Enterobacillus tribolii gen. nov., sp. nov., a novel member of the family Enterobacteriaceae, isolated from gut of a red flour beetle, Tribolium castaneum
Vikas S. Patil1#, Rahul C. Salunkhe2#, Ravindra H. Patil3, Husseneder C4, Yogesh S. Shouche1 and V. Venkata Ramana1*
1Microbial Culture Collection, National Centre for Cell Science, Pune, Maharastra 411007, India.
2Bombay Natural History Society Shaheed Bhagat Singh Road, Mumbai-400 001, Maharashtra, India.
3Department of Microbiology and Biotechnology, R.C.Patel Arts, Commerce and Science College, Shirpur, 425405, Maharashtra, India.
4 Department of Entomology, Louisiana State University Agricultural Center, Baton Rouge, Louisiana, United States of America
*Corresponding author
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# Authors equally contributed to the manuscript.
The GenBank / EMBL / DDBJ accession numbers for the 16S rRNA, rpoB and gyrBgene sequences of strains IG-V01T and IG-V01b are HG972968, LM993265, LM993263 and LK934679, LM993266, LM993264 respectively.
Key words: Enterobacteriaceae, Tribolium castaneum, phylogenetic analysis, polyphasic approach
Abstract
Two novel Gram-stain negative facultative anaerobic, motile rod shaped bacterial strains IG-V01T and IG-V01b were isolated from the gut of red flour beetles, Tribolium castaneum. The 16S rRNA gene sequences of strains IG-V01T and IG-V01bwas found to have their highest sequence similarity 96.5% and 96.4% with Serratia nematodiphilaDZ0503SBS1T(Enterobacteriaceae family) respectively. Strains IG-V01T and IG-V01b share 100 % 16S rRNA gene sequence similarity and exhibit very similar phenotypic characteristics. In addition, they show89.7 % genomic relatedness (DNA-DNA hybridisation). Major fatty acids were identified to be C16:0 (38.3%), C17:0cyclo (19.5-20%) and C14:0 (11.2-11.3%). Cells contain phosphatidylethanolamine (PE) and diphosphatidylglycerol (DPG) as predominant polar lipids. Genomic DNA G+C content (mol %) was determined to be 51.5 - 51.7. A polyphasic approach employing the study of morphological, physiological, chemotaxonomic, genomic and phylogenetic analysis revealed that the two newly isolated strains cannot be placed in any of the existing genera of the family Enterobacteriaceae. Therefore, it is proposed that strains IG-V01Tand IG-V01bbelong to a novel genuswithin the family Enterobacteriaceae, and represent a new speciesEnterobacillus tribolii gen. nov., sp. nov., with the type strain=IG-V01T=KCTC 42159T =MCC 2532T.
Introduction
As part of the taxonomic surveys onthe biodiversity of the microbial communities associated with insects(Dillon and Dillon 2004),the redflour beetles Triboliumcastaneum (Herbst) (Coleoptera: Tenebrionidae) have been investigated.Present study resulted in the isolation and identification oftwo novel bacterial strains belonging to the family Enterobacteriaceae, class Gammaproteobacteria( Bacteriaof this family comprise a large group of genetically related enterobacteria isolatedfrom diverse ecological habitats including the guts of different animal species. Currently familyEnterobacteriaceaeconsists of fifty three genera,which are differentiated based on 16S rRNA gene sequence similarities, physiological, biochemical, molecular characteristics and their association with the host (Brenner and Farmer 2005,Holmes and Farmer 2009). Here, we applied a polyphasic taxonomy approach (Vandamme et al. 1996), in order to clarify the taxonomic position of two newly isolated strains.
Materials and methods
Strains and culture conditions
Two strains IG-V01T and IG-V01b were isolated from the gut of red flour beetles T. castaneum. Ten red flour beetles were randomly collected from sesame seeds, wiped with70% ethanol and thoroughly rinsed using sterile distilled water. These specimens were dissected under a dissecting microscope(Goko) and whole gutswere extirpated. Guts (approx. 2.0-2.5 mg) from all the beetles were mixed and homogenized in 1.0 ml Phosphate Buffer Saline (PBS). The suspension was spread onto R2A, NA and TSA (HiMedia) media plates (100 µl each) followed by incubation at 37˚C for 24 to 48 h. Isolated colonies from each plate were streaked onto the R2A, NA and TSA (HiMedia) media plates. The fastest growth of isolated bacteria was observed on TSA and this medium was used for isolated strains growth and characterization. Putatively novel strains were selected based on 16S rRNA gene sequencing results as described in the following sections.
Strains IG-V01T, IG-V01b (from present study), Klebsiella pneumoniaeDSM 30104T, Citrobacter freundii DSM 30039Tand Escherichia coli DSM 30083T (obtained from DSMZ) were maintained in15 % glycerol stocks in -80˚C, liquid nitrogen (LN2), and in lyophilized form as well.All the trains procured from DSMZ, Germany, were used for the verification of phenotypic characteristics mentioned in Table 1 and Fig. S7.
Molecular and phylogenetic analysis
For 16S rRNA gene amplification, G+C content, ∆Tm analysis and DNA–DNA hybridization (DDH), DNA was extracted from log phase cultures by phenol-chloroform method (Marmur, 1961) with additional RNase treatment. The16S rRNA gene was amplified as described by elsewhere (Sambrook et al. 1989) by using eubacterial specific primers 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5’- ACG GCT ACC TTG TTA CGA CTT3′) (Lane, 1991). Amplified PCR products were purified using the polyethylene glycol (PEG)-NaCl method (Sambrook et al.1989). Both strands of the amplicons were sequenced on an ABI 3730 xl DNA analyser using the Big Dye terminator kit (Applied Biosystems, Inc., Foster City, CA). The sequences obtained were assembled using DNASTARPro (version 10) and analyzed by using the online database EZ‐Taxon of EZBIOCLOUD (Kim et al. 2012). All available sequences of 16S rRNA genes of the members of the family Enterobacteriaceaewere retrieved from NCBI. These sequences (including IG-V01T and IG-V01b) were aligned by using Clustal-W tool and MEGA5 software. Best fit model for phylogenetic analysis was identified through “Find best DNA/protein model” tool of MEGA5. Accordingly, two phylogenetic trees: one with type species of all the closest members (Fig. 1) and the second one with all most all the species of Enterobacteriaceae(Fig. S1), were constructed by using the neighbor joining method with Kimura-2-parameter as a model of nucleotide substitution and 1000 bootstrap replications with Gamma distribution (Tamura et al. 2011). Consistency of phylogenetic tree clustering pattern was confirmed by maximum parsimony (MP) and maximum likelihood (ML) methods (data not shown).
To support the phylogenetic position of strains IG-V01T and IG-V01b within the family Enterobacteriaceae, two protein encoding genes rpoB (RNA polymerase β-subunit) and gyrB (DNA gyrase β-subunit) were studied for comparative purposes, because these genes were suggested to be suitable for assessing phylogenetic affiliation of Enterobacteriaceae members (Brady et al. 2008; Dauga, 2002; Mollet et al. 1997).Gene fragments were amplified from total genomic DNA as described byBrady et al. (2008). Purification, sequencing and phylogenetic analysis of rpoB and gyrB genes (Fig. S2 and Fig. S3) were carried out using the same methods as used for 16S rRNA genes. Pairwise similarities of all the species used for 16S rRNA, rpoB and gyrB genes phylogenetic trees are given in the Supplementary Tables 1-3 respectively.
In order to analyze G+C content (mol %), DNA was suspended in 0.1X Saline Sodium Citrate buffer (SSC). Thermal denaturation was performed with 5µg of DNA in each well along with a fluorescent dye SYBR Green I (Invitrogen) at a final dilution of 1:100,000. Thermal conditions consisting of a ramp from 25˚C to 100˚C at 1˚C min-1 were achieved by using StepOnePlus Real-Time PCR system (Applied Biosystems) fitted with a 96 well thermal cycling block for running samples in96 well plates. Fluorescence readings were recorded at each step during the ramp. Tm based G+C analysis by fluorometric method was done in triplicates as described by Gonzalez and Saiz-Jimenez (2002). StepOnePlus Real-Time PCR and SYBR Green-I were also used for the analysis of ∆Tm and DNA-DNA relatedness.
Change in the melting temperature (∆Tm) of the homoduplex DNA (IG-V01T) and heteroduplex DNA (IG-V01b + IG-V01T) was estimated as described by Gonzalez and Saiz-Jimenez (2005). For this, DNA duplexes were prepared by denaturation followed by reassociation and the optimal denaturation temperature (Tor) was calculatedusing the equation, Tor = 0.51 × (% G+C) + 47.0 (DeLey et al. 1970;Gillis et al. 1970). SYBR Green-I, which is specific for binding to double stranded DNA (dsDNA), was used to analyze the melting profiles of homo-and heteroduplex DNAs.
DNA–DNA hybridization was carried out in triplicates as described by Loveland-Curtze et al. (2011).DNA-DNA relatedness in terms of relative binding ratio wascalculated as described elsewhere (DeLey et al.,1970). DNA was suspended in the 2X SSC and SYBR Green-I and sheared using ultrasonic bath to get uniformly small-sized fragments, approximately 400-1500bp length. Resulting DNA fragments were used in DNA-DNA hybridisations. The relative rate of reassociation of homoduplex and heteroduplex DNA was analysed by denaturation followed by optimum reassociation (Gillis et al. 1970).
Phenotypic study
Colony morphology was observed using light microscopy (Olympus, Magnus-MLX-DX). Cell size and shape were determined using a phase contrast microscope (Olympus-BX53F). Motility of strains was detected by hanging drop method and confirmed by using semi-solid agar method (Harley and Prescott 2002). Gram staining and spore staining were performed by using commercial kits (HiMedia) as per manufacturer’s guidelines. Strains were grown to exponential phase on TSA / TSB for physiological and biochemical characterization unless otherwise specified. Assimilation of different carbohydrates, nitrate reduction, enzyme production (gelatinase, urease, arginine dihydrolase and β-galactosidase) and acid production from various carbohydrates was tested by using API 20 NE, API ZYM and API 50 CHB/E test kits (BioMérieux) respectively, according to the manufacturer’s instructions. Catalase activity was tested by identifying the formation of oxygen bubbles after the addition of 3% (v/v) aqueous hydrogen peroxide solution. Oxidase activity was assessed by the addition of 1% oxidase test reagent N,N,Nl,Nl-tetramethyl-p-phenylenediamine dihydrochloride (HiMedia). Indole production and citrate utilization were tested with API 20 NE kits, and again confirmed during the IMViC tests, which were carried out in duplicates by conventional methods. Indole production from tryptophan was tested by adding Kovacs’ reagent into the 24 h-grown culture. Strains grown in MR-VP broth for 24-48 h were used for both methyl red (mixed acid fermentation) and Voges-Proskauer test (butanediol / acetoin production). Colour change by the addition of methyl red indicator and Barritt’s reagents (A and B) was observed for both the tests, respectively. The growth on the Simmons’ citrate agar slants wasobserved for their colour change due to the utilization of citrate. H2S production was tested on Triple Sugar Iron Agar. All the physiological tests were carried out in triplicates and growth was measured turbidometrically (OD) at 600 nm using a spectrophotometer (Spectra max plus 384) and cuvette of 1.0 cm path length. Growth at various concentrations of NaCl (0-10 %, w/v, at intervals of 1.0 %), different pH (pH 4.0-11, at intervals of 1.0 pH unit) and temperature (5-55 ºC, at intervals of 5 ºC) was investigated. Anaerobic growth was tested by inoculating the strains onto agar slants plugged with rubber Suba-Sealrubber septa followed by flushing with the inert gas argon (using needles) and incubation at 37˚C for 24 to 48 h.
Antibiotic susceptibility for both the strains IG-V01T and IG-V01b was tested in duplicates by disc diffusion method on TSA at 37˚C with filter paper discs (6 mm diameter, HiMedia) containing the following antibiotics with respective concentration: cefpodoxime (10 µg), chloramphenicol (30 µg), vancomycin (30 µg), streptomycin (10 µg), rifampicin (5 µg), levofloxacin (5 µg), cetriaxone (30 µg), clindamycin (2 µg), augmentin (30 µg), amikacin (30 µg), cefixime (5 µg), tetracycline (30 µg), co-trimoxazole (25 µg), colistin (10 µg), norfloxacin (10 µg), ceftriaxone(10 µg), ciprofloxacin (5 µg), cephotaxime (30 µg), centamicine (10 µg), curazolidone (50 µg), and amoxycillin (10 µg).
Chemotaxonomic characterization
For whole cell fatty acid analysis, strains were inoculated onto TSA medium (pH 7.0) and incubated at 28 ± 20C for 18 h. Cells were harvested and subjected to saponification, methylation, and extraction followed by base wash. Resulting methyl esters of fatty acids were analyzed by Gas Chromatography (Agilent Technologies; 7890 A) according to the rapid Microbial Identification System software (MIS, MIDI Inc., Newark, DE, USA; version 6.0;) and peaks were identified based on the RTSBA6 database (Sasser, 1990; revised- Polar lipids were extracted from freeze-dried culture in chloroform : methanol : 0.3% saline (1:2:0.8, v/v) as described by Bligh and Dyer (1959) considering the modifications of Card (1973). Lipids were separated on silica gel TLC (Kieselgel 60 F254; Merck) by two-dimensional chromatography using chloroform : methanol : water (65:25:4 v/v) in the first dimension and chloroform : methanol : acetic acid : water (80:12:15:4 v/v) in the second dimension (Tindall, 1990). Dried plates were stained with 5% ethanolic molybdophosphoric acid for total lipids. Lipid functional groups were identified by using spray reagents ninhydrin (specific for amino groups), molybdenum blue (specific for phosphates), Dragendorff (quaternary nitrogen) or α-naphthol (specific for sugars) for detection of lipids.
For MALDI-TOF-MS based ribosomal protein profiling, which was carried out as an additional supportive analysis, strains IG-V01T and IG-V01b along with strains of known species from different genera of the Enterobacteriaceae (Serratiamarcescens DSM 30121T, Pantoea calida DSM 22759T, Citrobacterfreundii DSM 30039T, Raoultellaornithinolytica DSM 7464T, Escherichia coli DSM 30083T, Enterobacterhormaechei DSM 12409T, KlebsiellapneumoniaeDSM 30104T) available in our culture collection were grown on TSA plates for 24 h. Whole cells were subjected to protein extraction by using ethanol, formic acid and acetonitrile as per manufacturer’s manual of Bruker Daltonics. Extracts were analyzed for ribosomal protein profiles (between 2.0 kD – 20.0 kD range) by using MALDI Biotyper 3.1. Resulting ribosomal protein profiles were used to generate Principal Component Analysis (PCA) dendrogram to check the phylogenetic position of novel strains within the family Enterobacteriaceae. Pseudomonasoleovorans DSM 1045Twas used as outgroup.
Results and discussion
Bacteria isolated from the gut of red-flour beetles was found to be phylogenetically related to bacteria of thegenera Staphylococcus, Bacillus and Serratia. The 16S rRNA gene sequences of two strains IG-V01T (1404 bp) and IG-V01b (1363 bp) was found to share 100 %, while showingclosest similarity (96.5 % and 96.4 %, respectively) to those of “Flavobacterium acidificum” LMG 8364T and Serratia nematodiphila DZ0503SBS1T followed by the members of the genera Pantoea and Cronobacter(Fig. S4). Furthermore, the sequence similarity of these two strains with other members of the family Enterobacteriacea was found to be below 96.4%. “Flavobacterium acidificum”LMG 8364T, the first closest match to strains IG-V01T and IG-V01b, was described by Steinhaus (1941) based on morphological, cultural and physiological characteristics and classified as a member of the family Flavobacteriaceae. Recently it was reported thatthe 16S rRNA gene sequence of the type strain “Flavobacterium acidificum” LMG 8364T shows 99.9 % similarity withPantoeaananatisATCC 33244Tof the family Enterobacteriaceae and therefore the taxonomic status of this bacterium requires revision (Yarza et al. 2013, In addition to 16S rRNA gene, protein encoding genes rpoB and gyrB were sequenced for the comparative phylogenetic analysis to conform the taxonomic affiliation of strains IG-V01T and IG-V01b. Phylogenetic analysis of sequence similarities of protein encoding genes rpoB and gyrB revealed that strains IG-V01T and IG-V01b share 100 % rpoB gene and 99.1 % gyrB gene sequence similarity between each other and comparatively low similarity between 92 % and 87 %,respectively,with other members of the family. Neighborjoining phylogenetic trees based on 16S rRNA gene sequences (Fig. 1, S1) and protein encoding genes [rpoB (Fig. S2) and gyrB (Fig. S3)] clearly showed that the two strains IG-V01T and IG-V01b consistently grouped together with high bootstrap support within a cluster with Cronobacter sakazakii ATCC 29544T and Escherichia coliDSM 30083T. Phylogenetic trees generated by using maximum-parsimony and maximum-likelihood methods showed similar tree topology (data not included) in support of the delineation of IG-V01T and IG-V01 as members of a novel genus of the Enterobacteriaceae family.
The novel strains are Gram-stain negative straight rods (Fig. S5), non-spore forming, facultative anaerobic, catalase positive, oxidase negative, and exhibited both respiratory and fermentative metabolism. Acid and visible gas production from the fermentation of D-glucose is observed.Among the 43 carbohydrates tested for acid production 17 were positive for both strains. Strain IG-V01T utilizes 6 of the 14 carbohydrates tested whereas IG-V01b shows positive results for 5 and weakly positive results for 1 out of the same 14 carbohydrates tested (Table 1).Both strains are susceptible to all tested antibiotics (as indicated in Material and Methods), grow at a broad range oftemperature (15-50 ˚C, optimum 35-37˚C), pH (5-10.0, optimum 8.0) and salinity (0-5.0 % (w/v NaCl), optimum 0.05 %). The genomic relatedness of two new isolates was found to be 89.7 % and ∆Tm <1.0˚C, confirming that that IG-V01T and IG-V01b belong to a single species Wayne et al. (1987).
To support the distincttaxonomic standing of two novel isolates, several phenotypic characteristicsof the strains IG-V01T and IG-V01 were compared with those of type species of twelve phylogenetically close genera of the family Enterobacteriaceae(Table 1 and 2).Both the strains could be distinguished from other closely related members with respect to their inability to reduce nitrates (differed from Serratia nematodiphilaDZ0503SBS1Tand Klebsiella pneumoniaeDSM 30104T), to produce gelatinase (differed from Serratia nematodiphilaDZ0503SBS1T, Serratia marcescensDSM 30121T, Pantoea agglomeransDSM 3493Tand Enterobacter cloacaeDSM 30054T), tryptophanase (differed from Leclercia adecarboxylataDSM 30081TandEscherichia coliDSM 30083T), urease (differed from Raoultella planticolaDSM 3069Tand K. pneumoniaeDSM 30104T), hydrogen sulfide(differed from C. freundiiDSM 30039T), citritase and acetoin (differed from S. nematodiphilaDZ0503SBS1T, S. marcescensDSM 30121T, C. sakazakiiATCC 29544T, P. agglomeransDSM 3493T, R. planticolaDSM 3069T, K. 1neumoniaDSM 30104T, E. cloacaeDSM 30054Tand Tatumella ptyseosLMG 7888T). Production of arginine dihydrolase is not observed in the novel isolates, but detected in S. nematodiphilaDZ0503SBS1T, C. sakazakiiATCC 29544T, E. cloacaeDSM 30054Tand Rosenbergiella nectereaDSM 24150T. Methyl Red (MR) test was positive for both the isolates indicating mixed acid fermentation which could not be observed with close relatives S. nematodiphilaDZ0503SBS1T, S. marcescensDSM 30121T, C. sakazakiiATCC 29544T and T. ptyseosLMG 7888T, whereas variable in P. agglomeransDSM 3493T(Table 1).The novel strains are capable of producing acids from D-xylose which could not be observed in their closest relatives S. nematodiphilaDZ0503SBS1T and S. marcescensDSM 30121T. Whereas, D-arabinose, inositol, lactose, cellobiose, D-arabitol, melibiose, which are utilized by most of the closest phylogenetic neighbors, are not utilized by strains IG-V01T and IG-V01b (Table 1).Differences in the utilization of carbohydrates between IG-V01T and IG-V01b and the type strains of phylogenetically closest members of the family Enterobacteriaceae are listed in Table 1. Two strains from the present study are able to grow at temperatures above 45˚C, in contrast to any other phylogenetically close neighbors included in Table 1. In contrast to strains IG-V01T and IG-V01b, S. nematodiphilaDZ0503SBS1T, P. agglomeransDSM 3493T, R. planticolaDSM 3069T, L. adecarboxylataDSM 30081Tand R. nectereaDSM 24150Tgrow at 5˚C (Table 1). The genomic G+C content (mol %) of the novel strains ranges from 51.5 to 51.7 %, the values, which are substantially low in comparisonto those of the phylogenetically closest neighbors (Table 1).