draft - university of toronto t-space · draft characterisation of ... 90 and api 20e system ......

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Characterisation of Klebsiella sp. S1: a bacterial producer of

secoisolariciresinol through biotransformation

Journal: Canadian Journal of Microbiology

Manuscript ID cjm-2016-0266.R2

Manuscript Type: Article

Date Submitted by the Author: 25-Jun-2016

Complete List of Authors: Zhou, Yu-Jie; Harbin Medical University Zhu, Songling ; Harbin Medical University Yang, Dong-Hui ; Peking University Zhao, Dan-Dan ; Harbin Medical University Li, Jia-Jing ; Harbin Medical University Liu, Shu-Lin; Harbin Medical University

Keyword: Klebsiella, secoisolariciresinol, biotransformation, flaxseed, lignan

https://mc06.manuscriptcentral.com/cjm-pubs

Canadian Journal of Microbiology

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Characterisation of Klebsiella sp. S1: a bacterial producer of secoisolariciresinol 1

through biotransformation 2

Yu-Jie Zhou1,2

, Songling Zhu1,2

, Dong-Hui Yang3, Dan-Dan Zhao

1,2, Jia-Jing Li

1,2, 3

Shu-Lin Liu1,2,4

* 4

1Systemomics Center, College of Pharmacy, and Genomics Research Center 5

(State-Province Key Laboratories of Biomedicine-Pharmaceutics of China), Harbin 6

Medical University, Harbin, China. 7

2HMU-UCFM Centre for Infection and Genomics, Harbin Medical University, Harbin, 8

China 9

3Department of Natural Medicines, School of Pharmaceutical Sciences, Peking University 10

Health Science Center, Beijing, China 11

4Department of Microbiology, Immunology and Infectious Diseases, University of Calgary, 12

Calgary, Canada. 13

*Corresponding author: Shu-Lin Liu 14

E-mail: [email protected] 15

Tel: +8645186692236, Fax: +8645187086735 16

17

18

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ABSTRACT 19

Secoisolariciresinol (SECO) is a lignan of potential therapeutical value for diseases such 20

as cancer, but its use has been limited by the lack of ideal production methods, even 21

though its precursors are abundant in plants, such as flaxseeds. Here we report the 22

characterization of a bacterial strain, S1, isolated from the human intestinal flora, which 23

could produce secoisolariciresinol by biotransformation of precursors in defatted flaxseeds. 24

This bacterium was a Gram-negative and facultatively anaerobic straight rod without 25

capsules. Biochemical assays showed that oxidase, lysine decarboxylase, orinithine 26

decarboxylase, arginine dihydrolase and β-glucolase were negative. The G+C content of 27

genomic DNA was 57.37 mol%. Phylogenetic analysis by 16S rRNA and rpoB gene 28

sequences demonstrated its close relatedness to Klebsiella. No homologues were found for 29

wzb or wzc (capsular genes), which may explain why Klebsiella S1 does not have the 30

capsule and was isolated from a healthy human individual. Based on the percentages of 31

homologous genes with identical nucleotide sequences between the bacteria in comparison, 32

we found that clear-cut genetic boundaries had been formed between S1 and any other 33

Klebsiella strains compared, dividing them into distinct phylogenetic lineages. This work 34

demonstrates that the intestinal Klebsiella, well known as important opportunistic 35

pathogens prevalent in potentially fatal nosocomial infections, may contain lineages that 36

are particularly beneficial to the human health. 37

38

Key words: Klebsiella; secoisolariciresinol; biotransformation; flaxseed; lignan 39

40

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INTRDUCTION 41

Secoisolariciresinol (SECO) is a lignan of potential therapeutical use, as suggested by 42

its preventive activities against colon and breast cancers (Thompson 1998), diabetes 43

(Prasad 2001; Prasad et al. 2000) and atherosclerosis (Prasad 1999). Additionally, it is also 44

an important intermediate in the biotransformation pathways from secoisolariciresinol 45

diglucoside (SDG) to enterodiol (END) and enterolactone (ENL). The two extensively 46

investigated phytoestrogens, END and ENL, have potent estrogenic as well as 47

anti-estrogenic activities in the human body and have been reported to be effective in the 48

prevention of several major human diseases such as osteoporosis, cardiovascular disorders, 49

hyperlipemia and menopausal syndrome in addition to cancers (Adlercreutz 2007; 50

Adlercreutz et al. 1992; Kitts et al. 1999; Lemay et al. 2002; Wang 2002). However, these 51

compounds have hitherto not been sufficiently applied in therapeutics, due largely to the 52

lack of efficient production methods. Although precursors of END and ENL are widely 53

distributed in nature and are especially abundant in plants like flaxseeds (Axelson et al. 54

1982; Borriello et al. 1985; Heinonen et al. 2001; Johnsson et al. 2000), they usually exist 55

as polymers and cannot be effectively extracted. Additionally, physicochemical methods 56

for the extraction or synthesis are expensive and in the meantime not environment-friendly. 57

Previously, we reported the isolation and preliminary characterization of a bacterial strain, 58

S1, which can produce enterolignans from precursors contained in defatted flaxseeds 59

through biotransformation by glycoside hydrolysis (see Fig. 1). Phylogenetic analysis 60

based on 16S rRNA comparison demonstrated its close relatedness to Klebsiella 61

pneumoniae (Wang et al. 2010). 62

Klebsiella are a large group of bacteria and contain clinically important species. The 63

genus Klebsiella, named in memory of the German microbiologist Edwin Klebs (Patrick 64

A.D. Grimont 2005), belongs to the family of Enterobacteriaceae and the type species is 65

Klebsiella pneumoniae. Bacteria in the genus of Klebsiella are genetically heterogeneous 66

and are divided into three clusters based on analysis of 16S rRNA and rpoB gene 67

sequences: clusterⅠcontains all subspecies of K. pneumoniae and also K. granulomatis; 68

cluster Ⅱ contains K. ornithinolytica, K. planticola, K. trevisanii and K. terrigena; and 69

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cluster Ⅲ contains K. oxytoca (Drancourt et al. 2001). In recent years, several novel 70

Klebsiella species have been reported such as K. singaporensis (Li et al. 2004), K. 71

variicola (Rosenblueth et al. 2004), K. alba (Xu et al. 2010) and K. michiganensis (Saha et 72

al. 2013). Klebsiellae are widely recognized as significant opportunistic pathogens in 73

hospitals, ranking second behind Escherichia coli as a major cause of nosocomial 74

Gram-negative bacteremia and representing 3–8% of all nosocomial infections (Podschun 75

and Ullmann 1998). The capsule was the first virulence factor described for Klebsiella 76

(Cryz et al. 1984; Mizuta et al. 1983; Williams et al. 1983). 77

In the present research, we provided in-depth phylogenetic analysis on bacterial 78

strain S1 using a polyphasic approach, including chemotaxonomic assays, 16S rRNA and 79

rpoB gene sequence analysis, and whole genome comparisons with representative 80

Klebsiella lineages. We demonstrated the existence of clear-cut genetic boundaries 81

between S1 and all Klebsiella strains compared and predicted 152 genes unique to S1. The 82

lack of two capsule genes may explain why S1 did not have the capsule. 83

MATERIALS AND METHODS 84

Isolation, morphology and physiology 85

Strain S1 (CGMCC No.3085) was cultured on Luria-Bertani (LB) nutrient agar 86

purchased from Beijing Land Bridge technology Co. Ltd (Beijing, China) at 37 . ℃ The 87

pure culture was preserved in 25% (v/v) glycerol at -80℃ (Wang et al. 2010). 88

The bacteria were characterized using Remel RapID ONE System (Kitch et al. 1994) 89

and Api 20E system (Butler et al. 1975; Holmes et al. 1978) following the manufacturer’s 90

recommendation. Remel RapID ONE System is a qualitative micromethod employing 91

chromogenic substrates for the identification of Enterobacteriaceae and other 92

gram-negative bacilli. It was comprised of panels and reagent. Reaction cavities contain 93

dehydrated reactants. A suspension of the Klebsiella S1 was used as the inoculums, which 94

rehydrated the reagents and initiated test reactions. After incubation of the panel, each test 95

cavity was examined for reactivity by the development of a color. Api 20E system is also 96

a standardized identification system for Enterobacteriaceae. The API 20E strip consisted 97

of 20 microtubes containing dehydrated substrates. These tests were inoculated with the 98

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suspension of Klebsiella S1 that reconstituted the media. During incubation, the bacterial 99

metabolism produced color changes. The results were read according to Reading Table 100

and the identification was obtained by referring to the Analytical Profile Index. 101

Genome sequencing and assembly 102

Genomic sequencing of S1 was conducted on the Illumina HiSeq 2000 platform, 103

which produced 620 Mb data. Library construction and sequencing were carried out 104

according to the manufacturer’s recommendation at the Illumina web site. The sequence 105

data from Illumina HiSeq 2000 were assembled with SOAPdenovo 2.04 software. The 106

draft genome sequences can be accessed under accession number LFOB00000000. We 107

predicted genes from the assembled sequences using Glimmer 3.02 (Delcher et al. 2007; 108

Delcher et al. 1999). 109

Phylogenetic analysis based on 16S rRNA and rpoB gene sequences 110

The 16S rRNA and rpoB gene sequences for comparisons with those of S1 were 111

obtained from NCBI http://www.ncbi.nlm.nih.gov/gene/. These sequences were aligned 112

using the Clustal X program (Thompson et al. 1997) and the phylogenetic tree was 113

constructed by MEGA6 (Tamura et al. 2013). The statistical significance of the groups 114

obtained was assessed by bootstrapping with 100 replicates. The accession number of 16S 115

rRNA of strain S1 is KT070083 and that of the rpoB gene of S1 is KT163260. 116

Digital DNA-DNA hybridization and BLAST alignment 117

The genome-to-genome distance calculator (GGDC) (Auch et al. 2006; Auch et al. 118

2010a; Auch et al. 2010b; Meier-Kolthoff et al. 2013; Meier-Kolthoff et al. 2014a; 119

Meier-Kolthoff et al. 2014b) from the website http://ggdc.dsmz.de/distcalc2.php was used 120

with the program of GBDP2_BLASTPLUS. Orthologs were determined by BLAST 121

alignment with the criteria that identity was larger than 70% and alignment length was 122

longer than 70% of the whole gene. When a sequence had multiple matches, genomic 123

locations were taken as the decisive criterion for the orthologous match. Concatenation of 124

conserved genes was done by using home-made Perl scripts as reported previously (Liu et 125

al. 2013). 126

ANI 127

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The average nucleotide identity (ANI) was based on the website 128

http://imedea.uib-csic.es/jspecies/index.html with the programs of BLAST and JSpecies 129

V1.2.1. 130

BLAST 131

Orthologs were determined by BLAST alignment with the criteria that identity was 132

larger than 70% and alignment length was longer than 70% of the whole gene. When a 133

sequence had multiple matches, genomic locations were taken as the decisive criterion for 134

the orthologous match. Concatenation of conserved genes was done by using home-made 135

Perl scripts. 136

RESULTS 137

Characteristics of strain S1 138

The bacterial cells of S1 were Gram-negative and facultatively anaerobic straight 139

rods without capsule. S1 bacteria could use glucose, sucrose, mannitol, inositol, galactose, 140

sorbitol, rhamnose, amygdalin, arabinose, melibiose and citrate as carbon sources. Urease 141

was positive but oxidase and lipase were negative. Indole was not produced. Gelatin was 142

not fermented. It was negative in the acid fast stain and it did not produce H2S. Lysine 143

decarboxylase, orinithine decarboxylase, arginine dihydrolase and β-glucolase were 144

negative, whereas ONPG, malonate and Voges-Proskauer tests were positive (Table 1). As 145

a potent SECO producer, S1 could yield an initial concentration of 34.97 ± 0.98 mg l -1

146

SECO within one day of incubation and a maximum concentration of 122.05 ± 7.67 mg l-1

147

with a longer incubation time (Wang et al. 2010). 148

Phylogenetic position of strain S1 149

We assembled the sequences of strain S1 into 50 contigs totaling 5.3Mp., and the GC 150

content was 57.37 mol %, which was within the range of bacteria in the genus Klebsiella 151

(53-58 mol%). The genome contained 4,987 predicted genes and the total length of genes 152

was 4,519,890 bp, which makes up 86.94% of genome. The number of tRNA was 74, and 153

there were eight genes for each of 16S and 23S ribosomal RNAs (rRNAs), and at least 154

eight 5S rRNA genes, which is consistent with our previous report that Klebsiella have 155

eight rrn operons [52]. A phylogenetic tree derived from 16S rRNA gene sequences 156

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indicated close relatedness of strain S1 with members of cluster I Klebsiella bacteria (Fig. 157

2). The phylogenetic position of Klebsiella on this tree matched the three clusters. The 158

novel species of Klebsiella michiganensis joined the cluster Ⅲ. The current cluster Ⅰ159

contained not only three subspecies of Klebsiella pneumoniae and Klebsiella granulomatis 160

but also Klebsiella singaporensis, Klebsiella variicola, Klebsiella alba and Klebsiella sp. 161

S1. These data demonstrated that strain S1 clearly belongs to the genus Klebsiella and that 162

the closest relative was K. pneumoniae subsp. pneumoniae ATCC13883 (99.7% 163

similarity). Strain S1 exhibited 98.6%-99.6% 16S rRNA gene sequence similarity to the 164

other species in Klebsiella cluster I except Klebsiella alba CW-D 3 (97.34%), 97.8–97.9% 165

similarity to Klebsiella cluster II and 97.4%-97.6% sequence similarity to Klebsiella 166

cluster III. It was obvious that strain S1 and cluster I shared a higher similarity than did 167

strain S1 and cluster II or III. C. granulomatis was demonstrated to be closely related to K. 168

pnuemoniae by sequence analysis of both 16S rRNA (Carter et al. 1999; Kharsany et al. 169

1999) and the phosphate porin (phoE) gene (Bastian and Bowden 1996), therefore it has 170

been suggested to be transferred to the genus Klebsiella as Klebsiella granulomatis (Carter 171

et al. 1999). A phylogenetic tree derived from rpoB gene sequences also demonstrated 172

similar evolutionary relationships (Fig. 3). Strain S1 showed 100% rpoB gene sequence 173

similarity with K. pneumoniae subsp. ozaenae ATCC 11296 and C. granulomatis but only 174

99.8% similarity with K. pneumoniae subsp. pneumoniae ATCC 13883. Consistent with 175

the 16S rRNA gene sequence results, strain S1 exhibited lower values of sequence 176

similarity with cluster II (93.8–94.5%) and cluster III (94.3%) Klebsiella lineages. 177

Evolutionary branching of Klebsiella based on whole genome information 178

To date, one of the criteria to categorize bacteria into taxonomic species, in addition 179

to 16S rRNA gene sequence similarity, is DNA-DNA hybridization (DDH), which, 180

however, is also arbitrary, due to the fact that the data, like the 16S rRNA gene sequence 181

similarity data, are continual without a border line for delineating bacteria into discrete 182

phylogenetic clusters. We used the genome-to-genome distance calculator (GGDC) to 183

estimate S1 for its evolutionary position in Klebsiella. This method works in three steps: 1, 184

the determination of a set of high-scoring segment pairs (HSPs) or maximally unique 185

matches (MUMs) between two genomes in comparison; 2, the calculation of distances; 186

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and 3, conversion of these distances in percent-wise similarities analogous to DDH (Auch 187

et al. 2010a; Auch et al. 2010b; Meier-Kolthoff et al. 2013). We compared S1 with other 188

Klebsiella strains using the whole genome sequences in the database by formula 2. S1 189

exhibited relatively high levels of DNA-DNA similarity to all K. pneumoniae strains 190

except K. pneumoniae subsp. pneumoniae KP5-1 and K. pneumoniae 342, to which the 191

DDH were only 58.1% and 58.3%, respectively (Table 2). These results demonstrate that 192

phylogenetic relationships among the Klebsiella lineages need to be re-evaluated based on 193

whole genome information; strain S1 is related to the majority of K. pneumoniae members 194

but some current taxonomic K. pneumoniae members, such as K. pneumoniae KP5-1 and 195

K. pneumoniae 342, should be transferred to other or novel Klebsiella species. Findings 196

from the average nucleotide identity (ANI) data were consistent with the DDH results 197

(Table 2). 198

Genetic boundaries separating Klebsiella S1 from other Klebsiella lineages 199

As the currently used golden standards for defining bacterial species, i.e., 16S 200

rRNA≥97% and DDH ≥70%, sometimes contradict each other, we have proposed the 3C 201

criteria for defining natural bacterial species (Tang and Liu 2012) and the concept of 202

genetic boundary to delineate bacteria into discrete phylogenetic clusters, which we treat 203

as natural species, based on the percentages of homologous genes with identical 204

nucleotide sequences between the bacteria in comparison (Tang et al. 2013a). We 205

compared the genomes of forty six strains from four Klebsiella species and Klebsiella sp. 206

S1, to reveal potentially important genomic differences that may clearly distinguish the 207

lineages on a phylogenetic basis. For this, we identified genes common to these genomes. 208

We found that most of the K. pneumoniae strains shared most of their genes and the 209

highest percentage was 99.7% between K. pneumoniae subsp. pneumoniae KPNIH10 and 210

K. pneumoniae subsp. pneumoniae KPNIH1. However we also found low percentages of 211

genes with identical sequence identity among strains of the same species, such as 14.6% 212

of shared genes between K. pneumoniae subsp. pneumoniae KPNIH1 and K. pneumoniae 213

subsp. pneumoniae KPNIH29; similar situations were seen also in other Klebsiella species. 214

There was a general tendency that closely related Klebsiella strains have 55.9% or greater 215

percentages of genes with identical sequences and Klebsiella strains in adjacent 216

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phylogenetic lineages have 34.7% or lower percentages of genes with identical sequences, 217

with a difference of ca. 20 percentage points reflecting the genetic boundary to separate 218

Klebsiella strains into discrete lineages (Fig. 4). Klebsiella S1 had very low percentages 219

(lower than 14.4%) of genes with identical nucleotide sequences compared with all other 220

sequenced Klebsiella strains compared here (Fig. 5). Furthermore, compared with 46 221

sequenced Klebsiella strains from Genbank, Klebsiella S1 had 152 unique genes (Table 222

S1), which would render sufficient biological uniqueness to Klebsiella sp. S1. 223

ββββ–glucosidase and capsular polysaccharide synthesis gene clusters of Klebsiella S1 224

We carried out further genetic and phenotypic characterizations, with a focus on 225

β–glucosidase and capsular polysaccharide synthesis (cps) genes. Previously, we had 226

isolated the bacterial strain Bacteroides uniformis ZL1 from the human intestinal flora 227

(Tao et al. 2014), which, like Klebsiella S1, could also convert SDG to SECO. Fifteen 228

genes (bgl1–bgl15) were amplified from strain ZL1, and we identified bgl8 as the gene 229

encoding the SDG-hydrolyzing β-glucosidase, which performs the biotransformation to 230

convert SDG to SECO. However, we did not find genes in Klebsiella S1 that were 231

homologous to any of bgl1–bgl15, so the functional gene to convert SDG to SECO in 232

Klebsiella S1 remains to be identified. 233

Many Klebsiella lineages like K. pneumoniae can produce capsular polysaccharides, 234

and the capsule is an important virulence factor (Cryz et al. 1984; Shu et al. 2009; 235

Williams et al. 1983). Typically, Klebsiella have a full set of capsular genes, including 236

galF, orf2, wzi, wza, wzb and wzc at the 5’ end of the cps cluster encoding proteins for cps 237

transportation and processing, and gnd, encoding gluconate-6-phosphate dehydrogenase, 238

at the 3’ end of the cps gene cluster. The central part of the cps gene cluster is highly 239

divergent, which encodes proteins for polymerization and assembly of the CPS subunits 240

(Shu et al. 2009). Compared with these genes of K. pneumoniae such as in strain NK8, 241

Klebsiella S1 homologues had high similarity with galF, orf2，gnd and wzi (91-95%) but 242

much lower similarity with wza (only 78%); no homologues were found for wzb or wzc 243

(Table 3), which may explain why Klebsiella S1 does not have the capsule and was 244

isolated from a healthy human individual. 245

DISCUSSION 246

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It has been a puzzling question why some plants or microbes would produce 247

anticancer agents while their close relatives may not? Systematic investigations on this 248

question, including especially whole genome analysis, may lead to discoveries about why 249

and how such organisms have evolved to acquire the anticancer capabilities, which in turn 250

may facilitate clinical application of more diverse or more specific natural anticancer 251

substances. In this study, we performed genome analysis on Klebsiella sp. S1, which 252

biotransforms SDG to an anticancer agent, SECO. As such, very closely related bacteria 253

need to be clearly distinguished to correlate special genomic traits to their unique 254

biological properties, such as producing anticancer agents de novo or through 255

biotransformation. For this purpose, in addition to analyzing the whole genome, we 256

identified genetic boundaries between Klebsiella sp. S1 and other Klebsiella strains based 257

on the concept and methodology reported earlier (Tang et al. 2013a; Tang et al. 2013b). 258

The genus Klebsiella contains diverse bacteria and the taxonomical positions of the 259

lineages need to be refined. For example, strains under the species name K. pneumoniae 260

appear at very different positions on the phylogenetic tree and in the meantime an isolate 261

of bacteria not having the K. pneumoniae species name, i.e., C. granulomatis, was 262

positioned very closely with K. pneumoniae strains on the same phylogenetic tree. 263

Although C. granulomatis was later proposed to be transferred to Klebsiella, the initial 264

naming of C. granulomatis as an independent bacterial species had robust biological 265

evidence (Carter et al. 1999; Kharsany et al. 1999). S1 has similar genetic distances to the 266

Klebsiella lineages as does C. granulomatis. 267

DDH remains to be one of the key parameters for defining taxonomic species of 268

bacteria, but to define natural species, such as one based on objective criteria that reflect 269

evolutionary relationships among the bacteria quantitatively, strictly reproducible 270

measurements according to a fundamental concept are needed for universal application for 271

all bacteria among different laboratories. The fundamental point in the concept that we 272

have proposed for defining natural species of bacteria is the clear-cut genetic boundary 273

that circumscribes bacteria into discrete phylogenetic clusters without intermediates 274

between the clusters and for the measurements we determine the percentage of 275

homologous genes with identical nucleotide sequences among closely related bacterial 276

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lineages (Tang et al. 2013a; Tang et al. 2013b). Our descriptions about defining the natural 277

species of bacteria had been based on previously existing bacterial taxa, such as 278

Salmonella serotypes. In this study, we for the first time applied this concept to define 279

Klebsiella based on both the genetic boundary criteria and on the unique biological 280

properties of the bacteria, e.g., production of secoisolariciresinol by biotransformation. To 281

encode this and other biological properties yet to be further characterized, some of the 152 282

unique genes of Klebsiella sp. S1 are anticipated to be involved, and some others of the 283

152 unique genes may encode certain unknown but important functions not present in 284

other investigated Klebsiella lineages. Therefore, we believe that the presence of over a 285

hundred unique and intact genes suffices to distinct Klebsiella sp. S1 from its close 286

relatives. 287

ACKNOWLEDGMENTS 288

This work was supported by a Heilongjiang Innovation Endowment Award for graduate 289

studies to YJZ (YJSCX2012-214HLJ) and grants of the National Natural Science 290

Foundation of China (NSFC81030029, 81271786) to SLL. 291

CONFLICT OF INTEREST: 292

No conflict of interest declared. 293

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419

420

421

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Table 1. Differential phenotypic characteristics of strain S1 and Klebsiella species. 422

Characteristics S1 1 2 3 4 5 6 7 8

Indole production - - - - + - d + -

Urea hydrolyzed + + d - + + + + -

Ornithine

decarboxylated

- - - - - - - + +

Lysine

decarboxylated

- + d - + + + + +

ONPG test + + + - + + + + +

Malonate test + + - + + + + + +

Voges-Proskauer test + + - - + + + + +

Taxa: 1, K. pneumoniae subsp. pneumoniae; 2, K. pneumoniae subsp. ozaenae; 3, K. 423

pneumoniae subsp. rhinoscleromatis; 4, K. oxytoca; 5, K. terrigena; 6, K. planticola; 7, K. 424

ornithinolytica; 8, K. mobilis. 425

Symbols and abbreviations: +, positive; -, negative; d, different reactions.426

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Table 2．．．．GGDC and ANI values between S1 and other Klebsiella strains. 427

Query

genome

Reference genome GGDC(%）））） ANI(%））））

S1 Klebsiella pneumoniae blaNDM-1 94.1 99.13

S1 Klebsiella pneumoniae 30660/NJST258_1 93.1 99.03

S1 Klebsiella pneumoniae 30660/NJST258_2 93.4 99.1

S1 Klebsiella pneumoniae 342 58.3 94.47

S1 Klebsiella pneumoniae CG43 93.9 99.16

S1 Klebsiella pneumoniae JM45 93.5 99.09

S1 Klebsiella pneumoniae KCTC 2242 93.5 99.08

S1 Klebsiella pneumoniae subsp. pneumoniae

HS11286

93.8 99.14


Kp5-1

58.1 94.33


KPNIH24

93.1 99.05


KPNIH27

94.1 99.19


KPR0928

93.2 99.05


KP13

93.7 99.13


MGH 78578

94 99.16


NTUH-K2044

93.9 99.14


PittNDMO1

93.3 99.09


1084

93.6 99.12

S1 Klebsiella pneumoniae Kp52.145 93.4 99.09

S1 Klebsiella pneumoniae subsp.pneumoniae

1158

93.3 99.08

S1 Klebsiella pneumoniae 34618 93 99.07

S1 Klebsiella pneumoniae HK787 93.8 99.16

S1 Klebsiella pneumoniae 32192 93.2 99.07


KPNIH31

93.7 99.05


KPNIH30

93.2 99.05


KPNIH29

93.8 99.07

S1 Klebsiella pneumoniae XH209 94.2 99.17

S1 Klebsiella pneumoniae subsp. pneumoniae 93.2 99.07

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Bold: the value of GGDC <97% or the value of ANI <95% 428

429

KPNIH32


KPNIH33

93.1 99.06

S1 Klebsiella pneumoniae PMK1 93.6 99.15


ATCC43816KPPR1

93.7 99.14

S1 Klebsiella pneumoniae ATCC BAA2146 93.1 98.99


KPNIH1

93.1 99.07


KPNIH10

93.1 99.07

S1 Klebsiella pneumoniae CAV1344 93.2 99.02




234-12

93.3 99.08

S1 Klebsiella variicola At-22 58.1 94.36

S1 Klebsiella variicola DSM 15968 58.4 94.41

S1 Klebsiella variicola DX120E 58.4 94.42

S1 Klebsiella oxytoca M1 27.1 83.16

S1 Klebsiella oxytoca E718 27.1 83.15

S1 Klebsiella oxytoca HKOPL1 27.3 83.22

S1 Klebsiella oxytoca KCTC 1686 27.2 83.15

S1 Klebsiella oxytoca KONIH1 27.3 83.12

S1 Klebsiella michiganensis RC10 21.2 76.16

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Table 3. Comparison of conserved cps genes between Klebsiella S1 and NK8. 430

431

432

433

Gene NK8(region）））） S1(region）））） Query coverage Identity

galF

orf2

wzi

wza

wzb

wzc

gnd

1-897

1290-1919

2798-4318

4463-5602

5602-6036

6054-8225

18097-19503

2427054-2426158

2425765-2425136

2424257-2422743

2422599-2421466

-

-

5187258-5188664

100%

100%

100%

99%

-

-

100%

95%

92%

91%

78%

-

-

95%

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Figure legends 434

Fig. 1 Biotransformation pathway of SECO from plant-derived lignan SDG 435

Fig. 2 Phylogenetic tree derived from 16S rRNA gene sequence analysis, showing the 436

position of S1 among representative members of Enterobacteriaceae. The tree was 437

generated by Kimura 2-parameter model and Neighbor-Joining method. Bootstrapping 438

was performed by using 100 replicates 439

Fig. 3 Phylogenetic tree derived from rpoB gene sequence analysis, showing the 440

relationship between Klebsiella sp.S1 and representative members of Enterobacteriaceae 441

based on partial rpoB gene sequences. The tree was generated by Kimura 2-parameter 442

model and Neighbor-Joining method. Bootstrapping was performed by using 100 443

replicates 444

Fig. 4 Genomic comparison among the Klebsiella strains. Sequences common to all 47 445

strains were concatenated and pair-wise aligned for the number of genes that have 100% 446

sequence identity 447

Fig. 5 Genomic comparison between Klebsiella sp.S1 and other Klebsiella strains 448

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Fig. 1 Biotransformation pathway of SECO from plant-derived lignan SDG

58x26mm (300 x 300 DPI)

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Fig. 2 Phylogenetic tree derived from 16S rRNA gene sequence analysis, showing the position of S1 among representative members of Enterobacteriaceae. The tree was generated by Kimura 2-parameter model and

Neighbor-Joining method. Bootstrapping was performed by using 100 replicates

164x94mm (300 x 300 DPI)

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Fig. 3 Phylogenetic tree derived from rpoB gene sequence analysis, showing the relationship between Klebsiella sp.S1 and representative members of Enterobacteriaceae based on partial rpoB gene sequences. The tree was generated by Kimura 2-parameter model and Neighbor-Joining method. Bootstrapping was

performed by using 100 replicates

79x65mm (300 x 300 DPI)

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Fig. 4 Genomic comparison among the Klebsiella strains. Sequences common to all 47 strains were concatenated and pair-wise aligned for the number of genes that have 100% sequence identity

27x30mm (600 x 600 DPI)

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Fig. 5 Genomic comparison between Klebsiella sp.S1 and other Klebsiella strains

46x16mm (300 x 300 DPI)

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