Erwinia teleogrylli sp. nov., a Bacterial Isolate Associated with a Chinese Cricket

A bacterial isolate (SCU-B244T) was obtained in China from crickets (Teleogryllus occipitalis) living in cropland deserted for approximately 10 years. The isolated bacteria were Gram-negative, facultatively anaerobic, oxidase-negative rods. A preliminary analysis of the 16S rRNA gene sequence indicated that the strain belongs to either the genus Erwinia or Pantoea. Analysis of multilocus sequence typing based on concatenated partial atpD, gyrB and infB gene sequences and physiological and biochemical characteristics indicated that the strain belonged to the genus Erwinia, as member of a new species as it was distinct from other known Erwinia species. Further analysis of the 16S rRNA gene showed SCU-B244T to have 94.71% identity to the closest species of that genus, Erwinia oleae (DSM 23398T), which is below the threshold of 97% used to discriminate bacterial species. DNA-DNA hybridization results (5.78±2.52%) between SCU-B244T and Erwinia oleae (DSM 23398T) confirmed that SCU-B244T and Erwinia oleae (DSM 23398T) represent different species combined with average nucleotide identity values which range from 72.42% to 74.41. The DNA G+C content of SCU-B244T was 55.32 mol%, which also differs from that of Erwinia oleae (54.7 to 54.9 mol%). The polyphasic taxonomic approach used here confirmed that the strain belongs to the Erwinia group and represents a novel species. The name Erwinia teleogrylli sp. nov. is proposed for this novel taxon, for which the type strain is SCU-B244T (= CGMCC 1.12772T = DSM 28222T = KCTC 42022T).


Introduction
Resistance to pesticides in insects is a serious concern worldwide [1] and often occurs approximately 10 years after the introduction of a new pesticide [2].The resistance mechanisms have been attributed to evolutionary changes in insect genomes, such as the alteration of drug target sites, up-regulation of degrading enzymes and the enhancement of drug excretion.Kikuchi In a recent study, we focused on the culturable strains associated with crickets (Teleogryllus occipitalis), a common pest living in a deserted cropland in China.Our aim was to explore the relationship between pesticide resistance and symbionts.Among 274 isolates cultured from Teleogryllus occipitalis, 27 strains of genera Lysinibacillus, Pseudomonas, Sphingobacterium, Exiguobacterium and Staphylococcus could evidently degrade chlorpyrifos, a common insecticide used in this field for many years.One isolate (SCU-B244 T ) that could degrade chlorpyrifos was cultured on TSA (tryptone soy agar) medium in August 2012 and could not be identified to the species level.A polyphasic taxonomic approach was used to investigate the strain, with the results suggesting that SCU-B244 T represents a novel species of the genus Erwinia.

Isolation procedures, culture conditions and initial microbiological characterization
Three crickets (collected near 30°33'N, 103°58'E; altitude 495 m) were added to 100 mL sterile 0.85% (w/v) NaCl solution in a 250 mL flask and shaken at 220 rpm for 30 min.The supernatant which contained bacteria was plated onto TSA plates (Tryptone 1.5%, Soy Peptone 0.5%, NaCl 0.5%, Agar 1.5%, w/v, pH 7.2) and subsequently incubated at 37°C for 5 days.Glycerol stock at -80°C was adopted for long term preservation of the isolates.Exponential phase cells cultured in TSB (Tryptone 1.5%, Soy Peptone 0.5%, NaCl 0.5%, pH 7.2) medium with shaking (200 rpm, 16 h) at 37°C were harvested for DNA G+C content and ANI analysis.Phase contrast and transmission electron microscopy were used to examine cellular morphology and motility after growth on TSA medium at 37°C for 24 h.Gram staining was performed as described by Gerhardt et al. [3].Oxidase and catalase activity were tested according to methods described by Smibert & Krieg [3].TSB medium with different NaCl concentration and pH were used to test tolerance to NaCl and pH range, and the growth was determined by OD 600.To confirm the results, each experiments mentioned above were performed three times.Erwinia oleae (DSM 23398 T ) was used as reference strain in this study.

16S rRNA gene sequence analysis
DNA extraction from strain SCU-B244 T , PCR amplification, primers used and DNA sequencing conditions of 16S rRNA gene were performed as previously described [4].The strain was analysed using the EzTaxon server [5] (www.ezbiocloud.net/eztaxon)by comparison with 16S rRNA gene sequence data.A neighbour-joining phylogenetic tree was constructed using the method of Saitou and Nei [6] with MEGA 5.2 software [7].Similarities were calculated using the Kimura 2-parameter [8,9] in MEGA 5.2.Maximum-likelihood phylogenetic trees were also constructed using 9] model and the method of Felsenstein [10] with MEGA 5.2 software.Robustness of the phylogenetic trees was evaluated by using the bootstrap resampling method of Felsenstein [11], with 1000 replicates.
The results of 16S rRNA gene sequence alignment on the EzTaxon server revealed that strain SCU-B244 T belongs to the family Enterobacteriaceae.Related 16S rRNA gene sequences for initial analysis were taken from the top 66 hits on the EzTaxon server (on May 2014, each representing a different species), with the highest similarity scores being 96.1% to 94.1%.Neighbour-joining and maximum-likelihood phylogenetic trees were constructed using the methods described above.
To refine the taxonomic position of SCU-B244 T , other neighbour-joining and maximumlikelihood phylogenetic trees based on 16S rRNA gene sequences were constructed, including the strain SCU-B244 T and all culturable type strains of the genera Erwinia (Candidatus Erwinia dacicola) and Pantoea, based on the same methods described above.

Multilocus sequence analysis
Multilocus sequence analysis (MLSA) of concatenated partial atpD, gyrB, infB and rpoB gene sequences enables the differentiation of the phylogenetically related genera Erwinia, Pantoea and Tatumella.A good congruence has previously been observed between DNA-DNA hybridization and MLSA [12][13][14][15][16].In the present study, to confirm the results observed with 16S rRNA gene sequencing data, three housekeeping genes, atpD, gyrB, and infB, were amplified and sequenced with the primers described by Brady et al. [12].Neighbour-joining and maximum-likelihood phylogenetic trees based on concatenated partial atpD, gyrB and infB gene sequences were constructed using the same method as described above.Strains from genera Erwinia, Pantoea and Tatumella were used in MLSA analysis, including SCU-B244 T .The partial sequence of atpD, gyrB and infB gene of related strains were obtained from GenBank and the accession numbers are indicated on the figures.

DNA-DNA hybridization
DNA-DNA hybridization between SCU-B244 T and Erwinia oleae DSM 23398 T was conducted as described by De Ley et al. [17] with hybridization temperature set at 67°C.Genomic DNA (OD 260/280 = 1.8 to 1.9) was extracted using the Omega Bacterial DNA kit (E.Z.N.A. 1 )

DNA G+C content
The DNA G+C mol% content of strain SCU-B244 T was determined by whole genome sequencing data.

Physiological and biochemical analysis
API 20E, API 50CHE acid production tests and API ZYM enzymatic characteristics test (Bio-Mérieux) were performed according to the manufacturer's instructions.BIOLOG GN2 carbon-source utilization analysis was conducted according to the instruction manual.Cellular fatty acids analysis was also performed.Biomass for fatty acid analysis was prepared by scraping growth from TSA plates after 24h incubation at 37°C.Lipids were extracted using the method of Folch et al. [18].Total lipids were converted to fatty acid methyl esters (FAMEs) with 4 mol/L HCl in 55% (V/V) methanol at 80±1°C for 10 mins.FAMEs were analyzed by GC-MS (Trace DSQII, Thermo Fisher), and detailed conditions of the fatty acids analysis by GC-MS have been previously described [19], The compounds were identified using NIST 05 database (NIST Mass Spectral Database, PC-Version 5.0, 2005, National Institute of Standardisation and Technology, Gaithersburg, MD, USA).To confirm the results, each experiment mentioned above was performed three times.

Genome Sequencing and Average Nucleotide Identity
For the genome sequencing of SCU-B244 T , Next Generation Sequencing (Illumina Miseq) was conducted at Majorbio Inc. and the data was used to calculate the Average Nucleotide Identity (ANI).DNA extraction method and purity were described above.ANI is a similarity measure between two genome sequences and it correlates well with DNA-DNA hybridization values.[20] A value of 70% DNA-DNA hybridization corresponds to about 95-96% ANI.[21] Genome data were taken from NCBI Genome and EzGenome web site.(www.ezbiocloud.net/ezgenome/browse_db).We used Orthologous Average Nucleotide Identity Tool (OAT software, Lee et al. 2015, Manuscript submitted, www.ezbiocloud.net/sw/oat)to calculate orthologous ANI values.This Whole Genome Shotgun project has been deposited at DDBJ/EMBL/ GenBank under the accession LLXO00000000.The version described in this paper is version LLXO01000000.

Results and Discussion
In total 274 isolates belonging to 29 genera were cultured from Teleogryllus occipitalis and a bacterial isolate (SCU-B244 T ) is herein described.Initial microbiological characterization of the strain revealed that the cells were Gram-negative, oxidase-negative, rod-shaped, catalasepositive and facultatively anaerobic, suggesting that the strain belongs to the family Enterobacteriaceae [22].
Neighbour The highest 16S rRNA gene identity of SCU-B244 T with species of genera Erwinia and Pantoea were 95.43% (Erwinia psidii LMG 7039 T ) and 95.42% (Pantoea calida 1400/07 T ) respectively, phylogenic closest strain was Erwinia oleae (DSM 23398 T ) and the identity was 94.71%, which is lower than the 97% threshold that has been established to discriminate species.Strains showing less than 97% 16S rRNA gene identity are unlikely to have more than 60 to 70% DNA -DNA relatedness [23], and this level of rRNA sequence identity strongly suggests that SCU-B244 T is a novel species.The DNA-DNA relatedness between SCU-B244 T and Erwinia oleae (DSM 23398 T ) was 5.79 ± 2.52%.The value is mean of six hybridizations ± SD, which is significantly lower than the 70% value considered to be the threshold for the delineation of bacterial species [24].ANI values between SCU-B244 T and related species are listed in Table 1.The values range from 72.42% to 74.41% which are lower than 95-96% ANI.
The DNA G+C content of strain SCU-B244 T was 55.32 mol% which could be discriminated from Erwinia oleae (DSM 23398 T ), which has a G+C content of 54.7 to 54.9 mol%.API 20E and API 50CHE (BioMérieux) tests were also carried out and the results were compared to related strains (Table 2).The results revealed that SCU-B244 T strain can be discriminated from each recognized species of the genus Erwinia by at least three characteristics, and from the phylogenetically most closely related species, Erwinia oleae, by 10 API 50CHE characteristics (Table 3).
API ZYM enzymatic characteristics and BIOLOG GN2 carbon-source utilization results are presented as supplementary data (S1 and S2 Tables).
Based on the genotypic data (16S rRNA gene sequence analysis and MLSA), it is clear that SCU-B244 T is a member of genus Erwinia.DNA-DNA hybridization results, ANI values and phenotypic data obtained in this study can separate this isolate from related species.In conclusion the bacterial strain isolated from a cricket (genus Teleogryllus) represents a novel species for which the name Erwinia teleogrylli sp.nov. is proposed.Strain SCU-B244 T (= CGMCC 1.12772 T = DSM 28222 T = KCTC 42022 T ) is the type strain.
Description of Erwinia teleogrylli sp.nov.
The colonies are milky white, circular and convex with entire margins.Cells are short Gram-negative, facultatively anaerobic rods that are oxidase-negative, catalase-positive and able to grow in 6% (w/v) NaCl.Growth pH range is 6 to 9, with optimum growth at 7. Nitrate is reduced to nitrite.
et al. have shown that infection with an insecticide-degrading bacterial symbiont immediately establishes insecticide resistance in insects [1], indicating bacteria play an important role in pesticide resistance.
-joining (S1 Fig) and maximum-likelihood (S2 Fig) phylogenetic trees of the first 66 hit with similarity values ranging from 96.1% to 94.1% revealed that strain SCU-B244 T and strains of the genera Erwinia and Pantoea cluster together.The diagram shows the phylogenetic relationship between SCU-B244 T and genera Erwinia and Pantoea.Further neighbour-joining (S3 Fig) and maximum-likelihood (Fig 1) phylogenetic trees, based on 16S rRNA gene sequences from members of the genera Erwinia and Pantoea, indicated that strain SCU-B244 T belongs to the genus Erwinia and is most closely related to Erwinia oleae (DSM 23398 T ), which is consistent with the results of the initial phenotypic analysis.In neighbour-joining (S4 Fig) and maximum-likelihood (Fig 2) phylogenetic trees based on concatenated partial atpD, gyrB and infB gene sequences, strain SCU-B244 T and Erwinia oleae (DSM 23398 T ) cluster together on a single branch, consistent with the previous results, suggesting strain SCU-B244 T is most closely related to Erwinia oleae (DSM 23398 T ).

Fig 1 .
Fig 1. Maximum-likelihood tree based on partial 16S rRNA gene sequences of the genera Erwinia and Pantoea.The diagram shows the phylogenetic relationship between Erwinia teleogrylli sp.nov.and related type species of the genera Erwinia and Pantoea except Candidatus Erwinia dacicola.Escherichia coli ATCC 11775 T was used as the outgroup.Bar, 0.5% nucleotide substitutions.Numbers at branching points are bootstrap percentage values based on 1000 replications.Only values >50% are shown.doi:10.1371/journal.pone.0146596.g001

Table 1 .
OrthoANI values between SCU-B244 T and related species.