Complete genome sequence analysis of the fish pathogen Flavobacterium columnare provides insights into antibiotic resistance and pathogenicity related genes

Accepted Manuscript Complete genome sequence analysis of the fish pathogen Flavobacterium columnare provides insights into antibiotic resistance and pathogenicity related genes Yulei Zhang, Lijuan Zhao, Wenjie Chen, Yunmao Huang, Ling Yang, V. Sarathbabu, Zaohe Wu, Jun Li, Pin Nie, Li Lin PII:

S0882-4010(17)30962-2

DOI:

10.1016/j.micpath.2017.08.035

Reference:

YMPAT 2425

To appear in:

Microbial Pathogenesis

Received Date: 5 August 2017 Revised Date:

29 August 2017

Accepted Date: 30 August 2017

Please cite this article as: Zhang Y, Zhao L, Chen W, Huang Y, Yang L, Sarathbabu V, Wu Z, Li J, Nie P, Lin L, Complete genome sequence analysis of the fish pathogen Flavobacterium columnare provides insights into antibiotic resistance and pathogenicity related genes, Microbial Pathogenesis (2017), doi: 10.1016/j.micpath.2017.08.035. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

1 / 27

1

Complete genome sequence analysis of the fish pathogen Flavobacterium columnare

2

provides insights into antibiotic resistance and pathogenicity related genes

3

Yulei Zhanga, Lijuan Zhaoa,b*, Wenjie Chenb, Yunmao Huangb, Ling Yangb, V. Sarathbabub,

4

Zaohe Wub, Jun Lib,d,e, Pin Niec*, Li Lina,b,e,f*

5

a

6

China

7

b

8

Guangdong Provincial Key Laboratory of Waterfowl Healthy Breeding, College of Animal

9

Sciences and Technology, Zhongkai University of Agriculture and Engineering, Guangzhou,

RI PT

College of Fisheries, Huazhong Agricultural University, Wuhan, Hubei Province, 430070,

SC

Guangzhou Key Laboratory of Aquatic Animal Diseases and Waterfowl Breeding,

10

Guangdong, 510225, China

11

c

12

Chinese Academy of Sciences, Wuhan, Hubei Province, 430072, China

13

d

14

USA

15

e

16

Laboratory for Marine Science and Technology, Qingdao, Shandong, 266071, China

17

f

18

Guangzhou, Guangdong, 510640, China

19

*

20

E-mail addresses:

21

[email protected] (Y. Zhang), [email protected] (L. Zhao),

22

[email protected] (W. Chen), [email protected] (Y. Huang),

23

[email protected] (L. Yang), [email protected] (V. Sarathbabu),

24

[email protected] (Z. Wu), [email protected] (J. Li), [email protected] (P. Nie),

25

[email protected] (L. Lin).

M AN U

State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology,

School of Biological Sciences, Lake Superior State University, Sault Ste. Marie, MI 49783,

TE D

Laboratory for Marine Fisheries Science and Food Production Processes, National

Agro-biological Gene Research Center, Guangdong Academy of Agricultural Sciences,

AC C

EP

Corresponding authors

ACCEPTED MANUSCRIPT 26

2 / 27

Abstract We analyzed here the complete genome sequences of a highly virulent Flavobacterium

28

columnare Pf1 strain isolated in our laboratory. The complete genome consists of a 3,171,081

29

bp circular DNA with 2,784 predicted protein-coding genes. Among these, 286 genes were

30

predicted as antibiotic resistance genes, including 32 RND-type efflux pump related genes

31

which were associated with the export of aminoglycosides, indicating inducible

32

aminoglycosides resistances in F. columnare. On the other hand, 328 genes were predicted as

33

pathogenicity related genes which could be classified as virulence factors, gliding motility

34

proteins, adhesins, and many putative secreted proteases. These genes were probably involved

35

in the colonization, invasion and destruction of fish tissues during the infection of F.

36

columnare. Apparently, our obtained complete genome sequences provide the basis for the

37

explanation of the interactions between the F. columnare and the infected fish. The predicted

38

antibiotic resistance and pathogenicity related genes will shed a new light on the development

39

of more efficient preventional strategies against the infection of F. columnare, which is a

40

major worldwide fish pathogen.

41

Keywords

44

SC

M AN U

TE D

related genes

EP

43

Flavobacterium columnare; complete genome; antibiotic resistance genes; pathogenicity

AC C

42

RI PT

27

ACCEPTED MANUSCRIPT 45

3 / 27

1. Introduction Flavobacterium columnare is a member of the family Flavobacteriaceae and the

47

causative pathogen of columnaris which is a world-wide bacterial fish disease [1-4]. The

48

major clinical signs of columnaris included frayed fins, rotted gills, and skin ulceration,

49

seriously with visceral injury and tissue necrosis [5-7]. According to FAO statistics [8], the

50

total production of fish and the freshwater fish in China reached 45,469 and 26,029.7 kilotons

51

in 2014, resulted in a ratio of 61.62% and 59.76% in the world, respectively. However, the

52

columnaris disease caused high mortality and great economic loss in many kinds of

53

freshwater fishes such as mandarin fish, channel catfish and grass carp in China [9-11].

54

However, currently, the effective prevention strategies against columnaris are still not

55

available.

M AN U

SC

RI PT

46

The research about the genetic basis for the molecular pathogenesis of F. columnare

57

infection will shed a new light on the development of a practical effective vaccine used for

58

combating fish columnaris. F. columnare has been classified into three genomovars, in

59

accordance with differences in 16S rRNA sequences and restriction fragment length

60

polymorphism (RFLP) of the 16S–23S rDNA spacer [12-15]. Five complete genome of F.

61

columnare strains were presented [16-19] and comparative genomic analysis were conducted

62

between Flavobacterium, revealing F. columnare is capable of denitrification, which would

63

enable anaerobic growth in aquatic pond sediments [16]. However, the systematical

64

researches of pathogenesis and antibiotic resistance mechanisms of F. columnare are

65

unavailable. We have isolated a highly virulent F. columnare Pf1 strain from the diseased

66

yellow catfish (Pelteobagrus fulvidraco) in our laboratory [20]. To illustrate the genetic

67

properties of the F. columnare Pf1 strain, here we utilized the single-molecule real-time

68

(SMRT) technology and Illumina sequencing to determine the complete genome sequences of

69

the strain. The results showed that F. columnare Pf1 strain belonged to genomovar I. Analysis

70

of the complete genome sequence of Pf1 revealed a number of genes declared in F. columnare

71

for adaptation to broad host niches and provided insights into the disease mechanisms and

72

drug targets in microorganism.

AC C

EP

TE D

56

ACCEPTED MANUSCRIPT 73

2. Materials and methods

74

2.1 Bacteria isolated, sequencing and annotation

4 / 27

F. columnare strain Pf1 was isolated from diseased yellow catfish (P. fulvidraco) in our

76

laboratory [20]. The genomic sequencing annotation methods were presented in our previous

77

brief announcement [19], with the GenBank accession number CP016277 We constructed a

78

circle map of Pf1 genome using the online software Circos (http://circos.ca/).

79

2.2 Construction of phylogenetic tree

RI PT

75

F. columnare belongs to Cytophaga-Flavobacterium-Bacteroides Taxa (CFB). The 16S

81

rDNA gene sequences of selected species including Flavobacterium, Cytophaga, Bacteroides

82

genus were obtained from the GenBank databases (Supplementary table S1). The sequences

83

were aligned using CLUSTALX, and the phylogenetic tree was obtained using MEGA 6

84

software of neighbor-joining method [21,22], with the bootstrap values of 1000 replicates.

85

The Bacteroides distasonis in the same family as outgroup.

86

2.3 Pathogenic factors and antibiotic resistance genes annotation

M AN U

SC

80

To identify the pathogenic factors and antibiotic resistance genes, all the predicted gene

87

and

sequences

were

blasted

to

virulence

factor

database

(VFDB)

89

(http://www.mgc.ac.cn/VFs/) [23,24] and comprehensive antibiotic resistance database

90

(CARD) (https://card.mcmaster.ca/) [25,26]. The VFDB is an integrated and comprehensive

91

online resource for curating information about virulence factors of bacterial pathogens. The

92

CARD database provided a centralized compendium of information on antibiotic resistance

93

and facilitated the consistent annotation of resistance information in newly sequenced

94

organisms and identification of new genes.

95

3. Results and discussion

96

3.1 General features of the complete chromosome sequence and comparison

AC C

EP

protein

TE D

88

97

The complete genome of F. columnare Pf1 strain consists of a single circular

98

chromosome of 3,171,081 bp, with an average G + C content of 31.6%. The relatively high

99

number of rRNA and tRNA genes, 18 and 81 respectively, is in consistence with the rather

100

rapid growth of the bacterium [27,28]. The chromosome is predicted to contain 2,784

ACCEPTED MANUSCRIPT

5 / 27

protein-coding genes with an average length of 982 bp, representing 86.19% of the genome.

102

Among them, 1,664 (59.77%) genes are assigned to a functional category of Cluster of

103

Orthologous Groups (COGs) and the remaining are annotated as hypothetical proteins or

104

proteins of unknown functions. We compared our obtained sequence data of F. columnare

105

strain with other Flavobacterium species (Table 1), Pf1 was nearly the same size and GC

106

content with F. columnare ATCC 49,512 (3.16 Mb) and TC 1,691 (3.03 Mb), which two

107

belong to genomovar I, but smaller than F. columnare 94-081 (3.32 Mb) and C#2 (3.33 Mb)

108

belong to genomovar II. All of the Flavobacterium strains have low G + C content. Only F.

109

psychrophilum JIP/86 and F. branchiophilum FL-15 carry plasmids. The number of total

110

genes, CDS, and RNAs were different to other genomes, revealed extensive sequence

111

diversity within the species.

M AN U

SC

RI PT

101

The circle map of F. columnare Pf1 genome was shown in Figure 1. Circle 1 (from

113

outside to inside) is the heat map of G+C percentage content, while the higher value makes

114

redder and the lower makes bluer. The histogram on circle 2 represents GC skew, when the

115

values of (G–C)/(G+C) is greater or less than zero was shown in red or blue. Circle 3 and 4

116

showed the plus and minus strands protein-coding genes according to COGs categories,

117

respectively. There are some multiple copy genes existed in the genome are linked by green

118

Bezier curve in the internal circle.

EP AC C

119

TE D

112

ACCEPTED MANUSCRIPT

1 / 27

Table 1. General features of the ten Flavobacterium genomes. Size

origin

(Mb)

F. columnare Pf1

China

3.17

31.6

2,883

2,784

982

F. columnare ATCC 49512

France

3.16

31.5

2,787

2,642

1,021

F. columnare TC 1691

China

3.03

31.6

2,676

2,576

–

F. columnare 94-081

United States

3.32

30.8

2,897

2,779

F. columnare C#2

United States

3.33

31.0

2,879

2,744

France

2.86

32.5

2,519

F. johnsoniae UW101

England

6.10

34.1

–

F. branchiophilum FL-15

Hungary

3.56

32.9

3,087

F. indicum GPTSA100-9T

India

2.99

31.8

2,787

F. enshiense DK69T

China

3.38

37.7

3,054

genes

Average CDS

%Coding

size (bp)

region

rRNA

tRNA

Plasmid

GenBank No.

86.2

18

81

None

CP016277.1

85.2

15

74

None

CP003222.2

–

22

75

None

CP018912.1

–

–

12

74

None

CP013992.1

–

–

39

93

None

CP015107.1

SC

CDS

2,432

1,003

84.5

18

49

pCP1

AM398681.2

5,056

1,061

87.3

18

62

None

CP000685.1

2,867

1,030

82.9

9

44

pFB1

FQ859183.1

2,671

–

–

12

55

None

HE774682

2,848

986

83.2

9

46

None

JRLZ00000000

TE D

121

Total

EP

F. psychrophilum JIP02/86

%GC

AC C

Bacteria strain

RI PT

Geographical

M AN U

120

ACCEPTED MANUSCRIPT

122

TE D

M AN U

SC

RI PT

1 / 27

Figure 1. Circular representation of the F. columnare Pf1 genome. The outer scale is in mega bases

124

(Mb). Circle 1 (from outside to inside), the heat map of G+C percentage content. Circle 2, GC skew.

125

Circle 3 and 4, plus and minus strands protein-coding genes according to COGs categories,

126

respectively. Multiple copies genes are linked by green Bezier curve in the internal circle. (COGs

127

classification in colors representative: C: Energy production and conversion; D: Cell division and

128

chromosome partitioning; E: Amino acid transport and metabolism; F: Nucleotide transport and

129

metabolism; G: Carbohydrate transport and metabolism; H: Coenzyme metabolism; I: Lipid

130

metabolism; J: Translation, ribosomal structure and biogenesis; K: Transcription; L: DNA replication,

131

recombination, and repair; M: Cell envelope biogenesis, outer membrane; N: Cell motility and

132

secretion; O: Post-translational modification, protein turnover, chaperones; P: Inorganic ion transport

133

and metabolism; Q: Secondary metabolites biosynthesis, transport, and catabolism; R: General

134

function prediction only; S: Function unknown; T: Signal transduction mechanisms; U: Intracellular

135

trafficking and secretion; V: Defense mechanisms; –: No COGs Annotation.)

AC C

EP

123

136

3.2 Phylogenetic analysis and genotyping

137

Based on the 16S rRNA gene sequences, a neighbor-joining phylogenetic tree was constructed

138

in the study. All the F. columnare strains were clustered into three branches and the strain Pf1

ACCEPTED MANUSCRIPT

2 / 27

139

was belonged to genomovar I (Figure 2). Our results were corresponding well to the previous

140

studies that F. columnare were divided into three genomovars (I, II, and III), respectively

141

[12-15]. 8 3

KE 02 XF

2

8239/ 97

7

3294/ 95

9

OS 03

23

Htan5 03 1397/ 00 52 10

F10-HK-A

F. columnare Genomovar I

ATCC 23463 ● Pf1

31

SC

59

RI PT

ATCC 49512

6

IAM 14301

99

FK 401

96

ARS-1

80

ATCC 49513

M AN U

76

LN1311 C5

60

8128/ 97

54 84

40

A8

BJ4

3

G4

1

G18

2

10

X1 Z0

99 100

TE D

72

60

LV339-01

51

EK-28

EP

PT-14-00-151 Ga-6-93 Z13

32

Z6

42

HJ

28

B2

22

H10

18

QJH-2

AC C

7 14

Z4 AU-98-24 GA-02-14

100

32

F. columnare Genomovar III

99

PH-97028

100

IFO 15970

F. johnsoniae

IFO 14942

F. johnsoniae

100

97 60

100

67

142 143

F. columnare Genomovar II

ALG-00-530 62

61

100

LP 8

DSM 2063

F. hydati

IAM 12365

F. hydati

CSF 259-93

F. psychrophilum

ATCC 49418

F. psychrophilum

IAM 12650

C. arvensicola

NCIMB 8628

C. aurantiaca

ATCC 8503

B. distasonis

ATCC 33236

B. gracilis

Figure 2. Phylogenetic tree based on 16S rDNA sequences of the species in Flavobacterium,

ACCEPTED MANUSCRIPT 144 145

3 / 27

Cytophaga, and Bacteroides.

3.3 Carbon-nitrogen metabolism and transports of F. columnare The first Flavobacterium metabolic pathways were constructed by Duchaud et al. [29]

147

with strain of F. psychrophilum JIP02/86. Carbon-nitrogen metabolism of F. columnare Pf1

148

was established using the Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway

149

Database annotation [30,31] (Figure 3). Among them, 33 genes encoding the enzymes of

150

glycolysis (except glucose kinase), gluconeogenesis, pentose phosphate pathway, citrate cycle,

151

and fructose and mannose metabolism were identified in this study. However, the genes

152

involved in sugar kinase and phosphotransferase systems were not existed, which are usually

153

used by bacteria for specific carbohydrate uptake. Actually, F. columnare is unable to use

154

cellulose [32] or starch [3] directly as sources of carbon and energy, but instead by other

155

pathways to acquire nutrients from their host for survival. The 6-phosphofructokinase 1

156

(Pf1_00390) and pyruvate kinase (Pf1_01219) are two important enzymes which can regulate

157

the rate of reaction in glycolysis. Moreover, compared genomic analysis showed that some

158

carbohydrate metabolism components and pathways are unique, such as starch and sucrose

159

utilization pathways are present only in F. johnsoniae and F. branchiophilum [16]. Diversity

160

pathways of amino acids biosynthesis and metabolism reflect a highly self-sufficient and

161

unique style in utilizing nutrients. Totally 70 genes in nitrogen metabolism pathways were

162

presented, including glycine, serine and threonine metabolism, phenylalanine, tyrosine and

163

tryptophan biosynthesis, valine, leucine and isoleucine degradation, alanine, aspartate and

164

glutamate metabolism, arginine and proline metabolism, and histidine metabolism. Genome

165

analysis of F. columnare ATCC 49512 indicated it is capable of denitrification, which would

166

enable anaerobic growth in aquatic pond sediments [16]. After the amino acid degradation, all

167

the nitrogen was excreted in the form of putrescine and NH3.

AC C

EP

TE D

M AN U

SC

RI PT

146

168

Beside the carbon-nitrogen metabolism in cytoplasm, the transporters are played

169

important roles in nutrition absorption and waste efflux. Gene clusters involved in substance

170

exchange on cytomembrane are shown in Figure 3. ATP-binding cassette transporters (ABC

171

transporters) utilize the energy of ATP binding and hydrolysis to transport various substrates

ACCEPTED MANUSCRIPT

4 / 27

across cellular membranes. Bacterial ABC transporters and secretion systems are essential in

173

cell viability, virulence, and pathogenicity [33,34]. Seven subclasses of ABC transporter

174

proteins were identified in Pf1 based on different substrates. Pathogens use siderophores to

175

scavenge iron that is in complex with high-affinity iron-binding proteins or erythrocytes [35].

176

Four iron complex transport system substrate-binding proteins (Pf1_00438, Pf1_02478,

177

Pf1_02477, Pf1_02475) were identified, indicating an efficient iron uptake system and

178

important virulence factors in Pf1.

179

AC C

EP

TE D

M AN U

SC

RI PT

172

180

Figure 3. Overview of metabolisms and transports in F. columnare Pf1. Different transport families

181

are distinguished by different colors and shapes. From top left going clockwise: ABC transporter

182

proteins (blue), secretion systems (orange), ions (green), nutrients (purple), polysaccharide export

183

(red), drug/metabolite efflux (pink). Arrows indicate the direction of transport. All the amino acid

184

biosynthesis genes and transports are listed in Supplementary table S2.

185

Total of 16 secretion related proteins were discerned including specialized secretion

186

systems of T1SS (H1yB), T2SS (Protein E), and T6SS (VgrG) in Gram negative bacteria.

187

Some secreted proteins are translocated across the cytoplasmic membrane by the Sec

ACCEPTED MANUSCRIPT

5 / 27

translocon, which requires the presence of an N-terminal signal peptide on the secreted

189

protein. Others are translocated across the cytoplasmic membrane by the twin-arginine

190

translocation pathway (Tat). Studies on type VI secretion suggested a broader physiological

191

role in defense against simple eukaryotic predators and its role in inter-bacteria interactions

192

[36].

RI PT

188

The breakdown of host proteins by the secreted proteases results in a mixture of amino

194

acids and oligopeptides that probably constitute the main source of carbon, nitrogen and

195

energy. Six importers probably involved in the uptake of host proteins degradation products

196

were identified, three of which were proton-dependent or proton-symport proteins

197

(Pf1_01932, Pf1_01931, and Pf1_00484). Except for organic matters absorption, inorganic

198

ions efflux through relevant transporters such as sodium, potassium, ferrous iron,

199

magnesium/cobalt, chloride, sulfate, phosphate, and nitrate were existed. TonB-dependent

200

receptors in gram-negative bacteria carry out high-affinity binding and energy-dependent

201

uptake of large substrates into the periplasmic space such as iron siderophore complexes

202

[37,38]. Plenty of ion channels on membrane provide sufficient nutrients for bacterial basic

203

requirements of growth.

TE D

M AN U

SC

193

The secretion of exopolysaccharides by bacterium with high viscosity provides a

205

beneficial environment for living and invading. F. columnare can secrete plenty of

206

exopolysaccharides under oligotrophic environment or during infection. Two polysaccharide

207

exporters (Pf1_103, Pf1_00125) were identified relevantly in the genome which must assist to

208

transporting polysaccharides from the cytoplasm to the periplasm with energy consumption.

209

Resistance-nodulation-division (RND) family and the major facilitator superfamily (MFS)

210

transporters are two categories of bacterial efflux pumps, especially identified in

211

Gram-negative bacteria and located in the cytoplasmic membrane, that actively transport

212

substrates [39,40]. The MFS family was originally believed to function primarily in the

213

uptake of sugars but subsequent studies revealed that drugs, metabolites, oligosaccharides,

214

amino acids and oxyanions were all transported by MFS family members [41]. Totally 13

215

drug/metabolite efflux genes were presented in Pf1 genome to drive transport utilizing the

AC C

EP

204

ACCEPTED MANUSCRIPT

6 / 27

electrochemical gradient of drugs. And on the other hand, 23 genes involved in antibiotic

217

biosynthesis genes in 7 KEGG pathways were find (Supplementary table S3), indicating that

218

the bacterium may secrete antibiotics to inhibit competitors and adapt to the environment

219

better.

220

3.4 Pathogenicity factors

RI PT

216

After all the annotated gene sequences were blasted to virulence factor database (VFDB),

222

328 genes were predicted as pathogenicity related genes which could be classified as

223

virulence factors, gliding motility proteins, adhesins and many putative secreted proteases.

224

These genes were probably involved in the colonization, invasion and destruction of fish

225

tissues during the infection of F. columnare.

226

3.4.1 Virulence factors

M AN U

SC

221

According to previous studies about pathogenic bacteria and the interactions with their

228

hosts, 13 putative factors involved in virulence in F. columnare were identified, which were

229

associated with bacteria invasion, nutrition scramble, and destruction of host physiology

230

during infection (Table 2). 8 of 13 genes are involved in iron absorption and utilization,

231

including two IucA/IucC family proteins (Pf1_02172, Pf1_02174), a siderophore biosynthetic

232

enzyme (Pf1_02176), three coproporphyrinogen III oxidases (Pf1_00890, Pf1_01643, and

233

Pf1_01775), and two hemolysins (Pf1_01169, Pf1_01214). Iron is a necessary nutritional

234

factor in the process of bacterial metabolism. The minimum concentration of iron intake for

235

bacterial growth is about 10-6 mol/L, but there is only about 10-18 mol/L in normal body fluid.

236

The capacity of bacteria acquired iron from the host is related to its pathogenicity. The

237

pathogenic E.coli uptakes iron from host by producing iron associative compounds (including

238

aerobactin and enterobactin) or secreting hemolysins. Aerobactin is a bacterial iron chelating

239

agent (siderophore) found in E. coli [42]. The aerobactin operon is roughly 8 kb long and

240

contains 5 genes (iucA, iucB, iucC, iucD, and iutA) in total [43]. Bacterial hemolysins are

241

cytolytic toxins that cause lysis of red blood cells by destroying their cell membrane, are

242

important virulence factors. In another fish pathogen Vibrio anguillarum, toxin VAH5 is able

243

to lyse rainbow trout erythrocytes and a VAH5 mutant showed attenuated virulence [44]. In

AC C

EP

TE D

227

ACCEPTED MANUSCRIPT

7 / 27

Gram-negative organisms, the coproporphyrinogen III oxidase is an enzyme participate in

245

heme synthesis, which is the second major point of regulation genes [45]. In Pf1, the two

246

hemolysins may have a similar role in pathogenicity and cooperate with secreted proteases for

247

tissue destruction. These genes annotated in F. columnare as virulent factors, suggesting a

248

typical strategy of bacteria in scrambling iron for survival and infecting host.

RI PT

244

Chondroitin AC lyase (chondroitinase, Pf1_01408) may cause the necrotic lesions by

250

degrading chondroitin in the extracellular matrix of fish tissue [46]. The F. columnare isolates

251

from cold water exhibited significantly greater chondroitinase activity than isolates from

252

warm water, suggested that chondroitinase activity may be correlated with virulence [47,48].

253

Internalin A (InlA, Pf1_01412) is a surface protein used by the bacteria to invade host cells

254

via cadherins transmembrane proteins [49]. Internalins include two forms (InlA and InlB) are

255

mainly surface-exposed virulence factors present in a number of gram-positive bacteria whose

256

role ranges from recognition of cellular receptors to aid in pathogen entry to escape from

257

autophagy [50].

M AN U

Table 2. Putative factors of F. columnare Pf1 involved in virulence. Product IucA/IucC family protein IucA/IucC family protein Siderophore biosynthetic enzyme Coproporphyrinogen III oxidase Coproporphyrinogen III oxidase Coproporphyrinogen III oxidase Hemolysin D Hemolysin Chondroitin AC lyase Thermolysin Peptidase M4, thermolysin Muramidase Internalin A

TE D

Length (bp) 1,812 2,418 1,323 1,365 906 1,056 954 1,812 2,286 2,988 2,709 942 942

AC C

Gene Pf1_02172 Pf1_02174 Pf1_02176 Pf1_00890 Pf1_01643 Pf1_01775 Pf1_01169 Pf1_01214 Pf1_01408 Pf1_00712 Pf1_01445 Pf1_02300 Pf1_01412

EP

258

SC

249

259 260

3.4.2 Motility and adhesion

261

In this study, 11 gld genes (gldA, -B, -D, -F, -G, -H, -I, -J, -K, -M, and -N) involved in

262

the gliding motility were identified (Table 3), identically with F. johnsoniae (except gldN or

ACCEPTED MANUSCRIPT

8 / 27

gldL) [51]. F. columnare infected on fish move rapidly over surfaces by two movement

264

patterns, one is gliding motility, an active movement that does not involve pili or flagella, and

265

another is swing one side, with another side attached. Cells with mutations in any of gliding

266

genes are completely nonmotile, and they form nonspreading colonies exhibit no movement

267

on agar or glass surfaces. Another two MotB-related proteins (Pf1_00564, Pf1_01212) were

268

also found, known as the component of the flagellar motor driving the flagella rotation [52].

269

But, no genes encoding flagella, pili or any motility organelles were found in the F. columnare

270

genome, indicating the two proteins may assist to swing or did other functions.

RI PT

263

We identified 8 genes probably involved in bacterial adhesion, including 3 fibronectins

272

and 5 adhesins (Table 3). Adhesion mechanisms are greatly diversified among bacteria and

273

are of particular importance in pathogenicity. Fibronectin plays a major role in cell adhesion,

274

growth, migration, and differentiation, and it is important for processes such as wound healing

275

and embryonic development [53]. SprD (Small pathogenicity island RNA D) is a non-coding

276

RNA expressed on pathogenicity islands in Staphylococcus aureus and significantly

277

contribute to causing disease in an animal model [54]. Those surface structures mediating

278

adherence and pathogen-host recognition seem to be the most important properties for the

279

initiation of infection process in F. columnare.

M AN U

TE D

Table 3. Putative genes of F. columnare Pf1 involved in motility and adhesion. Length (bp) 993 1,386 1,449 996 1,704 732 1,668 597 894 489 504 855 969 897 2,079

EP

Gene Pf1_01995 Pf1_02102 Pf1_02104 Pf1_02105 Pf1_02246 Pf1_00019 Pf1_00020 Pf1_00893 Pf1_01034 Pf1_01167 Pf1_01450 Pf1_00564 Pf1_01212 Pf1_01763 Pf1_02370

AC C

280

SC

271

Product Gliding motility protein GldB Gliding motility protein GldK Gliding motility protein GldM Gliding motility protein GldN Gliding motility lipoprotein GldJ Gliding motility protein GldF Gliding motility protein GldG Gliding motility lipoprotein GldI Gliding motility protein GldA Gliding motility protein GldD Gliding motility protein GldH Flagellar motor/chemotaxis (MotB)-related lipoprotein Flagellar motor/chemotaxis (MotB)-related protein Fibronectin type III domain-containing protein Fibronectin type III domain protein

ACCEPTED MANUSCRIPT Pf1_02377 Pf1_01525 Pf1_01526 Pf1_00314 Pf1_00870 Pf1_00889

255 1,479 4,035 783 4,452 1,827

9 / 27

Fibronectin type III domain-containing protein Adhesin precursor SprC Cell surface protein precursor SprD Putative adhesin Putative adhesin Adhesin SprB

282

RI PT

281

3.4.3 Putative secreted proteases

Twenty-five putative secreted proteases were identified in the F. columnare genome,

284

including 15 metalloproteases, 7 serine proteases and 3 endopeptidas (Table 4). In general,

285

pathogenic bacteria release proteases to break down proteins of the host’s extracellular matrix

286

when attaching onto host surfaces [55], thus causing necrotic lesions [56]. Studies in F.

287

psychrophilum also certified that proteases were among its most important virulence factors

288

and leaded to rapid and massive tissue destruction [29,57]. Some extracellular proteases and

289

membrane-associated zinc metalloproteases of F. columnare were characterized which was

290

considered virulence factors [58,59].

M AN U

SC

283

Most metalloproteases require zinc (such as Pf1_01415, Pf1_01566), but some use

292

cobalt (such as Pf1_00647). The metal ion is coordinated to the protein via three ligands,

293

which coordinating the metal ion can vary with histidine, glutamate, aspartate, lysine, and

294

arginine. The metalloprotease Fpp2 of F. psychrophilum hydrolyzes a broad range of

295

substrates, including basic elements of muscular tissues [60]. The oligopeptidase PepO from

296

Streptococcus thermophilus A was estimated to be a serine metallopeptidase and contains the

297

signature sequence of the metallopeptidase family [61]. Pathogenic fungi secrete

298

endopeptidases belonging to the fungalysin family of metalloproteases that cleave

299

extracellular matrix proteins and are involved in the breakdown of proteinaceous structural

300

barriers during host tissue colonization [62]. A similar mechanism may apply for F. columnare,

301

in that we identified a paralogous gene Pf1_0182, predicted to encode metalloprotease of the

302

fungalysin family. A correlation between proteolytic activity and the virulence of F.

303

psychrophilum was found in cold temperature, indicating that M43 cytophagalysin may be

304

involved in pathogenesis through destruction of host tissues [63].

305

AC C

EP

TE D

291

Serine proteases are enzymes that cleave peptide bonds in proteins, in which serine

ACCEPTED MANUSCRIPT

10 / 27

serves as the nucleophilic amino acid at the active site. We identified 7 serine proteases

307

including prolyl oligopeptidase in peptidase S9 family and subtilisin kexin sedolisin peptidase

308

S8/S53 family. Endopeptidase Clp hydrolyze proteins to small peptides in the presence of ATP

309

and Mg2+. This bacterial enzyme contains subunits of two types, ClpP (Pf1_01880), with

310

peptidase activity, and ClpA (Pf1_01712, Pf1_02751), with ATPase activity [64,65]. Table 4. Putative proteases of F. columnare Pf1 involved in the destruction of host tissues.

312 313

M AN U

SC

Product M1 family metalloprotease precursor M1 family metalloprotease precursor M13 family metallopeptidase PepO precursor M36 fungalysin family metalloprotease precursor M43 cytophagalysin family metalloprotease precursor M48 family metalloprotease YggG M48 family metalloprotease M50 family membrane-associated zinc metalloprotease precursor Peptidase membrane zinc metallopeptidase Cobaltochelatase, CobN subunit Metalloprotease Fpp2 precursor Metalloprotease Metalloprotease Metallopeptidase Metalloendopeptidase Endopeptidase ClpP Endopeptidase ClpA Endopeptidase ClpA Serine endopeptidase Serine endopeptidase Serine protease Prolyl oligopeptidase Peptidase S9 prolyl oligopeptidase Protease DegQ precursor Peptidase S8/S53 subtilisin kexin sedolisin

TE D

Length (bp) 1,956 1,641 2,013 2,733 954 825 816 1,350 699 3,735 2,832 906 903 942 2,040 657 2,469 2,610 1,614 1,611 1,452 2,118 1,851 1,386 6,408

AC C

Gene Pf1_01414 Pf1_02695 Pf1_01410 Pf1_01821 Pf1_00384 Pf1_01904 Pf1_02730 Pf1_01415 Pf1_01566 Pf1_00647 Pf1_00130 Pf1_00292 Pf1_00293 Pf1_02253 Pf1_02720 Pf1_01880 Pf1_01712 Pf1_02751 Pf1_00518 Pf1_01269 Pf1_02142 Pf1_02712 Pf1_01199 Pf1_02403 Pf1_01355

EP

311

RI PT

306

3.5 Antibiotic resistance

314

The treatment of infections is increasingly compromised by the ability of bacteria to

315

develop resistance to antibiotics through mutation or acquisition of resistance genes. Regular

316

blast DNA and protein sequences on CARD helped us to identify and annotate new potential

ACCEPTED MANUSCRIPT

11 / 27

resistance genes in F. columnare. In this report, 286 genes were predicted as antibiotic

318

resistance genes blasted with CARD. Among them, 32 genes were annotated as

319

resistance-nodulation-cell division (RND)-type efflux pumps (Table 5), which are tripartite

320

complexes form a channel to facilitate drug export across the inner and outer membranes,

321

driven by the proton motive force [66,67]. In particular, increased expression of adeABC and

322

adeIJK, the two RND-type efflux pumps, has been associated with a multidrug resistance

323

phenotype, specifically with the export of aminoglycosides [68,69]. AdeB is the multidrug

324

transporter of the adeABC efflux system, and adeI is the membrane fusion protein of the

325

adeIJK multidrug efflux complex. AdeABC expression is controlled positively by the

326

two-component regulatory system adeRS, whereas adeR inactivation leads to susceptibility of

327

aminoglycoside antibiotics [70]. In Pf1, 2 adeB, 6 adeI, 19 adeR, and adeS genes were

328

predicted in the genome, indicating an inducible aminoglycoside resistance in F. columnare.

329

Actually, a kanamycin resistance strain of F. columnare (strain Pf1-Ka) was induced by

330

gradual increasing kanamycin concentration in our lab, with the tolerance concentration 500

331

µg/mL. Transcriptome and proteome analysis between Pf1 and Pf1-Ka were conducted to

332

identify pivotal genes and proteins, as well as the biological pathways in kanamycin

333

resistance of microorganism (unpublished data). The current results support our genome

334

research analysis convincingly and lay a foundation for the further study on the mechanism of

335

drug resistance in F. columnare.

336

Table 5. The aminoglycoside resistant genes predicted in F. columnare Pf1.

EP

TE D

M AN U

SC

RI PT

317

Name acrF acrF/adeB acrF/adeB

Length (bp) 2,421 4,314 3,375

Pf1_00342 Pf1_00951 Pf1_01120 Pf1_01169 Pf1_01692 Pf1_01861 Pf1_00318

adeI adeI adeI adeI adeI adeI adeR

1,083 1,086 1,206 954 1,140 1,305 705

AC C

Gene Pf1_01128 Pf1_01119 Pf1_01693

Product Exporter Putative multidrug resistance protein Multidrug resistance protein precursor AcrB/AcrD/AcrF family protein Secretion protein HlyD family protein Secretion protein HlyD family protein RND family efflux transporter MFP subunit Hemolysin D Membrane fusion efflux protein precursor Membrane fusion efflux protein Two-component system response regulatory protein RprY

ACCEPTED MANUSCRIPT

696 720

Pf1_01370 adeR

684

Pf1_01428 Pf1_01434 Pf1_01443 Pf1_02641

adeR adeR adeR adeR

363 720 1,212 1,164

Pf1_02719 Pf1_00023 Pf1_00382 Pf1_00910 Pf1_02660 Pf1_00317

adeR adeR/adeS adeR/adeS adeR/adeS adeR/adeS adeS

1,545 2,202 3,975 2,178 2,193 1,548 978

Pf1_01430 Pf1_01432 Pf1_01652 Pf1_00950 Pf1_01168 Pf1_01691 Pf1_01860

1,938 1,047 1,377 1,320 1,419 1,323 1,446

337

4. Conclusions

AC C

338

EP

adeS adeS adeS tolC tolC tolC tolC

TE D

Pf1_01369 adeS

RI PT

Pf1_00864 adeR Pf1_01055 adeR

LytTR family two component transcriptional regulator Two-component system response regulatory protein LytTR family two component transcriptional regulator Two-component system response regulatory protein involved in phosphate regulation Two-component system response regulatory protein Two-component system response regulatory protein Putative glycosyltransferase Two component sigma-54 specific Fis family transcriptional regulator Two-component system response regulatory protein Integral membrane sensor hybrid histidine kinase Multi-sensor hybrid histidine kinase Two-component system sensor histidine kinase PAS/PAC sensor hybrid histidine kinase Two-component system sensor histidine kinase RprX Two-component system sensor histidine kinase involved in phosphate regulation Sensory transduction histidine kinase Two-component system sensor histidine kinase Two-component system sensor histidine kinase Putative outer membrane efflux protein Outer membrane efflux protein Outer membrane efflux protein precursor Putative outer membrane transport/efflux protein

SC

756

M AN U

Pf1_00560 adeR

12 / 27

339

Comprehensive sequence analysis of F. columnare, from the metabolism, virulence

340

factors, adhesion and antibiotic resistance, revealed a combination of strategies to colonize

341

and degrade fish tissues and to exploit proteinaceous compounds for growth. The various

342

candidate virulent factors together with its strong adhesive properties probably contribute to

343

its dissemination in aquacultures worldwide with strong virulence. Moreover, the antibiotic

344

resistance genes analysis provided the medication guidance for the bacterial infection. Thus,

345

the genetic analysis of F. columnare genome sequence should facilitate the understanding of

346

flavobacterial virulence mechanisms in fish and provide a basis for the development of better

ACCEPTED MANUSCRIPT 347

13 / 27

control strategies.

AC C

EP

TE D

M AN U

SC

RI PT

348

ACCEPTED MANUSCRIPT

14 / 27

349

Supplementary Materials:

350

Table S1. 16S rDNA sequences used in the present study with their derived bacteria and hosts.

351

Table S2. Amino acid biosynthesis genes and transports in F. columnare Pf1.

352

Table S3. Antibiotic biosynthesis related genes in Pf1 genome.

354

RI PT

353

Acknowledgements:

This study was jointly supported by National Natural Science Fund (31572657,

356

31372563, 31602190); Special funds from the Administration of Ocean and Fisheries of

357

Guangdong Province (A201512C003; 2015-115); Special fund for Science and technology

358

from Hubei Province (2015BBA228); Fund from Wuhan Science and Technology Bureau

359

(2016020101010089) and “Innovation and Strong Universities” special funds (KA170500G)

360

from the Department of Education of Guangdong Province; Chinese Academy of Sciences

361

(CAS) (XDA08010207).

M AN U

SC

355

362

Author Contributions: Li Lin, Pin Nie, Lijuan Zhao conceived and designed the

364

experiments; Yulei Zhang, Lijuan Zhao performed the experiments; Yulei Zhang, Lijuan Zhao,

365

Wenjie Chen, Yunmao Huang, Ling Yang, Zaohe Wu and Jun Li analyzed the data; Yulei

366

Zhang wrote the draft; Li Lin, Pin Nie, Jun Li and V. Sarathbabu finalized the manuscript.

369 370 371

EP

368

Conflicts of Interest:

The authors declare no conflict of interest.

AC C

367

TE D

363

ACCEPTED MANUSCRIPT

15 / 27

372

References

373

[1] Bernardet JF. ‘Flexibacter columnaris’: first description in France and comparison with

375 376

bacterial strains from other origins. Dis Aquat Organ 1989;6:37-44. [2] Decostere A, Haesebrouck F. Outbreak of columnaris disease in tropical aquarium fish. Vet Rec 1999;144:23-4.

RI PT

374

377

[3] Decostere A, Haesebrouck F, Devriese LA. Characterization of four Flavobacterium

378

columnare (Flexibacter columnaris) strains isolated from tropical fish. Vet Microbiol

379

1998;62:35-45.

[4] Figueiredo HCP, Klesius PH, Arias CR, Evans J, Shoemaker CA, Pereira Jr DJ, et al.

381

Isolation and characterization of strains of Flavobacterium columnare from Brazil. J Fish

382

Dis 2005;28(4):199-204.

M AN U

SC

380

383

[5] Davis HS. A new bacterial disease in freshwater fishes. US Bur Fish Bull 1922;38:37-63.

384

[6] Anacker RL, Ordal EJ. Studies on the myxobacterium Chondrococcus columnaris: I

385

Serological type. J Bacteriol 1959;78(1):25-32.

[7] Zhou W, Zhang Y, Wen Y, Ji W, Zhou Y, Ji Y, et al. Analysis of the transcriptomic

387

profilings of Mandarin fish (Siniperca chuatsi) infected with Flavobacterium columnare

388

with an emphasis on immune responses. Fish Shellfish Immunol 2015;43(1):111-9.

390

[8] FAO, Global fisheries and aquaculture status: contribute to comprehensively achievement of food and nutrition security, Rome, Italy, 2015.

EP

389

TE D

386

[9] Chen C, Shi W, Zhao G, Li M. Isolation and identification of pathogenic bacteria causing

392

roted gill disease in mandarinfish (Siniperca chuarsi basilewsky). J Huazhong Agricul

393

Univ 1995;14(3):263-6 (In Chinese).

AC C

391

394

[10] Liu LH, Li NQ, Shi CB, Pan HJ, Fu XZ, Wu SQ. Isolation and classification of

395

pathogenic bacterium caused by gill-rote disease in channel catfish (Ictalunes punctatus).

396

J Anhui Agri Sci 2008;36(17):7124-6 (In Chinese).

397

[11] Li N, Guo HZ, Jiao R, Zhang SH, Liu ZX, Yao WJ, et al. Identification and pathogenicity

398

of bacterial pathogens isolated in an outbreak on bacterial disease of Ctenopharngodon

399

idellus Acta. Hydrobiol Sinica 2011;35(6):980-6 (In Chinese).

ACCEPTED MANUSCRIPT 400 401

16 / 27

[12] Triyanto A, Wakabayashi H. Genotypic diversity of strains of Flavobacterium columnare from diseased fishes. Fish Pathol 1999;34:65-71. [13] Arias CR, Welker TL, Shoemaker CA, Abernathy JW, Klesius PH. Genetic fingerprinting

403

of Flavobacterium columnare isolates from cultured fish. J Appl Microbiol

404

2004;97(2):421-8.

RI PT

402

405

[14] Darwish AM, Ismaiel AA. Genetic diversity of Flavobacterium columnare examined by

406

restriction fragment length polymorphism and sequencing of the 16S ribosomal RNA

407

gene and the 16S-23S rDNA spacer. Mol Cell Probes 2005;19:267-74.

[15] Shoemaker CA, Olivares-Fuster O, Arias CR, Klesius PH. Flavobacterium columnare

409

genomovar influences mortality in channel catfish (Ictalurus punctatus). Vet Microbiol

410

2008;127:353-9.

M AN U

SC

408

411

[16] Tekedar HC, Karsi A, Reddy JS, Nho SW, Kalindamar S, Lawrence ML. Comparative

412

genomics and transcriptional analysis of Flavobacterium columnare strain ATCC 49512.

413

Front Microbiol 2017;8:588.

[17] Kumru S, Tekedar HC, Waldbieser GC, Karsi A, Lawrence ML. Genome sequence of the

415

fish pathogen Flavobacterium columnare Genomovar II strain 94-081. Genome Announc

416

2016;4(3):e00430-16.

TE D

414

[18] Bartelme RP, Newton RJ, Zhu Y, Li N, LaFrentz BR, McBride MJ. Complete genome

418

sequence of the fish pathogen Flavobacterium columnare strain C#2. Genome Announc

419

2016;4(3):e00624-16.

421

[19] Zhang Y, Nie P, Lin L. Complete genome sequence of the fish pathogen Flavobacterium

AC C

420

EP

417

columnare Pf1. Genome Announc 2016;4(5):e00900-16.

422

[20] Zhang YL, Zhao LJ, Zhou WD, Ai TS, Lin L. Characterization and pathogenicity of a

423

Flavobacterium columnare isolated from Pelteobagrus fulvidraco. J Huazhong Agricul

424

Univ 2016;35:27-33 (In Chinese).

425 426 427

[21] Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 2013;30(12):2725-9. [22] Saitou N, Nei M. The neighbor-joining method: a new method for reconstructing

ACCEPTED MANUSCRIPT

429 430 431 432

phylogenetic trees. Mol Biol Evol 1987;4:406-25. [23] Chen L, Yang J, Yu J, Yao Z, Sun L, Shen Y, et al. VFDB: a reference database for bacterial virulence factors. Nucleic Acids Res 2005;33(suppl_1):D325-8. [24] Yang J, Chen L, Sun L, Yu J, Jin Q. VFDB 2008 release: an enhanced web-based resource for comparative pathogenomics. Nucleic Acids Res 2007;36(suppl_1):D539-42.

RI PT

428

17 / 27

433

[25] McArthur AG, Waglechner N, Nizam F, Yan A, Azad MA, Baylay AJ, et al. The

434

Comprehensive Antibiotic Resistance Database. Antimicrob Agents Chemother

435

2013;57(7):3348-57.

438 439 440 441

SC

437

[26] McArthur AG, Wright GD. Bioinformatics of antimicrobial resistance in the age of molecular epidemiology. Curr Opin Microbiol 2015;27:45-50.

[27] Klappenbach JA, Dunbar J, Schmidt T. rRNA operon copy number reflects ecological

M AN U

436

strategies of bacteria. Appl Environ Microbiol 2000;66:1328-33. [28] Rocha EP. Codon usage bias from tRNA’s point of view: redundancy specialization and efficient decoding for translation optimization. Genome Res 2004;14:2279-86. [29] Duchaud E, Boussaha M, Loux V, Bernardet JF, Michel C, Kerouault B, et al. Complete

443

genome sequence of the fish pathogen Flavobacterium psychrophilum. Nat Biotechnol

444

2007;25(7):763-9.

447 448

of genes and genomes. Nucleic Acids Res 1999;27:29-34.

EP

446

[30] Ogata H, Goto S, Sato K, Fujibuchi W, Bono H, Kanehisa M. KEGG: kyoto encyclopedia

[31] Kanehisa M, Goto S. KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res 2000;28:27-30.

AC C

445

TE D

442

449

[32] Declercq AM, Haesebrouck F, Van den Broeck W, Bossier P, Decostere A. Columnaris

450

disease in fish: a review with emphasis on bacterium-host interactions. Vet Res

451

2013;44(1):27.

452 453 454 455

[33] Davidson AL, Dassa E, Orelle C, Chen J. Structure function and evolution of bacterial ATP-binding cassette systems. Microbiol Mol Biol R 2008;72(2):317-64. [34] Wooldridge K, Bacterial secreted proteins: secretory mechanisms and role in pathogenesis, Caister Academic Press, 2009.

ACCEPTED MANUSCRIPT

457 458 459 460 461

[35] Neilands JB. Siderophores: Structure and Function of Microbial Iron Transport Compounds. J Biol Chem 1995;270(45):26723-6. [36] Coulthurst S. The type VI secretion system - A widespread and versatile cell targeting system. Res Microbiol 2013;64(6):640-54. [37] Koebnik R, Locher KP, Van Gelder P. Structure and function of bacterial outer membrane

RI PT

456

18 / 27

proteins: barrels in a nutshell. Mol Microbiol 2000;37(2):239-53.

462

[38] Chimento DP, Kadner RJ, Wiener MC. The Escherichia coli outer membrane cobalamin

463

transporter BtuB: structural analysis of calcium and substrate binding and identification

464

of

465

2003;332(5):999-1014.

transporters

by sequence/structure

conservation.

SC

orthologous

J

Mol

Biol

[39] Tseng TT, Gratwick KS, Kollman J, Park D, Nies DH, Goffeau A, et al. The RND

467

permease superfamily: an ancient ubiquitous and diverse family that includes human

468

disease and development proteins. J Mol Microb Biotech 1999;1(1):107-125.

470 471 472

[40] Pao SS, Paulsen IT, Saier MH. Major facilitator superfamily. Microbiol Mol Biol R 1998;62(1):1-34.

[41] Marger MD, Saier MH. A major superfamily of transmembrane facilitators that catalyse

TE D

469

M AN U

466

uniport symport and antiport. Trends Biochem Sci 1993;18(1):13-20. [42] Johnson JR, Moseley SL, Roberts PL, Stamm WE. Aerobactin and other virulence factor

474

genes among strains of Escherichia coli causing urosepsis: association with patient

475

characteristics. Infect Immun 1988;56(2):405-12.

EP

473

[43] de Lorenzo V, Bindereif A, Paw BH, Neilands JB. Aerobactin biosynthesis and transport

477

genes of plasmid ColV-K30 in Escherichia coli K-12. J Bacteriol 1986;165(2):570-8.

478

[44] Rodkhum C, Hirono I, Crosa JH, Aoki T. Four novel hemolysin genes of Vibrio

479 480 481

AC C

476

anguillarum and their virulence to rainbow trout. Microb Pathog 2005;39:109-19. [45] Choby JE, Skaar EP. Heme synthesis and acquisition in bacterial pathogens. J Mol Biol 2016;428(17):3408-28.

482

[46] Pojasek K, Shriver Z, Kiley P, Venkataraman G, Sasisekharan R. Recombinant expression

483

purification and kinetic characterization of chondroitinase AC and chondroitinase B from

ACCEPTED MANUSCRIPT 484 485 486

19 / 27

Flavobacterium heparinum. Biochem Biophys Res Commun 2001;286(2):343-51. [47] Stringer-Roth KM, Yunghans W, Caslake LF. Differences in chondroitin AC lyase activity of Flavobacterium columnare isolates. J Fish Dis 2002;25:687-91. [48] Kunttu HMT, Jokinen EI, Valtonen ET, Sundberg LR. Virulent and nonvirulent

488

Flavobacterium columnare colony morphologies: characterization of chondroitin AC

489

lyase activity and adhesion to polystyrene. J Appl Microbiol 2011;111:1319-26.

RI PT

487

[49] Lecuit M, Ohayon H, Braun L, Mengaud J, Cossart P. Internalin of Listeria

491

monocytogenes with an intact leucine-rich repeat region is sufficient to promote

492

internalization. Infect Immun 1997;65(12):5309-19.

494 495 496

[50] Neves D, Job V, Dortet L, Cossart P, Dessen A. Structure of internalin InlK from the human pathogen Listeria monocytogenes. J Mol Biol 2013;425(22):4520-9.

M AN U

493

SC

490

[51] Nelson SS, McBride MJ. Mutations in Flavobacterium johnsoniae secDF result in defects in gliding motility and chitin utilization. J Bacteriol 2006;188:348-51. [52] Stader J, Matsumura P, Vacante D, Dean GE, Macnab R. Nucleotide sequence of the

498

Escherichia coli motB gene and site-limited incorporation of its product into the

499

cytoplasmic membrane. J Bacteriol 1986;166(1):244-52.

TE D

497

[53] Pankov R, Yamada KM. Fibronectin at a glance. J Cell Sci 2002;115(Pt 20):3861-3.

501

[54] Chabelskaya S, Gaillot O, Felden B. A Staphylococcus aureus small RNA is required for

502

bacterial virulence and regulates the expression of an immune-evasion molecule. PLoS

503

Pathog 2010;6(6):e1000927.

EP

500

[55] Durborow RM, Thune RL, Hawke JP, Camus AC. Columnaris disease: a bacterial

505

infection caused by Flavobacterium columnare. Southern Regional Aquaculture Centre

506

Publication 1998;479.

507 508

AC C

504

[56] Miyoshi S, Shinoda S. Microbial metalloproteases and pathogenesis. Microbes Infect 2000;2:91-8.

509

[57] Bertolini JM, Wakabayashi H, Watral VG, Whipple MJ, Rohovec JS. Electrophoretic

510

detection of proteases from selected strains of Flexibacter psychrophilus and assessment

511

of their variability. J Aquat Anim Health 1994;6(3):224-33.

ACCEPTED MANUSCRIPT

20 / 27

512

[58] Newton JC, Wood TM, Hartley MM. Isolation and partial characterization of

513

extracellular proteases produced by isolates of Flavobacterium columnare derived from

514

channel catfish. J Aquat Anim Health 1997;9(2):75-85. [59] Xie HX, Nie P, Sun BJ. Characterization of two membrane-associated protease genes

516

obtained from screening out-membrane protein genes of Flavobacterium columnare G4. J

517

Fish Dis 2004;27(12):719-29.

RI PT

515

[60] Secades P, Alvarez B, Guijarro JA. Purification and properties of a new psychrophilic

519

metalloprotease (Fpp2) in the fish pathogen Flavobacterium psychrophilum. FEMS

520

Microbiol Lett 2003;226:273-9.

SC

518

[61] Chavagnat F, Meyer J, Casey MG. Purification characterisation cloning and sequencing

522

of the gene encoding oligopeptidase PepO from Streptococcus thermophilus A. FEMS

523

Microbiol Lett 2000;191(1):79-85.

M AN U

521

524

[62] Brouta F, Descamps F, Fett T, Losson B, Gerday C, Mignon B. Purification and

525

characterization of a 435 kDa keratinolytic metalloprotease from Microsporum canis.

526

Med Mycol 2001;39(3):269-75. [63] Hesami

S,

Metcalf

DS,

Lumsden

TE D

527

JS,

MacInnes

JI.

Identification

of

528

cold-temperature-regulated genes in Flavobacterium psychrophilum. Appl Environ

529

Microb 2011;77(5):1593-600.

[64] Gottesman S, Clark WP, Maurizi MR. The ATP-dependent Clp protease of Escherichia

531

coli Sequence of clpA and identification of a Clp-specific substrate. J Biol Chem

532

1990;265:7886-93.

AC C

EP

530

533

[65] Maurizi MR, Clark WP, Katayama Y, Rudikoff S, Pumphrey J, Bowers B, et al. Sequence

534

and structure of Clp P the proteolytic component of the ATP-dependent Clp protease of

535

Escherichia coli. J Biol Chem 1990;265:12536-45.

536

[66] Magnet S, Courvalin P, Lambert T. Resistance-nodulation-cell division-type efflux pump

537

involved in aminoglycoside resistance in Acinetobacter baumannii strain BM4454.

538

Antimicrob Agents Chemother 2001;45:3375-80.

539

[67] Damier-Piolle L, Magnet S, Brémont S, Lambert T, Courvalin P. AdeIJK a

ACCEPTED MANUSCRIPT

21 / 27

540

resistance-nodulation-cell division pump effluxing multiple antibiotics in Acinetobacter

541

baumannii. Antimicrob Agents Chemother 2008;52:557-62.

542 543

[68] Fournier PE, Vallenet D, Barbe V, Audic S, Ogata H, Poirel L, et al. Comparative genomics of multidrug resistance in Acinetobacter baumannii. PLoS Genet 2006 2(1) e7. [69] Coyne S, Guigon G, Courvalin P, Périchon B. Screening and quantification of the

545

expression of antibiotic resistance genes in Acinetobacter baumannii with a microarray.

546

Antimicrob Agents Chemother 2010;54:333-40.

RI PT

544

[70] Marchand I, Damier-Piolle L, Courvalin P, Lambert T. Expression of the RND-type

548

efflux pump AdeABC in Acinetobacter baumannii is regulated by the AdeRS

549

two-component system. Antimicrob Agents Chemother 2004;48:3298-304.

SC

547

AC C

EP

TE D

M AN U

550

ACCEPTED MANUSCRIPT Highlights A complete genome map of F. columnare was constructed.

•

286 antibiotic resistance genes and 328 pathogenicity related genes were identified.

•

Inducible aminoglycosides resistances may occur in F. columnare.

•

Gene analysis demonstrated a strong virulent strain of Pf1 during infection.

AC C

EP

TE D

M AN U

SC

RI PT

•