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Genome of Serratia nematodiphila MB307 offers unique

insights into its diverse traits

Journal: Genome

Manuscript ID gen-2017-0250.R1

Manuscript Type: Article

Date Submitted by the Author: 09-Mar-2018

Complete List of Authors: Basharat, Zarrin; Fatima Jinnah Women University Tanveer, Faouzia; Quaid-i-Azam University Yasmin, Azra; Fatima Jinnah Women University, Shinwari, Zabta; Quaid-i-Azam University He, Tongtong; State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, China.

Tong, Yigang ; State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, China.

Keyword: Rhizobacteria, Antibiotic resistance., Serratia nematodiphila, Genome, Bioremediation

Is the invited manuscript for consideration in a Special

Issue? : N/A

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

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Genome of Serratia nematodiphila MB307 offers unique insights into its diverse traits 1

Zarrin Basharata, Faouzia Tanveerb, Azra Yasmina*, Zabta Khan Shinwarib, Tongtong Hec, Yigang 2

Tongc 3

a Microbiology & Biotechnology Research Lab, Department of Environmental Sciences, Fatima 4

Jinnah Women University, Rawalpindi 46000, Pakistan. 5

b Department of Biotechnology, Quaid-i-Azam University, Islamabad 44000, Pakistan. 6

c State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and 7

Epidemiology, Beijing 100071, China. 8

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*Corresponding author 10

[email protected] 11

Running Title: Genome of Serratia nematodiphila MB307 12

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Abstract 22

A pigment producing Serratia specie was isolated from the rhizosphere of a heavy metal resistant 23

Cannabis sativa plant, growing in effluent affected soil of Hattar Industrial Estate, Haripur, Pakistan. 24

Here, we report the genome sequence of this bacterium, which was demarcated as Serratia 25

nematodiphila using whole genome comparison based OrthoANI classification scheme. The 26

bacterium exhibited diverse traits, including plant growth promotion, antimicrobial, bioremediation 27

and pollutant tolerance capabilities, including metal tolerance, azo dye degradation and ibuprofen 28

degradation etc. Plant growth promoting exoenzyme production as well as phosphate solubilisation 29

properties were observed. Genes for phosphate solubilisation, siderophore production and chitin 30

destruction were identified in addition to other industrially important enzymes like nitrilase and 31

lipase. Secondary metabolite producing apparatus for high value chemicals in the whole genome was 32

also analysed. Number of antibiotic resistance genes was then profiled in silico, through a match with 33

Antibiotic resistant gene and CAR database. This is the first report of a Serratia nematodiphila 34

genome from polluted environment. This could significantly contribute to understand pollution 35

tolerance, antibiotic resistance, association with nematodes, bio-pesticide production, and role in plant 36

growth promotion. 37

Keywords: Rhizobacteria, Serratia nematodiphila, Genome, Bioremediation, Antibiotic 38

resistance. 39

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1. Introduction 46

Members of the genus Serratia have been isolated from diverse habitats and include both pathogenic 47

and non-pathogenic strains with varying traits (Basharat and Yasmin 2016). A new member of this 48

genus, having a symbiotic association with the entomopathogenic nematode Heterorhabditidoides 49

chongmingensis was first reported in 2009 and the name Serratia nematodiphila was proposed for this 50

novel bacterium (Zhang et al. 2009). It showed a symbiotic-pathogenic life cycle and a multifaceted 51

relationship with entomopathogenic nematodes and insect pests. Insecticidal activity of this bacterium 52

against larvae of three mosquito species illustrates significance of this bacterium to control mosquito-53

borne diseases (Patil et al. 2012). 54

It has been implicated in the synthesis of silver nanoparticles, with bactericidal activity against 55

Bacillus subtilis, Klebsiella planticola and Pseudomonas aeruginosa (Malarkodi et al. 2013a). 56

Semiconductor cadmium sulfide nanoparticles have also been produced with the aid of this bacterium 57

(Malarkodi et al. 2013b). These type of nanoparticles have applications in solar cell fabrication, 58

photovoltaics, field effect transistor and biobased environmental sensor assembly. Lipases that work 59

as biocatalysts for transesterification of fatty acids during biodiesel production and have been 60

successfully sequestered from Serratia nemtodiphila. Whole-cell culture immobilization led to an 61

enhanced recovery of fatty acids of methyl oleate, methyl linoleate, and methyl palmitate, with 62

prospective for high quality biodiesel production (Boonmahome et al. 2015). Biodegradation of 2,4,6-63

trinitrotoulene has also been accomplished by this bacterium (Wu et al. 2013). 64

Gibberellin and abscissic acid producing Serratia nematodiphila has been reported to promote plant 65

growth and ameliorate cold tolerance in Capsicum annuum L. (Kang et al. 2015). A Serratia 66

nematodiphila strain NII-0928 showed improved plant growth in Piper nigrum L. by solubilisation of 67

phosphates, production of indole acetic acid (IAA) and siderophores (Dastager et al. 2011). Serratia 68

nematodiphila has also been isolated as a heavy metal resistant endophytic bacteria, from the 69

cadmium hyperaccumulator plant Solanum nigrum L. and showed plant growth promotion traits with 70

phosphate solubilizing activity, IAA and siderophore production (Chen et al. 2010). This indicates 71

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that the bacterium could be useful in phytoremediation as well. Khoa et al. (2016) tested Serratia 72

nemtodiphila for activity against rice blight pathogen Xanthomonas oryzae, and it demonstrated 73

strong antagonistic effects against Xanthomonas oryzae via antibiosis and siderophore production, 74

highlighting importance of this bacterium as a biocontrol agent. These diverse traits of Serratia 75

nematodiphila make it imperative to sequence its species from various sources (Environmental and 76

other) to explore genomics of these mechanisms in detail and to identify new and linked phenomenon 77

with these processes. 78

Despite such tremendous progress in genomic sequencing, decrease in cost and sophistication of 79

instrumentation, algorithms and softwares to process data, only data for three genomes of this 80

important bacterium was available publicly (in the National Centre for Biotechnology database) at the 81

time of sequencing of our Serratia nemtodiphila isolate. First genome sequence of this bacterium was 82

announced in 2015 by Kwak et al. (2015). Here, we report the sequencing, preliminary analysis and 83

comparison to the two type strains of Serratia nematodiphila of this bacterium, to fill up this 84

knowledge gap. Our strain was isolated from the rhizosphere of Cannabis sativa plant, growing in 85

extremely polluted and acidic environment (at a soil pH of 2), which heralds its remarkable potential 86

for bioremediation. 87

2. Material and methods 88

2.1. Isolation and characterization of the bacterium 89

Various parameters of the sampling site affected with effluent of multiple industries i.e. 90

pharmaceutical, chemical and textile industry, were recorded. Cannabis sativa plant growing in the 91

effluent polluted soil of Hattar industrial site (identified by a taxonomist at the Quaid-i-Azam 92

University, Islamabad) was uprooted, placed in polythene sealed bag along with adhering soil and 93

carried to the lab, where samples were processed. One gram soil adhering to the roots was air dried, 94

added in 10 ml autoclaved distilled water and left on the bench for 2-4 hrs. 50 µl of suspension was 95

plated on metal supplemented nutrient agar plates (100 µg/ml of Ni2+, Pb2+, Cd2+ and Cr6+). Plates 96

were incubated at 37 °C for 3-5 days. Morphological and biochemical characterization of the isolated 97

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strain MB307 was done following Bergey’s Manual of Determinative Bacteriology (Holt et al. 1994). 98

Tolerance to eleven different metals at varying concentrations was tested. Metals included NiCl2, 99

Pb(NO3)2, K2CrO4, CdSO4, CuSO4.5H2O, CoCl2, MnCl2, FeCl3, As2O3, HgCl2 and ZnCl2 100

(concentration: 50-1000 µg/ml or higher). Azo dye degradation (Methyl Orange, Congo Red) and 101

Ibuprofen tolerance assays (at a concentration of 100 µg/ml) were also attempted (Marchlewicz et al. 102

2017). All chemicals used in the study were of analytical grade and purchased from Sigma-Aldrich. 103

2.2. Plant growth promotion parameters 104

Bacterium was investigated for production of IAA (Gordon and Weber 1951) in nutrient medium 105

supplemented with tryptophan (500 µg/ml) and either with or without Pb (200 µg/ml). After 24 hours 106

of incubation, culture was centrifuged at 10,000 rpm and supernatant was taken. Salkowski’s reagent 107

(2 ml) was added to the supernatant and kept for 25 minutes in the dark. This reagent (FeCl3 10mM 108

/perchloric acid 35%) is often used for detection of indolic substances. Absorbance was recorded at 109

530 nm to quantify IAA production. Phosphate solubilisation ability was checked using NBRIP 110

(National Botanical Research Institute Phosphate) medium (Nautiyal 1999) amended with tricalcium 111

phosphate (5g/l) and with or without Pb (200 µg/ml). Plates were prepared, spot inoculated and kept 112

at 37 °C for a week. Halo formation around the colony was taken as a positive indication of phosphate 113

solubilisation. Protease activity was measured on nutrient agar medium supplemented with 2% w/v 114

skimmed milk powder. Cellulase production was tested on a minimal medium supplemented with 2% 115

carboxy methyl cellulose. Gelatin and agar were then added after pH treatment and medium was then 116

heated for complete dissolution of all ingredients (Hendrick et al. 1995). Pectinase production was 117

checked according to Kumar and Sharma (2012), with polygalacturonic acid as substrate. Siderophore 118

production was determined on Chrome Azurole S agar medium (Alexander and Zubeber 1991). 119

Ammonia (Marques et al. 2010) and aminocyclopropane-1-carboxylate (ACC) deaminase enzyme 120

production (Penrose and Glick 2003) were also evaluated. Antifungal activity was investigated 121

against two major phytopathogens i.e. Aspergillus niger (Accession No. 1109) and Fusarium 122

oxysporum (Accession No 1114). Cultures were obtained from Fungal Culture Bank, University of the 123

Punjab, Pakistan and accession numbers refer to the ones specified by the ‘First Culture Bank of 124

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Pakistan, University of Punjab, Lahore, Pakistan’. Fungal strains were cultured on Potato Dextrose 125

and Tryptic Soy Agar (1:1). Bacterium was streaked 2 cm away from fungal plug in the centre of petri 126

plate and incubated at 30˚C for 7 days (Kumar et al. 2012). 127

2.3. Genome sequencing and analysis 128

For genome sequencing, DNA was extracted using high pure PCR template preparation kit (Roche, 129

Switzerland) and sequencing was carried out on MiSeq PE300 sequencer with 2×300bp pair-end 130

library. A total of 1,042,516 reads were generated (57× coverage of the genome). The raw reads were 131

cleaned, trimmed and assembled into 31 scaffolds using A5-miseq pipeline (v. 20160825) with default 132

assembly parameters (Coil et al. 2014), and evaluated with QUAST (Gurevich et al. 2013). The 133

sequence reads have been deposited in the NCBI SRA database and allocated the accession number: 134

SRR5816373. Genome annotation was performed using the NCBI prokaryotic genome annotation 135

pipeline. Genomes were visualized using CGView (Grant and Stothard 2008) with the parameters: 136

Global Blast Settings: query_split_size=50000; overlap_split_size=0; Query: MB307.fasta; Blast 1: 137

DSM 21420.fasta; blastn expect=0.1; Bacterial and Plant Plastid, filter=Yes; alignment_cutoff=70; 138

Blast 2: CGMCC.fasta. Alignment with reference sequences i.e. type strains of Serratia 139

nematodiphila CGMCCT (GenBank Accession no: JPUX01000001-JPUX01000002) and DSM 140

21420T (GenBank Accession no: JELU01000001-JELU01002516) was carried out with 141

progressiveMAUVE (Darling et al. 2010). Alignment was carried out to get an overview of broad 142

rearrangements in closely related genomes, due to recombination and display of any segmental gain or 143

loss. Secondary metabolite producing operons/gene clusters were studied using antiSMASH 3.0 144

(Weber et al. 2015). Contigs in .gbk format were taken and gene cluster as well as sub-cluster BLAST 145

analysis was performed. Gene function was discerned through smCOG. BlastKOALA was used for 146

pathway mapping against KEGG database (Kanehisa et al. 2004). 147

Whole genome was blasted against the manually curated ‘Antibiotic Resistance Genes Database’ (Liu 148

et al. 2009) to compare the Serratia nematodiphila MB307 antibiotic resistance profile with the 149

reference microbial antibiotic resistance genes. Genes from 1737 bacterial species (conferring 150

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resistance to 249 antibiotics) were aligned with Serratia nematodiphila MB307 genes (parameters: 151

program: blastp; percent identity cut-off = 40; E-value cut-off:1 x 10-4). Antimicrobial resistance of 152

genes was also profiled via BLAST search against the manually curated CARD database (Jia et al. 153

2016). It consists of high quality experimental reference data on antimicrobial resistance. This 154

database is updated on a monthly basis and based on genome sequence analysis, manual literature 155

curation along with computational text mining. Visualization of antibiotic resistance genes following 156

strict and loose criterion was then done through a heatmap plot. 157

3. Results and Discussion 158

3.1. Physicochemical characteristics 159

Our isolate MB307 was a gram negative, rod-shaped, endospore forming, motile, and facultative 160

anaerobe. Colonies appeared as circular (0.1-0.4 mm in diameter), creamy, raised with entire margin. 161

Colour was off-white and appeared reddish brown under the microscope but was observed to change 162

from off-white to pinkish red and then complete red over time. It was able to reduce nitrate to nitrite 163

while showing positive catalase and oxidase activity. The strain could utilize glucose, lactose, sucrose 164

and citrate as carbon source. H2S production was also observed indicated by the blackening of the butt 165

in Triple sugar iron test. Similarly, it was able to grow as well as change the colour of the Eosin 166

Methylene Blue (EMB) agar medium to purple further confirming lactose fermenting ability. 167

3.2. Pollutant tolerance and biodegradation potential 168

We are in continuous search of isolates with better pollutant tolerance characteristics. Our isolate 169

showed remarkable pollutant tolerance and degradation aptitude to various pollutants i.e. metals, azo 170

dyes and pharmaceutical Ibuprofen. An excellent tolerance to eleven heavy metals was observed 171

(Table 1). Least tolerance was observed for Cd2+, while the strain was most tolerant to Mn2+. Heavy 172

metal pollution due to anthropogenic activities is a serious threat to the environment and this strain 173

could serve as a source material for cloning of metal resistance genes. Azo dyes are another menace 174

that are hazardous and pose environmental and health problems. Sulphonated azo dyes are difficult to 175

degrade and very expensive to process through physical and chemical methods. Bioremediation 176

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efficacy of the strain was tested against two sulphonated azo dyes, both qualitatively and 177

quantitatively. It exhibited azo dye degradation capability of 40 and 77.97% after 72 hours for Methyl 178

orange and Congo red respectively (pH:7; Temperature:37ºC), with no inhibitory effect on the growth 179

of the bacteria. 180

With the passage of time, reports of pharmaceutical pollutant toxicity have been published from many 181

countries, with occurrence in sewage treatment plants, surface/drinking water, effluents, seawater and 182

groundwater. Serratia nematodiphila MB307 manifested remarkable tolerance to Ibuprofen, with an 183

optical density value of λ600=0.58 (cell count=4.64 x 108) after incubation time of 24 hours in 184

Ibuprofen supplemented nutrient broth at a temperature of 37 °C. Azo dye and Ibuprofen degrading 185

enzymes need to be further studied in this bacterium for understanding molecular basis of dye 186

degradation. The authors are currently working on micropollutant (including azo dyes and Ibuprofen) 187

degradation by this bacterium using several parameters and state of the art techniques. 188

3.3. Plant growth promotion study 189

The bacteria possessing multiple plant growth promoting traits have a better chance to act as 190

successful inoculants for the field studies. These bacteria may utilize different traits at different times 191

in their life cycles e.g. ACC deaminase enzyme activity may be important during initial growth for 192

root development, while siderophore and auxin production may be involved in nutrient acquisition to 193

maintain a hormonal balance (Dell’Amico et al. 2008). In the present study, plant associated bacteria 194

Serratia nematodiphila MB307 exhibited growth promoting traits such as Indole Acetic Acid (IAA) 195

production and phosphate solubilisation (with and without metal stress). It also showed the ability to 196

produce 1-aminocyclopropane-1-carboxylate (ACC) deaminase enzyme and iron chelating 197

siderphores. ACC deaminase enzyme production is important for ameliorating the stress induced by 198

higher ethylene levels in plants in response to various external stresses such as pollutants, radiation, 199

phytopathogens, extremes of temperature and high salt concentration (Glick 2012). Siderophore 200

production by bacteria makes iron accessible for plant uptake as well as increases competition for 201

minerals with phytopathogens (Glick 2003; Pieterse et al. 2001). The biocontrol potential of Serratia 202

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MB307 was confirmed by its ability to produce cell wall degrading enzyme i.e. protease, production 203

of ammonia that inhibits the growth of pathogens and positive anti-fungal activity against plant 204

pathogens Aspergillus niger & Fusarium oxysporum. Proteases and chitinases are enzymes with 205

ability to degrade cell wall of fungal pathogens (Kim et al. 2008). Cellulase and pectinase production 206

indicate ability of Serratia nematodiphila MB307 to degrade cellulose and pectin respectively (Fig. 1, 207

Table 2). 208

3.4. Overview of Serratia nematodiphila MB307 genome 209

Total genome length was estimated as 5,172,349 bp, having a GC content of 59.6% (Fig. 2A). Total 210

4,913 genes were annotated in Serratia nematodiphila MB307 with 4,794 as coding and the rest as 211

RNA genes. A total of 41 pseudogenes, 82 tRNAs and 14 ncRNAs were predicted. OrthoANI value 212

of 99.30% was obtained after comparison to Serratia nematodiphila DSM 21420T. An OrthoANI 213

value of greater than or equal to 95 means that the strain belongs to same bacterial species and this is 214

why the isolated strain MB307 was demarcated as Serratia nematodiphila. Alignment to the two 215

sequenced type strains of Serratia nematodiphila showed a change in the overall arrangement of 216

locally collinear blocks with transitions, transversions and deletions (Fig. 2B). A large number of 217

unique genes (360) were present in MB307 as compared to the type strains CGMCCT and DSM 218

21420T present in the NCBI database. 219

Antiholin and phage shock proteins were present in the genome, suggesting involvement in bacterial 220

protection from phages. Type I and VI secretion system proteins were also identified. Cellulose 221

synthase indicates biofilm formation capability while presence of phenazine biosynthesis protein 222

implicates dye production. Streptomycin producing enzyme sequence was also identified. Apart from 223

cell growth, division, amino acid metabolism, organic hyperoxide resistance, fatty acid 224

transformation, acid shock, cold shock, heat shock, flagellar assembly, pilin and polysaccharide 225

synthesis genes were detected. Siderophore, hemophore, ACC deaminase important for stress 226

amelioration, mineral phosphate solubilizing pyrroloquinoline quinone synthase, thioesterase and 227

industrially important enzyme like phytase, lipase, nitrilase and chitinase were also identified. Phytase 228

is involved in dephosphorylation of seeds and grains to usable phosphate form. Chitinase destroys the 229

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exoskeleton of worms and fungi, thus making this bacterium detritivorous. The genome also 230

comprised of exotoxins for biocontrol of pests, which makes it an eco-friendly substitute for chemical 231

based pesticides and conspicuous for enhancing crop productivity and maintain soil quality in agro-232

ecosystems. 233

Cyanate degradation and atrazine degradation operons were found in addition to tellurite and heavy 234

metal resistance apparatus. Three copies of azoreductase were observed with variation in sequence, 235

which suggest a paralogous origin of enzymes conferring capability of this bacterium to degrade and 236

survive in genotoxic azo dye polluted environment. Fourteen different monooxygenases and 5 copies 237

of different dioxygenases were present in the genome. Dioxygenases with only decarboxylation and 238

not cleavage or post-processing functionality were mined. KEGG reaction modules portrayed their 239

connection with dihydroxylation of aromatic ring type 1 i.e. dioxygenase and decarboxylating 240

dehydrogenase reactions. These genes help resist various pollutants and complement the phenotypic 241

characteristics of pollution tolerance traits of Serratia nematodiphila MB307 at genomic level. 242

Out of 634 genes responsible for metabolism in Serratia nematodiphila MB307 genome, 225 were 243

categorized as specific to microbial metabolism in diverse environments. Around 24 genes were 244

specialized for 2-oxocarboxylic acid metabolism and 22 genes for degradation of aromatic 245

compounds. Aromatic compounds are natural substrates of bacteria and also form recalcitrant class of 246

pollutants predominant in the environment (Seo et al. 2009). 178 genes involved in the synthesis and 247

metabolism of co-factors and vitamins were also mined. Nineteen fatty acid metabolism genes and 15 248

pathways linked with xenobiotics biodegradation and metabolism were surveyed in the genome 249

(Table 3). 250

Antibiotics are responsible for an ever-changing core molecular landscape of bacterial resistance 251

mechanism and a large number of genes conferring resistance to antibiotics were found after search 252

against ‘Antibiotic Resistance Genes Database’. Seven genes conferring resistance to different classes 253

of antibiotics had a similarity value above (Table 4), while the value for 43 genes was below cut-off 254

point. A resistance similarity to 19 genes (including beta-lactams and drug-efflux pumps) was found 255

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with strict and 36 with loose criteria after BLAST search against the ‘CARD’ record (Fig. 3). A 256

detailed antibiogram and resistome of this bacterium is being mapped by our group i.e. association 257

with mobile elements such as transposons, insertion sequence-based transposable units, integrons and 258

synteny of resistance gene operons for Serratia MB307. 259

3.5. Secondary metabolite producing gene cluster analysis 260

Nonribosomal peptide synthetases (NRPS) are independent of mRNA and each synthetase produces 261

one type of peptide secondary metabolite only. NR peptides possess various types of pharmacological 262

action and diverse biological activities. A total of six NRPS operons with peptide metabolite 263

producing capability were mined. Other metabolites included fatty acids, thiopeptides and 264

saccharides. NRPS Enterobactin, ravidomycin, prodigiosin, turnerbactin and xantholipin producing 265

clusters in the Serratia nematodiphila MB307 genome depicted considerable similarity to the 266

previously annotated gene clusters in the database. A similarity of 93% of the genes was observed 267

among the cluster/operon for prodigiosin production, 20% for microcin, 15% for turnerbactin, 12% 268

for enterobactin, 5% for ravidomycin and 4% for xantholipin producing gene cluster. Gene clusters 269

entailed biosynthetic, transport-related, regulatory and other genes. Hidden Markov Model based 270

smCOG analysis showed that at least one biosynthetic gene in all the mined NRPS clusters belonged 271

to condensation domain containing protein family. Condensation domain usually occurs in multi-272

domain enzymes that produce peptide antibiotics and has a role in catalysis of peptide bond forming 273

condensation reaction in NRPS (Stachelhaus et al. 1998). 274

Other predicted metabolite producing operons did not share a close homolog in MIBiG database and 275

the predicted chemical structure scaffold was therefore, blasted against Norine database in order to 276

find nature of produced chemical structure. In one operon, it resembled an antibiotic (penicillin and 277

cephalosporin precursor, Norine ID: NOR00006) and a siderophore (chrysobactin, Norine ID: 278

NOR00210), based on the fingerprint distance(s) between the predicted scaffold composition and the 279

Norine peptides. In the other operons, coronatine family toxins N-coronafacoyl-L-valine (Norine ID: 280

NOR00748) and N-coronafacoyl-L-threonine (Norine ID: NOR00751) production was projected. 281

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Most of the genes in the cluster showed ~90% or more similarity to the gene clusters in other Serratia 282

species (Fig. 4), which portends similar toxins in them but they have remained unexplored and 283

understudied still now. These metabolites are intermediates of the phytotoxin coronatine and first 284

isolated from Pseudomonas syringae pv. glycinea. Coronatine functions in plant function regulation 285

i.e. stomatal re-opening after pathogen attack on plant and during infection (Melotto et al. 2008) so 286

basically it has a role in mitigating stress. This suggests a putative role of Serratia nematodiphila 287

phytotoxins in plant protection against infection and stress but further experimental validation is 288

required for detailed derivation of the mechanism. These type of toxins need to be studied further as 289

they have potential for transgenic plant development, with resistance against pathogens and infection. 290

4. Conclusion 291

We sequenced Serratia nematodiphila MB307 to explore a native pollutant and stress tolerant 292

bacterium, harboring potential eco-friendly traits (plant growth promoting, biocontrol and 293

bioremediation). It has biostimulation properties and could serve as sustainable alternative to the 294

existing chemical fertilizers/pesticides. Important secondary metabolite gene operons were mined and 295

analysed in relation to other Serratia spp genomes. This strain could be utilized in various functional 296

genomic studies which could in return, lead to a better understanding of the concerted action of 297

enzymes and metabolic routes for tolerance to pollutants and biodegradation of various xenobiotics. It 298

could also prove useful for industrially and agriculturally important enzyme production. Further work 299

on the understanding and improvement of enzymatic activity regarding various aspects of this 300

remarkable strain is being carried out by our group. 301

Genome accession number 302

The genome sequence of Serratia nematodiphila MB307 has been deposited in NCBI GenBank 303

(Accession number: MTBJ01000001-MTBJ01000031). 304

305

References 306

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Alexander, D. B., and Zuberer, D. A. 1991. Use of chrome azurol S reagents to evaluate siderophore 307

production by rhizosphere bacteria. Biol. Fertil. Soils, 12(1): 39-45. 308

Aziz, R. K., Bartels, D., Best, A. A., DeJongh, M., Disz, T., Edwards, R. A., Formsma, K., Gerdes, S., 309

Glass, E.M., Kubal, M., and Meyer, F. 2008. The RAST Server: rapid annotations using 310

subsystems technology. BMC Genomics, 9(1): 1. 311

Basharat, Z., and Yasmin, A. 2016. Pan-genome analysis of the genus Serratia. arXiv preprint 312

arXiv:1610.04160. 313

Boonmahome, P., Suwannasom, P., Leesing, R., Ruangviriyachai, C., and Mongkol Thanaruk, W. 314

2016. Characterization of Serratia nematodiphila lipase for biocatalyst in fatty acid methyl 315

esters production. Walailak J. Sci. Technol., 14(3). 316

Chen, L., Luo, S., Xiao, X., Guo, H., Chen, J., Wan, Y., Li, B., Xu, T., Xi, Q., Rao, C., and Liu, C. 317

2010. Application of plant growth-promoting endophytes (PGPE) isolated from Solanum 318

nigrum L. for phytoextraction of Cd-polluted soils. Appl. Soil Ecol., 46(3): 383-389. 319

Coil, D., Jospin, G., and Darling, A. E. 2014. A5-miseq: an updated pipeline to assemble microbial 320

genomes from Illumina MiSeq data. Bioinformatics, btu661. 321

Darling, A. E., Mau, B., and Perna, N. T. 2010. progressiveMauve: multiple genome alignment with 322

gene gain, loss and rearrangement. PloS One, 5(6): e11147. 323

Dastager, S. G., Deepa, C. K., and Pandey, A. 2011. Potential plant growth-promoting activity of 324

Serratia nematodiphila NII-0928 on black pepper (Piper nigrum L.). World J. Microbiol. 325

Biotechnol., 27(2): 259-265. 326

Dell’Amico, E., Cavalca, L., and Andreoni, V. 2008. Improvement of Brassica napus growth under 327

cadmium stress by cadmium-resistant rhizobacteria. Soil Biol. Biochem., 40(1): 74-84. 328

Glick, B. R. 2003. Phytoremediation: synergistic use of plants and bacteria to clean up the 329

environment. Biotechnol. Adv., 21(5): 383-393. 330

Glick, B. R. 2012. Plant growth-promoting bacteria: mechanisms and applications. Scientifica, 1-15. 331

Gordon, S. A., and Weber, R. P. 1951. Colorimetric estimation of indoleacetic acid. Plant 332

Physiol., 26(1): 192. 333

Page 13 of 26


Genome

Draft

14

Grant, J.R. and Stothard, P. 2008. The CGView Server: a comparative genomics tool for circular 334

genomes. Nucleic Acids Res., 36(suppl_2): W181-W184. 335

Gurevich, A., Saveliev, V., Vyahhi, N., and Tesler, G. 2013. QUAST: quality assessment tool for 336

genome assemblies. Bioinformatics, 29(8): 1072-1075. 337

Hendricks, C. W., Doyle, J. D., and Hugley, B. 1995. A new solid medium for enumerating cellulose-338

utilizing bacteria in soil. Appl. Environ. Microbiol., 61(5): 2016-2019. 339

Holt, J. G. K., Sneath, N. R., Staley, P. H., Williams, J. T., and Stanley, T. 1994. Bergey's manual of 340

determinative bacteriology (No. QR81 S6 1994). 341

Jia, B., Raphenya, A. R., Alcock, B., Waglechner, N., Guo, P., Tsang, K. K., Lago, B.A., Dave, B.M., 342

Pereira, S., Sharma, A.N., and Doshi, S. 2017. CARD 2017: expansion and model-centric 343

curation of the comprehensive antibiotic resistance database. Nucleic Acids Res., 45(D1): 344

D566-D573. 345

Kanehisa, M., Goto, S., Kawashima, S., Okuno, Y., and Hattori, M. 2004. The KEGG resource for 346

deciphering the genome. Nucleic Acids Res., 32(suppl_1): D277-D280. 347

Kang, S. M., Khan, A. L., Waqas, M., You, Y. H., Hamayun, M., Joo, G. J., Shahzad, R., Choi, K.S., 348

and Lee, I. J. 2015. Gibberellin-producing Serratia nematodiphila PEJ1011 ameliorates low 349

temperature stress in Capsicum annuum L. Eur. J. Soil Biol., 68: 85-93. 350

Khoa, N. Đ., Giàu, N. Đ. N., and Tuấn, T. Q. 2016. Effects of Serratia nematodiphila CT-78 on rice 351

bacterial leaf blight caused by Xanthomonas oryzae pv. oryzae. Biol. Control, 103: 1-10. 352

Kim, Y. C., Jung, H., Kim, K. Y., and Park, S. K. 2008. An effective biocontrol bioformulation 353

against Phytophthora blight of pepper using growth mixtures of combined chitinolytic 354

bacteria under different field conditions. Eur. J. Plant Pathol., 120(4): 373-382. 355

Kumar, A., and Sharma, R. 2012. Production of alkaline pectinase by bacteria (Cocci sps.) isolated 356

from decomposing fruit materials. J. Phytol., 4(1): 01-05. 357

Kumar, P., Dubey, R. C., and Maheshwari, D. K. 2012. Bacillus strains isolated from rhizosphere 358

showed plant growth promoting and antagonistic activity against phytopathogens. Microbiol. 359

Res., 167(8): 493-499. 360

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Draft

15

Kwak, Y., Khan, A. R., and Shin, J. H. 2015. Genome sequence of Serratia nematodiphila DSM 361

21420 T, a symbiotic bacterium from entomopathogenic nematode. J. Biotechnol., 193: 1-2. 362

Liu, B., and Pop, M. 2009. ARDB-Antibiotic resistance genes database. Nucleic Acids Res., 363

37(Database issue): D443-7. 364

Malarkodi, C., and Annadurai, G. 2013b. A novel biological approach on extracellular synthesis and 365

characterization of semiconductor zinc sulfide nanoparticles. Appl. Nanosci., 3(5): 389-395. 366

Malarkodi, C., Rajeshkumar, S., Paulkumar, K., Vanaja, M., Jobitha, G. D. G., and Annadurai, G. 367

2013a. Bactericidal activity of bio mediated silver nanoparticles synthesized by Serratia 368

nematodiphila. Drug Invent. Today, 5(2): 119-125. 369

Marchlewicz, A., Guzik, U., Hupert-Kocurek, K., Nowak, A., Wilczyńska, S., and Wojcieszyńska, D. 370

2017. Toxicity and biodegradation of ibuprofen by Bacillus thuringiensis B1 (2015b). 371

Environ. Sci. Pollut. Res., 1-13. 372

Marques, A. P., Pires, C., Moreira, H., Rangel, A. O., and Castro, P. M. 2010. Assessment of the plant 373

growth promotion abilities of six bacterial isolates using Zea mays as indicator plant. Soil 374

Biol. Biochem., 42(8): 1229-1235. 375

Melotto, M., Underwood, W., and He, S. Y. 2008. Role of stomata in plant innate immunity and foliar 376

bacterial diseases. Annu. Rev. Phytopathol., 46: 101-122. 377

Nautiyal, C. S. 1999. An efficient microbiological growth medium for screening phosphate 378

solubilizing microorganisms. FEMS Microbiol. Lett.170(1): 265-270. 379

Patil, C. D., Patil, S. V., Salunke, B. K., and Salunkhe, R. B. 2012. Insecticidal potency of bacterial 380

species Bacillus thuringiensis SV2 and Serratia nematodiphila SV6 against larvae of 381

mosquito species Aedes aegypti, Anopheles stephensi, and Culex quinquefasciatus. Parasitol. 382

Res., 110(5): 1841-1847. 383

Penrose, D. M., and Glick, B. R. 2003. Methods for isolating and characterizing ACC deaminase‐384

containing plant growth‐promoting rhizobacteria. Physiol. Plant., 118(1): 10-15. 385

Pieterse, C. M., Ton, J., and Van Loon, L. C. 2001. Cross-talk between plant defence signalling 386

pathways: boost or burden?. AgBiotechNet, 1-8. 387

Page 15 of 26


Genome

Draft

16

Seo, J. S., Keum, Y. S., and Li, Q. X. 2009. Bacterial degradation of aromatic compounds. Int. J. Env. 388

Res. Public Health, 6(1): 278-309. 389

Stachelhaus, T., Mootz, H. D., Bergendahl, V., and Marahiel, M. A. (1998). Peptide bond formation 390

in nonribosomal peptide biosynthesis catalytic role of the condensation domain. J. Biol. 391

Chem., 273(35): 22773-22781. 392

Weber, T., Blin, K., Duddela, S., Krug, D., Kim, H. U., Bruccoleri, R., Lee, S.Y., Fischbach, M.A., 393

Müller, R., Wohlleben, W., and Breitling, R. 2015. antiSMASH 3.0—a comprehensive 394

resource for the genome mining of biosynthetic gene clusters. Nucleic Acids Res., 43(W1): 395

W237-W243. 396

Wu, Y., Yan, H., Zhao, D., Yin, X., Jia, B., and Gao, Y. 2013. Biodegradation of 2, 4, 6-397

trinitrotoluene by a novel bacterium Serratia nematodiphila-YW: isolation, biodegrading 398

characterisation, and products. Fresenius Environ. Bull., 22(6): 1710-1718. 399

Zhang, C. X., Yang, S. Y., Xu, M. X., Sun, J., Liu, H., Liu, J. R., Liu, H., Kan, F., Sun, J., Lai, R., and 400

Zhang, K. Y. 2009. Serratia nematodiphila sp. nov., associated symbiotically with the 401

entomopathogenic nematode Heterorhabditidoides chongmingensis (Rhabditida: 402

Rhabditidae). Int. J. Syst. Evol. Microbiol., 59(7): 1603-1608. 403

404

405

406

407

408

409

410

411

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412

413

414

415

416

Table 1. Maximum tolerable capability of Serratia nematodiphila MB307 to selected metals (µg/ml) 417

after 24 hrs of incubation. Assays were performed in triplicate and an average was calculated from the 418

obtained values. 419

Substrate Ni2+ Pb2+ Cr6+ Cu2+ Cd2+ Co2+ Mn2+ As3+ Hg2+ Fe3+ Zn2+

Tolerance (µg/ml) 600 1400 900 700 500 400 2700 300 100 700 500

420

421

422

423

424

425

426

427

428

429

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430

431

432

433

434

435

Table 2. Plant growth promoting activities of Serratia nematodiphila MB307. 436

PSI means Phosphate Solublization Index calculated as colony diameter + zone diameter/ colony 437

diameter, Ca3(PO4)2 provided as 5g/L, metal selected was Pb2+ at a concentration of 200µg/ml. Zone 438

sizes mentioned after subtracting colony diameter from halo diameter. + in case of antifungal assay 439

indicates positive anti-fungal activity by Serratia nematodiphila MB307. Mentioned values are 440

average of the assays done in triplicate. 441

IAA (µg/ml) PSI Sideropho

re

productio

n

(zone

sizes in

mm)

Enzyme Activity

(zone sizes in mm)

ACC

deaminas

e activity

(α-

ketobutyr

ate

µmol/mg/

h)

Anti-fungal activity

With

Metal

Without

Metal

Witho

ut

Metal

Wit

h

Met

al

2nd

day

5th

day

Protea

se

Cellula

se

Pectina

se

0.1 Aspergill

us niger

Fusariu

m

oxysporu

m

8.089±1.2

32

7.285±0.7

86

2.66 3.5 8 12 11.3 15 27 +++ +

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442

443

Table 3. Xenobiotics biodegradation and metabolism pathways for Serratia nematodiphila 444

MB307. 445

Serial No. KEGG map ID Degradation pathway No. of genes mapped to

degradation pathway

1. 00362 Benzoate degradation 18

2. 00627 Aminobenzoate degradation 4

3. 00364 Fluorobenzoate degradation 7

4. 00625 Chloroalkane and chloroalkene

degradation

3

5. 00361 Chlorocyclohexane and chlorobenzene

degradation

3

6. 00623 Toluene degradation 3

7. 00622 Xylene degradation 5

8. 00633 Nitrotoluene degradation 1

9. 00642 Ethylbenzene degradation 1

10. 00643 Styrene degradation 1

11. 00791 Atrazine degradation 3

12. 00930 Caprolactam degradation 4

13. 00621 Dioxin degradation 1

14. 00626 Naphthalene degradation 3

15. 00980 Metabolism of xenobiotics by cytochrome

P450

3

446

447

448

449

450

451

452

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453

454

Table 4. Antibiotic resistance profile of Serratia nematodiphila MB307 genome blasted against 455

the ‘Antibiotic Resistance Genes Database’. 456

Matching

gene

Definition of matched gene Resistance to antibiotic

type/class

aac6ic Aminoglycoside N-acetyltransferase, which modifies aminoglycosides by acetylation.

isepamicin netilmicin tobramycin amikacin sisomicin dibekacin

acrb Resistance-nodulation-cell division transporter system. Multidrug resistance efflux pump.

aminoglycoside glycylcycline macrolide beta_lactam acriflavin

baca Undecaprenyl pyrophosphate phosphatase, which consists in the sequestration of Undecaprenyl pyrophosphate.

bacitracin

bl1_sm Class C beta-lactamase. This enzyme breaks the beta-lactam antibiotic ring open and deactivates the molecule's antibacterial properites.

cephalosporin

ksga Specifically dimethylates two adjacent adenosines in the loop of a conserved hairpin near the 3'-end of 16S rRNA in the 30S particle. Its inactivation leads to kasugamycin resistance.

kasugamycin

qnrb Pentapeptide repeat family, which protects DNA gyrase from the inhibition of quinolones.

fluoroquinolone

rosb Efflux pump/potassium antiporter system. RosA: Major facilitator superfamily transporter. RosB: Potassium antiporter.

fosmidomycin

457

458

459

460

461

462

463

464

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465

466

Figure Captions 467

Fig. 1. (A) Siderophore, (B) pectinase, (C) cellulase, (D) protease production in Serratia 468

nematodiphila MB307. (E) Plate showing positive phosphate solubilisation activity of Serratia 469

nematodiphila MB307. 470

Fig. 2(A). Circular representation of the Serratia nematodiphila MB307 genome. BLAST 1 and 2 471

search mentioned in the legend is for Serratia nematodiphila CGMCCT and Serratia nematodiphila 472

DSM 21420T respectively. OriC replication region of 380 nt (position 270583-270962 nt) is shown by 473

black divider ring with 3’ leader to the left and 5’ trailer at its right side. (B) Alignment of Serratia 474

nematodiphila MB307 (shown in the middle) with Serratia nematodiphila DSM 21420T (shown at the 475

top) and Serratia nematodiphila strain CGMCCT (shown below MB307). Nucleotide positions are 476

shown above the chromosomal segments. Locally collinear blocks shown in same colours and 477

connected by vertical lines in the studied genomes depict homologous regions. Phylogenetically both 478

type strains were equidistant to the strain MB307. 479

Fig. 3. Antibiotic Resistance Ontology (ARO) heatmap plot showing similarity of strict matches (blue 480

colour) for antibiotic resistance genes of our strain Serratia nematodiphila MB307, compared with 481

other bacterial species in the database. ARO categories mentioned at the bottom indicate resistance 482

mechanism i.e. drug target modification, replacement, protection, enzymatic destruction and transport 483

etc. 484

Fig. 4. N-coronafacoyl-L-valine and N-coronafacoyl-L-threonine analog producing gene clusters in 485

the Serratia nematodiphila. Similarity percentage of genes is mentioned above each operon of other 486

Serratia species relative to Serratia nematodiphila MB307. Putative biosynthetic genes are shown in 487

teal and purple, additional biosynthetic in grey and transcription regulation-related genes in aqua 488

colour. 489

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490

491

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Fig. 1.

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Fig. 2.

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