performed using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) [19] and the DOE-JGI Microbial Genome Annotation Pipeline version 4.10.5 [20]. The prediction was done using Glimmer 3.02 [21]. The nontranslated genes were predicted by tRNAscan-SE [22], RNAmmer [23], and RFAM [24]. Functional classification of the predicted genes was carried out using RPSBLAST program v. 2.2.15 [25]. Analysis of the functional annotation was further performed using the Integrated Microbial Genomes & Microbiomes system v.5.0 [26] and the Pathosystems Resource Integration Center [27]. CDS count for these strains was derived. Comparative genome analysis was performed using EDGAR platform (http://edgar.computational.bio) [28]. The core genome and the singletons for the 4 related S. algae genomes were generated for Prokka-annotated genomes using EDGAR (http://edgar.computational.bio). We compared the S. algae genomes using the MUMmer software package [29] together with the Circos visualization engine [30].


## 3. Results

### 3.1. Genome Sequencing and Assembly

The genomic sequencing consisted of 250 bp paired-end reads, yielding approximately 0.88 Gbp to 1.24 Gbp for each isolate. The de novo assembly of genome sequence data revealed that the number of contigs (>200 bp) varied from 27 to 74 for each genome. The maximum contig size among the genomes was 976,090 bp aligned to YHL. The GC content ranged from 52.96% for CHL to 53.08% for ACCC. Table1 shows the descriptive statistics of the genomic characteristics for the strains in this study. The sequence data were publicly available in NCBI SRA database (accession number: ACCC [LVCY00000000.1], CHL [LVDF00000000.1], and YHL [LVDU00000000.1]).


### 3.2. Genome-Based Phylogenetic Analysis

The average nucleotide identity (ANI) was calculated and revealed that testedS. algae strains were identical in terms of nucleotide sequences, as shown in Figure 1.Figure 1
Whole-genome phylogeny ofS. algae in the study.


### 3.3. Comparative Genomics

We constructed a pan-genome dataset using whole-genome sequence of sequencedS. algae strains. Figure 2 shows orthologous genes shared among strains and depicts the position and color-coded function of the S. algae genes. The numbers of orthologous and strain-specific unique genes are shown in the Venn diagram. Core genome for the S. algae strains consists of 1354 coding sequences (Figure 3). The set of unique genes harbored by each strain varies from 335 for S. algae YHL to 466 for S. algae CHL. Following genome map construction, we conducted genome mapping among the S. algae strains in the study. In this comparison, colored arcs indicate regions of high similarity as revealed by the NUCmer script from the MUMmer software package. As shown in Figure 4, the alignment revealed an obvious syntenic relationship in these strains.Figure 2
Circular genomes representation map and genome comparison ofShewanella algae (CHL, ACCC, MARS 14, and YHL). Predicted coding sequences (CDSs) are assigned various colors with respect to cellular functions. Circles show, from the outermost to the innermost, (1) DNA coordinates; (2, 3) function-based color-coded mapping of the CDSs predicted on the forward and reverse strands of the S. algae CHL genome, respectively; (4) orthologous CDSs shared between S. algae CHL and S. algae ACCC; (5) S. algae CHL-specific CDSs, compared with S. algae ACCC; (6) orthologous CDSs shared between S. algae CHL and S. algae MARS 14; (7) S. algae CHL-specific CDSs, compared with S. algae MARS 14; (8) orthologous CDSs shared between S. algae CHL and S. algae YHL; (9) S. algae CHL-specific CDSs, compared with S. algae YHL; (10) GC plot with regions above and below average in green and violet; (11) GC skew showing regions above and below average in yellow and light blue. This figure was plotted in Scalable Vector Graphics format via an in-house script, which calculates the radius and ribbon width according to the BLAST alignments and adds colors by COG classification of all orthogonal genes.Figure 3
Comparison of the gene contents of theShewanella algae in this study, Venn diagram showing the numbers of conserved and strain-specific coding sequences (CDSs).Figure 4
Genomes mapping between strains in the study. Each colored arc indicates an orthologous match between two species. The color segments in the outer circle are randomly displayed and do not correspond to a particular scheme. A minimum seed match size of 500 bp was used.


### 3.4. Analysis of Putative-Virulence-Related Genes

As illustrated in Table2, genes encoded exbBD, galU, and htpB are shared with S. algae genomes. Heat shock protein gene clpP and hemolysis homologous genes, hlyA, hlyD, hlyIII, and tolC, were found in each S. algae genome. Gene cluster cheYZA operon and cheW involved in chemotaxis were detected in all tested S. algae. Flagellar gene operons are present in all tested S. algae genome.Table 2
Virulence genes shared withS. algae strains in this study.
Gene
locus_tag
Length
locus_tag
Length
locus_tag
Length
locus_tag
Length
Strains
MARS 14
YHL
CHL
ACCC
hlyA
BN1227_RS19795
443
AYI97_RS17645
443
AYI82_RS07480
443
AYI77_RS13890
443
hlyD
BN1227_RS18765
352
AYI97_RS03440
352
AYI82_RS00925
352
AYI77_RS05040
352
BN1227_RS19395
349
AYI97_RS04065
349
AYI82_RS01570
349
AYI77_RS08410
349
BN1227_RS08585
314
AYI97_RS05045
314
AYI82_RS17560
314
AYI77_RS14620
314
hlyIII
BN1227_RS10295
226
AYI97_RS09385
226
AYI82_RS06545
226
AYI77_RS04320
226
tolC
BN1227_RS02290
424
AYI97_RS03690AYI97_RS12455AYI97_RS17535AYI97_RS18425
466438467491
AYI82_RS01185AYI82_RS03510AYI82_RS07370
466438467
AYI77_RS01075AYI77_RS01845AYI77_RS02720AYI77_RS04785AYI77_RS13995
491
BN1227_RS02895
438
438
BN1227_RS03705
491
424
BN1227_RS12395
438
466
BN1227_RS19025
466
467
BN1227_RS19685
467
htpB (groL)
BN1227_RS18535
546
AYI97_RS03170
546
AYI82_RS00700
546
AYI77_RS05265
546
galU
BN1227_RS14240
303
AYI97_RS19990
303
AYI82_RS13425
303
AYI77_RS03360
303
AYI82_RS18495
294
AYI77_RS16730
294
exbB
BN1227_RS13275
175
AYI97_RS02650
238
AYI82_RS00200
238
AYI77_RS07050
175
BN1227_RS13280
451
AYI97_RS04225
164
AYI82_RS01760
164
AYI77_RS07055
451
BN1227_RS17925
238
AYI97_RS14790
175
AYI82_RS21410
175
AYI77_RS08255
164
BN1227_RS19555
164
AYI97_RS14795
451
AYI82_RS21415
451
AYI77_RS08875
238
exbD
BN1227_RS13270
134
AYI97_RS02655
135
AYI82_RS00205
135
AYI77_RS07045
134
BN1227_RS17930
135
AYI97_RS04230
135
AYI82_RS01765
135
AYI77_RS08250
135
BN1227_RS19560
135
AYI97_RS14785
134
AYI82_RS21405
134
AYI77_RS08870
135
cheY
BN1227_RS07095
127
AYI97_RS06385
127
AYI82_RS05630
127
AYI77_RS20450
127
cheZ
BN1227_RS07100
245
AYI97_RS06380
245
AYI82_RS05625
245
AYI77_RS20455
245
cheA
BN1227_RS01115
701
AYI97_RS06375
776
AYI82_RS05620
770
AYI77_RS12010
696
BN1227_RS07105
776
AYI97_RS16805
696
AYI82_RS09360
701
AYI77_RS20925
754
cheW
BN1227_RS07130
164
AYI97_RS06350
164
AYI82_RS05595
164
AYI77_RS20950
164
BN1227_RS01120
183
AYI97_RS16800
183
AYI82_RS05600
336
AYI77_RS12015
183
BN1227_RS07125
336
AYI97_RS06355
335
AYI82_RS09355
183
AYI77_RS20945
336
clpP
BN1227_RS08465
202
AYI97_RS05170
202
AYI82_RS17685
202
AYI77_RS14495
202
FlgA
BN1227_RS06885
235
AYI97_RS06595
235
AYI82_RS05050
248
AYI77_RS09380
248
BN1227_RS21260
248
AYI97_RS14310
248
AYI82_RS05840
235
AYI77_RS20850
235
FlgB
BN1227_RS06900
132
AYI97_RS06580
132
AYI82_RS05045
116
AYI77_RS09385
116
BN1227_RS21255
116
AYI97_RS14305
116
AYI82_RS05825
132
AYI77_RS20835
132
FlgC
BN1227_RS06905
138
AYI97_RS06575
138
AYI82_RS05040
136
AYI77_RS09390
136
BN1227_RS21250
136
AYI97_RS14300
136
AYI82_RS05820
138
AYI77_RS20830
138
FlgD
BN1227_RS21245
221
AYI97_RS06570
227
AYI82_RS05035
221
AYI77_RS09395
221
AYI97_RS14295
221
AYI82_RS05815
227
AYI77_RS20825
227
FlgE
BN1227_RS06915
453
AYI97_RS06565
453
AYI82_RS05810
453
AYI77_RS20820
453
FlgF
BN1227_RS06920
247
AYI97_RS06560
247
AYI82_RS05805
247
AYI77_RS20815
247
FlgG
BN1227_RS06925
262
AYI97_RS06555
262
AYI82_RS05020
261
AYI77_RS09410
261
BN1227_RS21230
261
AYI97_RS14280
261
AYI82_RS05800
262
AYI77_RS20810
262
FlgH
BN1227_RS06930
224
AYI97_RS06550
363
AYI82_RS05015
223
AYI77_RS09415
223
BN1227_RS21225
223
AYI97_RS14275
224223
AYI82_RS05795
224
AYI77_RS20805
224
FlgI
BN1227_RS06935
363
AYI97_RS06545
363
AYI82_RS05010
373
AYI77_RS09420
359
BN1227_RS21220
373
AYI97_RS14270
373
AYI82_RS05790
363
AYI77_RS20800
363
FlgJ
BN1227_RS06940
336
AYI97_RS06540
336
AYI82_RS05785
336
AYI77_RS20795
336
FlgK
BN1227_RS06945
641
AYI97_RS06535
641
AYI82_RS05000
456
AYI77_RS09430
456
BN1227_RS21210
456
AYI97_RS14260
456
AYI82_RS05780
641
AYI77_RS20790
641
FlgL
BN1227_RS06950
401
AYI97_RS06530
401
AYI82_RS05775
401
AYI77_RS20785
FlgM
BN1227_RS06880
106
AYI97_RS06600
106
AYI82_RS05055
94
AYI77_RS09375
94
BN1227_RS21265
94
AYI97_RS14315
94
AYI82_RS05845
106
AYI77_RS20855
106
FlgN
BN1227_RS06875
143
AYI97_RS06605
143
AYI82_RS05060
171
AYI77_RS09370
171
AYI82_RS05850
143
AYI77_RS20860
143
FlgP
BN1227_RS06870
155
AYI97_RS06610
155
AYI82_RS05855
155
AYI77_RS20865
155
FlgT
BN1227_RS06860
385
AYI97_RS06620
385
AYI82_RS05865
385
AYI77_RS20875
385
FliA
BN1227_RS07090
239
AYI97_RS06390
239
AYI82_RS04955
236
AYI77_RS20445
239
BN1227_RS21165
236
AYI97_RS14215
236
AYI82_RS05635
239
AYI77_RS09475
236
FliD
BN1227_RS06970
456
AYI97_RS06510
456
AYI82_RS04980
445
AYI77_RS20325
451
BN1227_RS21190
445
AYI97_RS14240
445
AYI82_RS05755
456
FliE
BN1227_RS07000
110
AYI97_RS06480
110
AYI82_RS05090
111
AYI77_RS09340
111
BN1227_RS21300
111
AYI97_RS14350
111
AYI82_RS05725
110
AYI77_RS20355
110
FliF
BN1227_RS07005
569
AYI97_RS06475
569
AYI82_RS05085
555
AYI77_RS09345
555
BN1227_RS21295
555
AYI97_RS14345
555
AYI82_RS05720
569
AYI77_RS20360
569
FliG
BN1227_RS07010
347
AYI97_RS06470
347
AYI82_RS05080
328
AYI77_RS09350
324
BN1227_RS21290
328
AYI97_RS14340
328
AYI82_RS05715
347
AYI77_RS20365
347
FliH
BN1227_RS07015
322
AYI97_RS06465
324
AYI82_RS05710
324
AYI77_RS20370
324
FliI
BN1227_RS07020
446
AYI97_RS06460
446
AYI82_RS05070
441
AYI77_RS09360
441
BN1227_RS21280
441
AYI97_RS14330
441
AYI82_RS05705
446
AYI77_RS20375
446
FliJ
BN1227_RS07025
149
AYI97_RS06455
149
AYI82_RS05700
149
AYI77_RS20380
149
FliL
BN1227_RS00740
135
AYI97_RS06445
174
AYI82_RS04960
145
AYI77_RS11650
135
BN1227_RS07035
174
AYI97_RS14220
145
AYI82_RS05690
174
AYI77_RS20390
174
BN1227_RS21170
145
AYI97_RS17155
135
AYI82_RS09710
135
FliM
BN1227_RS07040
342
AYI97_RS06440
342
AYI82_RS05685
342
AYI77_RS18030
238
BN1227_RS21315
300
AYI97_RS14365
300
AYI77_RS20395
342
FliN
BN1227_RS07045
126
AYI97_RS06435
126
AYI82_RS05110
114
AYI77_RS18025
114
BN1227_RS21320
114
AYI97_RS14370
114
AYI82_RS05680
126
AYI77_RS20400
126
FliO
BN1227_RS07050
119
AYI97_RS06430
119
AYI82_RS05675
119
AYI77_RS20405
119
FliP
BN1227_RS07055
247
AYI97_RS06425
247
AYI82_RS05115
265
AYI77_RS18020
265
BN1227_RS21325
265
AYI97_RS14375
265
AYI82_RS05670
247
AYI77_RS20410
247
FliQ
BN1227_RS07060
89
AYI97_RS06420
89
AYI82_RS05120
89
AYI77_RS18015
89
BN1227_RS21330
89
AYI97_RS14380
89
AYI82_RS05665
89
AYI77_RS20415
89
FliR
BN1227_RS07065
265
AYI97_RS06415
265
AYI82_RS05125
259
AYI77_RS18010
259
BN1227_RS21335
259
AYI97_RS14385
259
AYI82_RS05660
265
AYI77_RS20420
265
FliS
BN1227_RS06980
136
AYI97_RS06500
136
AYI82_RS04975
126
AYI77_RS09455
126
BN1227_RS21185
126
AYI97_RS14235
126
AYI82_RS05745
136
AYI77_RS20335
136
flhA
BN1227_RS21345
692
AYI97_RS14395
692
AYI82_RS05135
692
AYI77_RS18000
692
BN1227_RS07075
701
AYI97_RS06405
701
AYI82_RS05650
701
AYI77_RS20430
701
flhB
BN1227_RS07140
105
AYI97_RS06340
105
AYI82_RS05585
105
AYI77_RS20960
105
BN1227_RS21340
376
AYI97_RS14390
376
AYI82_RS05130
376
AYI77_RS18005
376
BN1227_RS07070
378
AYI97_RS06410
378
AYI82_RS05655
378
AYI77_RS20425
378
flhF
BN1227_RS07080
458
AYI97_RS06400
458
AYI82_RS05645
458
AYI77_RS20435
458


## 3.1. Genome Sequencing and Assembly

The genomic sequencing consisted of 250 bp paired-end reads, yielding approximately 0.88 Gbp to 1.24 Gbp for each isolate. The de novo assembly of genome sequence data revealed that the number of contigs (>200 bp) varied from 27 to 74 for each genome. The maximum contig size among the genomes was 976,090 bp aligned to YHL. The GC content ranged from 52.96% for CHL to 53.08% for ACCC. Table1 shows the descriptive statistics of the genomic characteristics for the strains in this study. The sequence data were publicly available in NCBI SRA database (accession number: ACCC [LVCY00000000.1], CHL [LVDF00000000.1], and YHL [LVDU00000000.1]).


## 3.2. Genome-Based Phylogenetic Analysis

The average nucleotide identity (ANI) was calculated and revealed that testedS. algae strains were identical in terms of nucleotide sequences, as shown in Figure 1.Figure 1
Whole-genome phylogeny ofS. algae in the study.


## 3.3. Comparative Genomics

We constructed a pan-genome dataset using whole-genome sequence of sequencedS. algae strains. Figure 2 shows orthologous genes shared among strains and depicts the position and color-coded function of the S. algae genes. The numbers of orthologous and strain-specific unique genes are shown in the Venn diagram. Core genome for the S. algae strains consists of 1354 coding sequences (Figure 3). The set of unique genes harbored by each strain varies from 335 for S. algae YHL to 466 for S. algae CHL. Following genome map construction, we conducted genome mapping among the S. algae strains in the study. In this comparison, colored arcs indicate regions of high similarity as revealed by the NUCmer script from the MUMmer software package. As shown in Figure 4, the alignment revealed an obvious syntenic relationship in these strains.Figure 2
Circular genomes representation map and genome comparison ofShewanella algae (CHL, ACCC, MARS 14, and YHL). Predicted coding sequences (CDSs) are assigned various colors with respect to cellular functions. Circles show, from the outermost to the innermost, (1) DNA coordinates; (2, 3) function-based color-coded mapping of the CDSs predicted on the forward and reverse strands of the S. algae CHL genome, respectively; (4) orthologous CDSs shared between S. algae CHL and S. algae ACCC; (5) S. algae CHL-specific CDSs, compared with S. algae ACCC; (6) orthologous CDSs shared between S. algae CHL and S. algae MARS 14; (7) S. algae CHL-specific CDSs, compared with S. algae MARS 14; (8) orthologous CDSs shared between S. algae CHL and S. algae YHL; (9) S. algae CHL-specific CDSs, compared with S. algae YHL; (10) GC plot with regions above and below average in green and violet; (11) GC skew showing regions above and below average in yellow and light blue. This figure was plotted in Scalable Vector Graphics format via an in-house script, which calculates the radius and ribbon width according to the BLAST alignments and adds colors by COG classification of all orthogonal genes.Figure 3
Comparison of the gene contents of theShewanella algae in this study, Venn diagram showing the numbers of conserved and strain-specific coding sequences (CDSs).Figure 4
Genomes mapping between strains in the study. Each colored arc indicates an orthologous match between two species. The color segments in the outer circle are randomly displayed and do not correspond to a particular scheme. A minimum seed match size of 500 bp was used.


## 3.4. Analysis of Putative-Virulence-Related Genes

As illustrated in Table2, genes encoded exbBD, galU, and htpB are shared with S. algae genomes. Heat shock protein gene clpP and hemolysis homologous genes, hlyA, hlyD, hlyIII, and tolC, were found in each S. algae genome. Gene cluster cheYZA operon and cheW involved in chemotaxis were detected in all tested S. algae. Flagellar gene operons are present in all tested S. algae genome.Table 2
Virulence genes shared withS. algae strains in this study.
Gene
locus_tag
Length
locus_tag
Length
locus_tag
Length
locus_tag
Length
Strains
MARS 14
YHL
CHL
ACCC
hlyA
BN1227_RS19795
443
AYI97_RS17645
443
AYI82_RS07480
443
AYI77_RS13890
443
hlyD
BN1227_RS18765
352
AYI97_RS03440
352
AYI82_RS00925
352
AYI77_RS05040
352
BN1227_RS19395
349
AYI97_RS04065
349
AYI82_RS01570
349
AYI77_RS08410
349
BN1227_RS08585
314
AYI97_RS05045
314
AYI82_RS17560
314
AYI77_RS14620
314
hlyIII
BN1227_RS10295
226
AYI97_RS09385
226
AYI82_RS06545
226
AYI77_RS04320
226
tolC
BN1227_RS02290
424
AYI97_RS03690AYI97_RS12455AYI97_RS17535AYI97_RS18425
466438467491
AYI82_RS01185AYI82_RS03510AYI82_RS07370
466438467
AYI77_RS01075AYI77_RS01845AYI77_RS02720AYI77_RS04785AYI77_RS13995
491
BN1227_RS02895
438
438
BN1227_RS03705
491
424
BN1227_RS12395
438
466
BN1227_RS19025
466
467
BN1227_RS19685
467
htpB (groL)
BN1227_RS18535
546
AYI97_RS03170
546
AYI82_RS00700
546
AYI77_RS05265
546
galU
BN1227_RS14240
303
AYI97_RS19990
303
AYI82_RS13425
303
AYI77_RS03360
303
AYI82_RS18495
294
AYI77_RS16730
294
exbB
BN1227_RS13275
175
AYI97_RS02650
238
AYI82_RS00200
238
AYI77_RS07050
175
BN1227_RS13280
451
AYI97_RS04225
164
AYI82_RS01760
164
AYI77_RS07055
451
BN1227_RS17925
238
AYI97_RS14790
175
AYI82_RS21410
175
AYI77_RS08255
164
BN1227_RS19555
164
AYI97_RS14795
451
AYI82_RS21415
451
AYI77_RS08875
238
exbD
BN1227_RS13270
134
AYI97_RS02655
135
AYI82_RS00205
135
AYI77_RS07045
134
BN1227_RS17930
135
AYI97_RS04230
135
AYI82_RS01765
135
AYI77_RS08250
135
BN1227_RS19560
135
AYI97_RS14785
134
AYI82_RS21405
134
AYI77_RS08870
135
cheY
BN1227_RS07095
127
AYI97_RS06385
127
AYI82_RS05630
127
AYI77_RS20450
127
cheZ
BN1227_RS07100
245
AYI97_RS06380
245
AYI82_RS05625
245
AYI77_RS20455
245
cheA
BN1227_RS01115
701
AYI97_RS06375
776
AYI82_RS05620
770
AYI77_RS12010
696
BN1227_RS07105
776
AYI97_RS16805
696
AYI82_RS09360
701
AYI77_RS20925
754
cheW
BN1227_RS07130
164
AYI97_RS06350
164
AYI82_RS05595
164
AYI77_RS20950
164
BN1227_RS01120
183
AYI97_RS16800
183
AYI82_RS05600
336
AYI77_RS12015
183
BN1227_RS07125
336
AYI97_RS06355
335
AYI82_RS09355
183
AYI77_RS20945
336
clpP
BN1227_RS08465
202
AYI97_RS05170
202
AYI82_RS17685
202
AYI77_RS14495
202
FlgA
BN1227_RS06885
235
AYI97_RS06595
235
AYI82_RS05050
248
AYI77_RS09380
248
BN1227_RS21260
248
AYI97_RS14310
248
AYI82_RS05840
235
AYI77_RS20850
235
FlgB
BN1227_RS06900
132
AYI97_RS06580
132
AYI82_RS05045
116
AYI77_RS09385
116
BN1227_RS21255
116
AYI97_RS14305
116
AYI82_RS05825
132
AYI77_RS20835
132
FlgC
BN1227_RS06905
138
AYI97_RS06575
138
AYI82_RS05040
136
AYI77_RS09390
136
BN1227_RS21250
136
AYI97_RS14300
136
AYI82_RS05820
138
AYI77_RS20830
138
FlgD
BN1227_RS21245
221
AYI97_RS06570
227
AYI82_RS05035
221
AYI77_RS09395
221
AYI97_RS14295
221
AYI82_RS05815
227
AYI77_RS20825
227
FlgE
BN1227_RS06915
453
AYI97_RS06565
453
AYI82_RS05810
453
AYI77_RS20820
453
FlgF
BN1227_RS06920
247
AYI97_RS06560
247
AYI82_RS05805
247
AYI77_RS20815
247
FlgG
BN1227_RS06925
262
AYI97_RS06555
262
AYI82_RS05020
261
AYI77_RS09410
261
BN1227_RS21230
261
AYI97_RS14280
261
AYI82_RS05800
262
AYI77_RS20810
262
FlgH
BN1227_RS06930
224
AYI97_RS06550
363
AYI82_RS05015
223
AYI77_RS09415
223
BN1227_RS21225
223
AYI97_RS14275
224223
AYI82_RS05795
224
AYI77_RS20805
224
FlgI
BN1227_RS06935
363
AYI97_RS06545
363
AYI82_RS05010
373
AYI77_RS09420
359
BN1227_RS21220
373
AYI97_RS14270
373
AYI82_RS05790
363
AYI77_RS20800
363
FlgJ
BN1227_RS06940
336
AYI97_RS06540
336
AYI82_RS05785
336
AYI77_RS20795
336
FlgK
BN1227_RS06945
641
AYI97_RS06535
641
AYI82_RS05000
456
AYI77_RS09430
456
BN1227_RS21210
456
AYI97_RS14260
456
AYI82_RS05780
641
AYI77_RS20790
641
FlgL
BN1227_RS06950
401
AYI97_RS06530
401
AYI82_RS05775
401
AYI77_RS20785
FlgM
BN1227_RS06880
106
AYI97_RS06600
106
AYI82_RS05055
94
AYI77_RS09375
94
BN1227_RS21265
94
AYI97_RS14315
94
AYI82_RS05845
106
AYI77_RS20855
106
FlgN
BN1227_RS06875
143
AYI97_RS06605
143
AYI82_RS05060
171
AYI77_RS09370
171
AYI82_RS05850
143
AYI77_RS20860
143
FlgP
BN1227_RS06870
155
AYI97_RS06610
155
AYI82_RS05855
155
AYI77_RS20865
155
FlgT
BN1227_RS06860
385
AYI97_RS06620
385
AYI82_RS05865
385
AYI77_RS20875
385
FliA
BN1227_RS07090
239
AYI97_RS06390
239
AYI82_RS04955
236
AYI77_RS20445
239
BN1227_RS21165
236
AYI97_RS14215
236
AYI82_RS05635
239
AYI77_RS09475
236
FliD
BN1227_RS06970
456
AYI97_RS06510
456
AYI82_RS04980
445
AYI77_RS20325
451
BN1227_RS21190
445
AYI97_RS14240
445
AYI82_RS05755
456
FliE
BN1227_RS07000
110
AYI97_RS06480
110
AYI82_RS05090
111
AYI77_RS09340
111
BN1227_RS21300
111
AYI97_RS14350
111
AYI82_RS05725
110
AYI77_RS20355
110
FliF
BN1227_RS07005
569
AYI97_RS06475
569
AYI82_RS05085
555
AYI77_RS09345
555
BN1227_RS21295
555
AYI97_RS14345
555
AYI82_RS05720
569
AYI77_RS20360
569
FliG
BN1227_RS07010
347
AYI97_RS06470
347
AYI82_RS05080
328
AYI77_RS09350
324
BN1227_RS21290
328
AYI97_RS14340
328
AYI82_RS05715
347
AYI77_RS20365
347
FliH
BN1227_RS07015
322
AYI97_RS06465
324
AYI82_RS05710
324
AYI77_RS20370
324
FliI
BN1227_RS07020
446
AYI97_RS06460
446
AYI82_RS05070
441
AYI77_RS09360
441
BN1227_RS21280
441
AYI97_RS14330
441
AYI82_RS05705
446
AYI77_RS20375
446
FliJ
BN1227_RS07025
149
AYI97_RS06455
149
AYI82_RS05700
149
AYI77_RS20380
149
FliL
BN1227_RS00740
135
AYI97_RS06445
174
AYI82_RS04960
145
AYI77_RS11650
135
BN1227_RS07035
174
AYI97_RS14220
145
AYI82_RS05690
174
AYI77_RS20390
174
BN1227_RS21170
145
AYI97_RS17155
135
AYI82_RS09710
135
FliM
BN1227_RS07040
342
AYI97_RS06440
342
AYI82_RS05685
342
AYI77_RS18030
238
BN1227_RS21315
300
AYI97_RS14365
300
AYI77_RS20395
342
FliN
BN1227_RS07045
126
AYI97_RS06435
126
AYI82_RS05110
114
AYI77_RS18025
114
BN1227_RS21320
114
AYI97_RS14370
114
AYI82_RS05680
126
AYI77_RS20400
126
FliO
BN1227_RS07050
119
AYI97_RS06430
119
AYI82_RS05675
119
AYI77_RS20405
119
FliP
BN1227_RS07055
247
AYI97_RS06425
247
AYI82_RS05115
265
AYI77_RS18020
265
BN1227_RS21325
265
AYI97_RS14375
265
AYI82_RS05670
247
AYI77_RS20410
247
FliQ
BN1227_RS07060
89
AYI97_RS06420
89
AYI82_RS05120
89
AYI77_RS18015
89
BN1227_RS21330
89
AYI97_RS14380
89
AYI82_RS05665
89
AYI77_RS20415
89
FliR
BN1227_RS07065
265
AYI97_RS06415
265
AYI82_RS05125
259
AYI77_RS18010
259
BN1227_RS21335
259
AYI97_RS14385
259
AYI82_RS05660
265
AYI77_RS20420
265
FliS
BN1227_RS06980
136
AYI97_RS06500
136
AYI82_RS04975
126
AYI77_RS09455
126
BN1227_RS21185
126
AYI97_RS14235
126
AYI82_RS05745
136
AYI77_RS20335
136
flhA
BN1227_RS21345
692
AYI97_RS14395
692
AYI82_RS05135
692
AYI77_RS18000
692
BN1227_RS07075
701
AYI97_RS06405
701
AYI82_RS05650
701
AYI77_RS20430
701
flhB
BN1227_RS07140
105
AYI97_RS06340
105
AYI82_RS05585
105
AYI77_RS20960
105
BN1227_RS21340
376
AYI97_RS14390
376
AYI82_RS05130
376
AYI77_RS18005
376
BN1227_RS07070
378
AYI97_RS06410
378
AYI82_RS05655
378
AYI77_RS20425
378
flhF
BN1227_RS07080
458
AYI97_RS06400
458
AYI82_RS05645
458
AYI77_RS20435
458


## 4. Discussion

S. algae has become an emerging marine zoonotic pathogen world-wide [5]. The spectrum of S. algae infection is broad with considerable morbidity and mortality in compromised hosts [31, 32]. Thus, understanding genomic characterization of S. algae is important for determining molecular epidemiology, understanding its pathogenesis, identifying specific biomarkers, tracing evolution of these strains, and developing control strategy of these pathogens in host reservoirs. In this study, we investigated the core genetic structure underlying S. algae virulence. The pathogenicity and distribution patterns of the S. algae strains extended our understanding of their pathogenic potential.Previous attempts have been made to report the basic features of the genome ofS. algae from various sources [33, 34]. In the present study, we used comparative genomics to analyze chromosomal sequence of four isolates to determine the common genetic content and organization, unique virulence attributes, and evolutionary relationship with other strains. Whole-genome sequence analysis of S. algae detected the presence of chemotaxis gene cluster cheYZA operon that is conserved in the chemotactic bacteria [35]. Chemotaxis is a directed motility in response to concentration gradients of signals. The cheA was demonstrated to be essential for chemotaxis using a two-component pathway [36]. In brief, CheA phosphorylates cheY and then is dephosphorylated by the phosphatase cheZ [37]. Previous studies revealed that CheW and CheA share structural homology and bind to the same site on chemoreceptors [37]. CheW is essential to the activation of CheA and the formation of CheA-CheW complex [38]. Owing to the wide range of S. algae habitats, the drivers of its chemotaxis could be very diverse. Previous studies have demonstrated that pathogenic bacteria use chemotaxis to localize reservoirs. Further study would be needed to identify the microenvironments suit for S. algae and the trigger of its chemotaxis.Biliary tract infection is main manifestation ofS. algae infection, and bile resistance has been noted in pathogenic strains [31]. In the study we also identified genes associated with bile adaption. The exbBD gene encodes Ton energy transduction system implicated in the response to bile [39, 40]. We also detected galU, htpB, and wecA involved in bile resistance [41–43]. The results support an earlier genomic study suggesting a common mechanism of bile resistance in Shewanella.Motility is one characteristic ofS. algae [3]. We identified series of flagellar gene operons in S. algae genomes. These flagellar systems are unique and require more study regarding the evolution and organization. Hemolysis is a main pathogenic feature in S. algae [44]. The gene hlyA encodes RTX pore-forming toxin α-hemolysin, which alters membrane permeability and causes cell lysis in a variety of human and animal hosts [45].


## 5. Conclusions

In conclusion, this is one of the few studies tracking genetic background of putative virulence-related genes inS. algae. Although the number of strains was limited, we highlight the unique characteristics of core virulence determinants in these strains, as a high level of genomic conservation.



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*Source: 9205197-2020-03-30.xml*