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Enterobacillus tribolii gen. nov., sp. nov., a novel member of the family
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Enterobacteriaceae, isolated from gut of a red flour beetle, Tribolium
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castaneum
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Vikas S. Patil1#, Rahul C. Salunkhe2#, Ravindra H. Patil3, Husseneder C4, Yogesh S. Shouche1 and V.
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Venkata Ramana1*
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India.
Microbial Culture Collection, National Centre for Cell Science, Pune, Maharastra 411007,
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India.
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College, Shirpur, 425405, Maharashtra, India.
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4
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Louisiana, United States of America
Bombay Natural History Society Shaheed Bhagat Singh Road, Mumbai-400 001, Maharashtra,
Department of Microbiology and Biotechnology, R.C.Patel Arts, Commerce and Science
Department of Entomology, Louisiana State University Agricultural Center, Baton Rouge,
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*Corresponding author
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E_mail: ramanabiotechv@yahoo.com, venkat.vemuluri@gmail.com
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Phone: +91-20-25329034
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Fax: +91-20-25329001
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#
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The GenBank / EMBL / DDBJ accession numbers for the 16S rRNA, rpoB and gyrB gene
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sequences of strains IG-V01T and IG-V01b are HG972968, LM993265, LM993263 and
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LK934679, LM993266, LM993264 respectively.
Authors equally contributed to the manuscript.
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Key words: Enterobacteriaceae, Tribolium castaneum, phylogenetic analysis, polyphasic
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approach
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Abstract
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Two novel Gram-stain negative facultative anaerobic, motile rod shaped bacterial strains
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IG-V01T and IG-V01b were isolated from the gut of red flour beetles, Tribolium castaneum. The
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16S rRNA gene sequences of strains IG-V01T and IG-V01b was found to have their highest
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sequence similarity 96.5% and 96.4% with Serratia nematodiphila DZ0503SBS1T
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(Enterobacteriaceae family) respectively. Strains IG-V01T and IG-V01b share 100 % 16S rRNA
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gene sequence similarity and exhibit very similar phenotypic characteristics. In addition, they
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show 89.7 % genomic relatedness (DNA-DNA hybridisation). Major fatty acids were identified
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to be C16:0 (38.3%), C17:0 cyclo (19.5-20%) and C14:0 (11.2-11.3%). Cells contain
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phosphatidylethanolamine (PE) and diphosphatidylglycerol (DPG) as predominant polar lipids.
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Genomic DNA G+C content (mol %) was determined to be 51.5 - 51.7. A polyphasic approach
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employing the study of morphological, physiological, chemotaxonomic, genomic and
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phylogenetic analysis revealed that the two newly isolated strains cannot be placed in any of the
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existing genera of the family Enterobacteriaceae. Therefore, it is proposed that strains IG-V01T
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and IG-V01b belong to a novel genus within the family Enterobacteriaceae, and represent a new
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species Enterobacillus tribolii gen. nov., sp. nov., with the type strain= IG-V01T =KCTC
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42159T =MCC 2532T.
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Introduction
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As part of the taxonomic surveys on the biodiversity of the microbial communities associated
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with insects (Dillon and Dillon 2004), the red flour beetles Tribolium castaneum (Herbst)
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(Coleoptera: Tenebrionidae) have been investigated. Present study resulted in the isolation and
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identification of two novel bacterial strains belonging to the family Enterobacteriaceae, class
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Gammaproteobacteria (http://www.bacterio.net/enterobacteriaceae.html). Bacteria of this
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family comprise a large group of genetically related enterobacteria isolated from diverse
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ecological habitats including the guts of different animal species. Currently family
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Enterobacteriaceae consists of fifty three genera, which are differentiated based on 16S rRNA
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gene sequence similarities, physiological, biochemical, molecular characteristics and their
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association with the host (Brenner and Farmer 2005, Holmes and Farmer 2009). Here, we
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applied a polyphasic taxonomy approach (Vandamme et al. 1996), in order to clarify the
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taxonomic position of two newly isolated strains.
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Materials and methods
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Strains and culture conditions
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Two strains IG-V01T and IG-V01b were isolated from the gut of red flour beetles T. castaneum.
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Ten red flour beetles were randomly collected from sesame seeds, wiped with 70% ethanol and
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thoroughly rinsed using sterile distilled water. These specimens were dissected under a
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dissecting microscope (Goko) and whole guts were extirpated. Guts (approx. 2.0-2.5 mg) from
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all the beetles were mixed and homogenized in 1.0 ml Phosphate Buffer Saline (PBS). The
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suspension was spread onto R2A, NA and TSA (HiMedia) media plates (100 µl each) followed
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by incubation at 37˚C for 24 to 48 h. Isolated colonies from each plate were streaked onto the
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R2A, NA and TSA (HiMedia) media plates. The fastest growth of isolated bacteria was observed
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on TSA and this medium was used for isolated strains growth and characterization. Putatively
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novel strains were selected based on 16S rRNA gene sequencing results as described in the
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following sections.
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Strains IG-V01T, IG-V01b (from present study), Klebsiella pneumoniae DSM 30104T, Citrobacter
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freundii DSM 30039Tand Escherichia coli DSM 30083T (obtained from DSMZ) were maintained in 15
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% glycerol stocks in -80˚C, liquid nitrogen (LN2), and in lyophilized form as well. All the trains
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procured from DSMZ, Germany, were used for the verification of phenotypic characteristics
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mentioned in Table 1 and Fig. S7.
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Molecular and phylogenetic analysis
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For 16S rRNA gene amplification, G+C content, ∆Tm analysis and DNA–DNA hybridization
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(DDH), DNA was extracted from log phase cultures by phenol-chloroform method (Marmur,
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1961) with additional RNase treatment. The16S rRNA gene was amplified as described by
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elsewhere (Sambrook et al. 1989) by using eubacterial specific primers 27F (5′-
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AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5’- ACG GCT ACC TTG TTA CGA CTT3′)
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(Lane, 1991). Amplified PCR products were purified using the polyethylene glycol (PEG)-NaCl
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method (Sambrook et al.1989). Both strands of the amplicons were sequenced on an ABI 3730 xl
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DNA analyser using the Big Dye terminator kit (Applied Biosystems, Inc., Foster City, CA). The
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sequences obtained were assembled using DNASTARPro (version 10) and analyzed by using the
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online database EZ‐Taxon of EZBIOCLOUD (Kim et al. 2012). All available sequences of 16S
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rRNA genes of the members of the family Enterobacteriaceae were retrieved from NCBI. These
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sequences (including IG-V01T and IG-V01b) were aligned by using Clustal-W tool and MEGA5
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software. Best fit model for phylogenetic analysis was identified through “Find best
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DNA/protein model” tool of MEGA5. Accordingly, two phylogenetic trees: one with type
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species of all the closest members (Fig. 1) and the second one with all most all the species of
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Enterobacteriaceae (Fig. S1), were constructed by using the neighbor joining method with
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Kimura-2-parameter as a model of nucleotide substitution and 1000 bootstrap replications with
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Gamma distribution (Tamura et al. 2011). Consistency of phylogenetic tree clustering pattern
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was confirmed by maximum parsimony (MP) and maximum likelihood (ML) methods (data not
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shown).
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To support the phylogenetic position of strains IG-V01T and IG-V01b within the family
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Enterobacteriaceae, two protein encoding genes rpoB (RNA polymerase β-subunit) and gyrB
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(DNA gyrase β-subunit) were studied for comparative purposes, because these genes were
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suggested to be suitable for assessing phylogenetic affiliation of Enterobacteriaceae members
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(Brady et al. 2008; Dauga, 2002; Mollet et al. 1997).Gene fragments were amplified from total
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genomic DNA as described by Brady et al. (2008). Purification, sequencing and phylogenetic
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analysis of rpoB and gyrB genes (Fig. S2 and Fig. S3) were carried out using the same methods
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as used for 16S rRNA genes. Pairwise similarities of all the species used for 16S rRNA, rpoB
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and gyrB genes phylogenetic trees are given in the Supplementary Tables 1-3 respectively.
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In order to analyze G+C content (mol %), DNA was suspended in 0.1X Saline Sodium Citrate
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buffer (SSC). Thermal denaturation was performed with 5µg of DNA in each well along with a
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fluorescent dye SYBR Green I (Invitrogen) at a final dilution of 1:100,000. Thermal conditions
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consisting of a ramp from 25˚C to 100˚C at 1˚C min-1 were achieved by using StepOnePlus Real-
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Time PCR system (Applied Biosystems) fitted with a 96 well thermal cycling block for running
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samples in 96 well plates. Fluorescence readings were recorded at each step during the ramp. Tm
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based G+C analysis by fluorometric method was done in triplicates as described by Gonzalez
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and Saiz-Jimenez (2002). StepOnePlus Real-Time PCR and SYBR Green-I were also used for
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the analysis of ∆Tm and DNA-DNA relatedness.
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Change in the melting temperature (∆Tm) of the homoduplex DNA (IG-V01T) and heteroduplex
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DNA (IG-V01b + IG-V01T) was estimated as described by Gonzalez and Saiz-Jimenez (2005).
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For this, DNA duplexes were prepared by denaturation followed by reassociation and the optimal
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denaturation temperature (Tor) was calculated using the equation, Tor = 0.51 × (% G+C) + 47.0
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(De Ley et al. 1970; Gillis et al. 1970). SYBR Green-I, which is specific for binding to double
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stranded DNA (dsDNA), was used to analyze the melting profiles of homo-and heteroduplex
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DNAs.
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DNA–DNA hybridization was carried out in triplicates as described by Loveland-Curtze et al.
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(2011). DNA-DNA relatedness in terms of relative binding ratio was calculated as described
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elsewhere (De Ley et al., 1970). DNA was suspended in the 2X SSC and SYBR Green-I and
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sheared using ultrasonic bath to get uniformly small-sized fragments, approximately 400-1500
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bp length. Resulting DNA fragments were used in DNA-DNA hybridisations. The relative rate
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of reassociation of homoduplex and heteroduplex DNA was analysed by denaturation followed
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by optimum reassociation (Gillis et al. 1970).
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Phenotypic study
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Colony morphology was observed using light microscopy (Olympus, Magnus-MLX-DX). Cell
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size and shape were determined using a phase contrast microscope (Olympus-BX53F). Motility
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of strains was detected by hanging drop method and confirmed by using semi-solid agar method
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(Harley and Prescott 2002). Gram staining and spore staining were performed by using
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commercial kits (HiMedia) as per manufacturer’s guidelines. Strains were grown to exponential
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phase on TSA / TSB for physiological and biochemical characterization unless otherwise
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specified. Assimilation of different carbohydrates, nitrate reduction, enzyme production
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(gelatinase, urease, arginine dihydrolase and β-galactosidase) and acid production from various
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carbohydrates was tested by using API 20 NE, API ZYM and API 50 CHB/E test kits
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(BioMérieux) respectively, according to the manufacturer’s instructions. Catalase activity was
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tested by identifying the formation of oxygen bubbles after the addition of 3% (v/v) aqueous
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hydrogen peroxide solution. Oxidase activity was assessed by the addition of 1% oxidase test
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reagent N,N,Nl,Nl -tetramethyl-p-phenylenediamine dihydrochloride (HiMedia). Indole
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production and citrate utilization were tested with API 20 NE kits, and again confirmed during
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the IMViC tests, which were carried out in duplicates by conventional methods. Indole
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production from tryptophan was tested by adding Kovacs’ reagent into the 24 h-grown culture.
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Strains grown in MR-VP broth for 24-48 h were used for both methyl red (mixed acid
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fermentation) and Voges-Proskauer test (butanediol / acetoin production). Colour change by the
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addition of methyl red indicator and Barritt’s reagents (A and B) was observed for both the tests,
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respectively. The growth on the Simmons’ citrate agar slants was observed for their colour
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change due to the utilization of citrate. H2S production was tested on Triple Sugar Iron Agar. All
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the physiological tests were carried out in triplicates and growth was measured turbidometrically
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(OD) at 600 nm using a spectrophotometer (Spectra max plus 384) and cuvette of 1.0 cm path
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length. Growth at various concentrations of NaCl (0-10 %, w/v, at intervals of 1.0 %), different
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pH (pH 4.0-11, at intervals of 1.0 pH unit) and temperature (5-55 ºC, at intervals of 5 ºC) was
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investigated. Anaerobic growth was tested by inoculating the strains onto agar slants plugged
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with rubber Suba-Seal rubber septa followed by flushing with the inert gas argon (using needles)
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and incubation at 37˚C for 24 to 48 h.
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Antibiotic susceptibility for both the strains IG-V01T and IG-V01b was tested in duplicates by
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disc diffusion method on TSA at 37˚C with filter paper discs (6 mm diameter, HiMedia)
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containing the following antibiotics with respective concentration: cefpodoxime (10 µg),
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chloramphenicol (30 µg), vancomycin (30 µg), streptomycin (10 µg), rifampicin (5 µg),
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levofloxacin (5 µg), cetriaxone (30 µg), clindamycin (2 µg), augmentin (30 µg), amikacin (30
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µg), cefixime (5 µg), tetracycline (30 µg), co-trimoxazole (25 µg), colistin (10 µg), norfloxacin
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(10 µg), ceftriaxone (10 µg), ciprofloxacin (5 µg), cephotaxime (30 µg), centamicine (10 µg),
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curazolidone (50 µg), and amoxycillin (10 µg).
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Chemotaxonomic characterization
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For whole cell fatty acid analysis, strains were inoculated onto TSA medium (pH 7.0) and
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incubated at 28 ± 20C for 18 h. Cells were harvested and subjected to saponification,
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methylation, and extraction followed by base wash. Resulting methyl esters of fatty acids were
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analyzed by Gas Chromatography (Agilent Technologies; 7890 A) according to the rapid
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Microbial Identification System software (MIS, MIDI Inc., Newark, DE, USA; version 6.0;) and
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peaks were identified based on the RTSBA6 database (Sasser, 1990; revised-www.midi-
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inc.com). Polar lipids were extracted from freeze-dried culture in chloroform : methanol : 0.3%
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saline (1:2:0.8, v/v) as described by Bligh and Dyer (1959) considering the modifications of Card
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(1973). Lipids were separated on silica gel TLC (Kieselgel 60 F254; Merck) by two-dimensional
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chromatography using chloroform : methanol : water (65:25:4 v/v) in the first dimension and
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chloroform : methanol : acetic acid : water (80:12:15:4 v/v) in the second dimension (Tindall,
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1990). Dried plates were stained with 5% ethanolic molybdophosphoric acid for total lipids.
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Lipid functional groups were identified by using spray reagents ninhydrin (specific for amino
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groups), molybdenum blue (specific for phosphates), Dragendorff (quaternary nitrogen) or α-
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naphthol (specific for sugars) for detection of lipids.
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For MALDI-TOF-MS based ribosomal protein profiling, which was carried out as an additional
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supportive analysis, strains IG-V01T and IG-V01b along with strains of known species from
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different genera of the Enterobacteriaceae (Serratia marcescens DSM 30121T, Pantoea calida
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DSM 22759T, Citrobacter freundii DSM 30039T, Raoultella ornithinolytica DSM 7464T,
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Escherichia coli DSM 30083T, Enterobacter hormaechei DSM 12409T, Klebsiella pneumoniae
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DSM 30104T) available in our culture collection were grown on TSA plates for 24 h. Whole cells
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were subjected to protein extraction by using ethanol, formic acid and acetonitrile as per
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manufacturer’s manual of Bruker Daltonics. Extracts were analyzed for ribosomal protein
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profiles (between 2.0 kD – 20.0 kD range) by using MALDI Biotyper 3.1. Resulting ribosomal
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protein profiles were used to generate Principal Component Analysis (PCA) dendrogram to
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check the phylogenetic position of novel strains within the family Enterobacteriaceae.
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Pseudomonas oleovorans DSM 1045T was used as outgroup.
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Results and discussion
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Bacteria isolated from the gut of red-flour beetles was found to be phylogenetically related to
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bacteria of the genera Staphylococcus, Bacillus and Serratia. The 16S rRNA gene sequences of
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two strains IG-V01T (1404 bp) and IG-V01b (1363 bp) was found to share 100 %, while
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showing closest similarity (96.5 % and 96.4 %, respectively) to those of “Flavobacterium
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acidificum” LMG 8364T and Serratia nematodiphila DZ0503SBS1T followed by the members
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of the genera Pantoea and Cronobacter (Fig. S4). Furthermore, the sequence similarity of these
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two strains with other members of the family Enterobacteriacea was found to be below 96.4%.
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“Flavobacterium acidificum” LMG 8364T, the first closest match to strains IG-V01T and IG-
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V01b, was described by Steinhaus (1941) based on morphological, cultural and physiological
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characteristics and classified as a member of the family Flavobacteriaceae. Recently it was
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reported that the 16S rRNA gene sequence of the type strain “Flavobacterium acidificum” LMG
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8364T shows 99.9 % similarity with Pantoea ananatis ATCC 33244T of the family
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Enterobacteriaceae and therefore the taxonomic status of this bacterium requires revision
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(Yarza et al. 2013, http://www.bacterio.net/flavobacterium.html#acidificum). In addition to 16S
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rRNA gene, protein encoding genes rpoB and gyrB were sequenced for the comparative
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phylogenetic analysis to conform the taxonomic affiliation of strains IG-V01T and IG-V01b.
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Phylogenetic analysis of sequence similarities of protein encoding genes rpoB and gyrB revealed
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that strains IG-V01T and IG-V01b share 100 % rpoB gene and 99.1 % gyrB gene sequence
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similarity between each other and comparatively low similarity between 92 % and 87 %,
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respectively, with other members of the family. Neighborjoining phylogenetic trees based on 16S
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rRNA gene sequences (Fig. 1, S1) and protein encoding genes [rpoB (Fig. S2) and gyrB (Fig.
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S3)] clearly showed that the two strains IG-V01T and IG-V01b consistently grouped together
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with high bootstrap support within a cluster with Cronobacter sakazakii ATCC 29544T and
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Escherichia coli DSM 30083T . Phylogenetic trees generated by using maximum-parsimony and
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maximum-likelihood methods showed similar tree topology (data not included) in support of the
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delineation of IG-V01T and IG-V01 as members of a novel genus of the Enterobacteriaceae
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family.
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The novel strains are Gram-stain negative straight rods (Fig. S5), non-spore forming, facultative
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anaerobic, catalase positive, oxidase negative, and exhibited both respiratory and fermentative
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metabolism. Acid and visible gas production from the fermentation of D-glucose is observed.
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Among the 43 carbohydrates tested for acid production 17 were positive for both strains. Strain
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IG-V01T utilizes 6 of the 14 carbohydrates tested whereas IG-V01b shows positive results for 5
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and weakly positive results for 1 out of the same 14 carbohydrates tested (Table 1). Both strains
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are susceptible to all tested antibiotics (as indicated in Material and Methods), grow at a broad
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range of temperature (15-50 ˚C, optimum 35-37˚C), pH (5-10.0, optimum 8.0) and salinity (0-5.0
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% (w/v NaCl), optimum 0.05 %). The genomic relatedness of two new isolates was found to be
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89.7 % and ∆Tm <1.0˚C, confirming that that IG-V01T and IG-V01b belong to a single species
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Wayne et al. (1987).
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To support the distinct taxonomic standing of two novel isolates, several phenotypic
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characteristics of the strains IG-V01T and IG-V01 were compared with those of type species of
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twelve phylogenetically close genera of the family Enterobacteriaceae (Table 1 and 2). Both the
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strains could be distinguished from other closely related members with respect to their inability
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to reduce nitrates (differed from Serratia nematodiphila DZ0503SBS1T and Klebsiella
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pneumoniae DSM 30104T), to produce gelatinase (differed from Serratia nematodiphila
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DZ0503SBS1T, Serratia marcescens DSM 30121T, Pantoea agglomerans DSM 3493T and
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Enterobacter cloacae DSM 30054T), tryptophanase (differed from Leclercia adecarboxylata
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DSM 30081T and Escherichia coli DSM 30083T), urease (differed from Raoultella planticola
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DSM 3069T and K. pneumoniae DSM 30104T), hydrogen sulfide (differed from C. freundii DSM
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30039T), citritase and acetoin (differed from S. nematodiphila DZ0503SBS1T, S. marcescens
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DSM 30121T, C. sakazakii ATCC 29544T, P. agglomerans DSM 3493T, R. planticola DSM
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3069T, K. 11neumonia DSM 30104T, E. cloacae DSM 30054T and Tatumella ptyseos LMG
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7888T). Production of arginine dihydrolase is not observed in the novel isolates, but detected in
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S. nematodiphila DZ0503SBS1T, C. sakazakii ATCC 29544T, E. cloacae DSM 30054T and
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Rosenbergiella necterea DSM 24150T. Methyl Red (MR) test was positive for both the isolates
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indicating mixed acid fermentation which could not be observed with close relatives S.
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nematodiphila DZ0503SBS1T, S. marcescens DSM 30121T, C. sakazakii ATCC 29544T and T.
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ptyseos LMG 7888T, whereas variable in P. agglomerans DSM 3493T (Table 1). The novel
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strains are capable of producing acids from D-xylose which could not be observed in their
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closest relatives S. nematodiphila DZ0503SBS1T and S. marcescens DSM 30121T. Whereas, D-
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arabinose, inositol, lactose, cellobiose, D-arabitol, melibiose, which are utilized by most of the
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closest phylogenetic neighbors, are not utilized by strains IG-V01T and IG-V01b (Table 1).
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Differences in the utilization of carbohydrates between IG-V01T and IG-V01b and the type
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strains of phylogenetically closest members of the family Enterobacteriaceae are listed in Table
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1. Two strains from the present study are able to grow at temperatures above 45˚C, in contrast to
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any other phylogenetically close neighbors included in Table 1. In contrast to strains IG-V01T
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and IG-V01b, S. nematodiphila DZ0503SBS1T, P. agglomerans DSM 3493T, R. planticola DSM
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3069T, L. adecarboxylata DSM 30081T and R. necterea DSM 24150T grow at 5˚C (Table 1). The
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genomic G+C content (mol %) of the novel strains ranges from 51.5 to 51.7 %, the values, which
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are substantially low in comparison to those of the phylogenetically closest neighbors (Table 1).
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Fatty acids of the novel strains comprise C16:0 (38.3 %), C17:0 cyclo (19.5-20.4 %), C14:0 (11.2-
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11.3 %), C19:0 cyclo ω8c (8.3-8.7 %), Sum In Feature 2 (8.1 %), Sum In Feature 8 (7.1-7.4 %)
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and trace amount of Sum In Feature 3 (2.7-3.1 %) and C12:0 (1.1 %) and this profile appeared to
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be different from those of other genera (Table 2). For example, fatty acids, C16:0, C17:0 cyclo and
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C14:0, are the major fatty acids and present in relatively high proportion amongst all other closest
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members of the family Enterobacteriaceae, except L. adecarboxylata DSM 30081T (for C17:0
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cyclo). Sum In Feature 8 and Sum In Feature 3, which are the major fatty acids of most of the
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closest members, except S. nematodiphila DZ0503SBS1T, were found to be the minor fatty acids
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of strain IG-V01T. Phosphatidylethanolamine (PE) and diphosphatidylglycerol (DPG) were
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identified as major polar lipids in both the strains (Fig. S6). An unidentified aminolipid (UAL)
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was also observed as a minor polar lipid in IG-V01T which could not found in IG-V01b.
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Through MALDI-TOF-MS based ribosomal protein profiling, it is shown both strains exhibit a
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highly similar pattern of strains IG-V01T and IG-V01b confirming their close relatedness, it was
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further found that strains IG-V01T and IG-V01b exhibit peaks (2.0 – 20.0 KDa m/z) at 3919
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(3918), 4351 (4350), 5296 (5295), 6244 (6257), 7141 (7024) and 9566 (9567) representing major
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ribosomal proteins [Fig. S7 (a)]. The cumulative number of qualitative and quantitative
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variations in the ribosomal protein profiles is shown in a Principle Component Analysis (PCA)
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dendrogram (Fig. S7 (b)). The PCA dendrogram generated based on ribosomal protein patterns
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of all the tested strains clearly demarcated the distant relatedness of the novel strains to all other
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investigated members of Enterobacteriaceae family. Thus, morphological, physiological,
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biochemical, chemotaxonomic, genetic, phylogenetic, and MALDI-TOF (MS) based ribosomal
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protein profiling strongly supportdistinct taxonomic standing of strains IG-V01T and IG-V01b as
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a new species of the novel genus of the family Enterobacteriaceae with the proposed name
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Enterobacillus tribolii.
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Description of Enterobacillus gen. nov.
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Enterobacillus (En.te.ro.ba.cil’lus. Gr. n. enteron, gut; N.L. masc. n. bacillus, a rod; N.L. masc.
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n. Enterobacillus, a rod from a gut).
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Cells are Gram-stain negative, non-spore forming, motile, straight rods, facultative anaerobic,
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catalase positive and oxidase negative. Negative for nitrate reduction, gelatinase, urease,
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tryptophanase, arginine dihydrolase, acetoin and H2S production. Major cellular fatty acids are
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C16:0, C17:0 cyclo and C14:0 with minor amount of C19:0 cyclo ω8c, Sum In Feature 2 and Sum In
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Feature 8. Phosphatidylethanolamine (PE) and Diphosphatidylglycerol (DPG) are major polar
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lipids. DNA G+C content (mol %) ranges from 51.5 - 51.7. Type species of the genus is
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Enterobacillus tribolii IG-V01T.
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Description of Enterobacillus tribolii sp. nov.
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Enterobacillus tribolii sp. nov. (tri.bo’li.i. N.L. gen. n. tribolii, of Tribolium, the genus name of
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T. castaneum, the red flour beetle from which the type strain has been isolated).
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Colonies are white, round to irregular, convex with smooth edges and 3.0-3.5 mm in diameter
302
when grown on TSA for 48 h at 37˚C. Optimum growth at 35-37˚C, pH 8.0 and 0.05 % NaCl.
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Do not produce gas from the following carbohydrates tested: Cellobiose, D-arabinose, D-
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arabitol, D-fucose, dulcitol, inositol, lactose, L-arabitol, L-fucose, melibiose, melizitose, methyl
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β-D-xyloside, raffinose, xylitol and D-xylose. Type strain utilize D-fructose, D-xylose,
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gluconate, L-arabinose, L-rhamnose, malate, gluconate and could not utilize most of the
307
carbohydrates tested by 50CHB/E (Table 1). PE and DPG are major phospholipids. Sensitive to
308
all 22 antibiotics tested (listed in methodology). Except methyl red, strains are negative for
309
remaining IMViC tests such as Indole, Voges-Proskauer and citrate utilization. Genomic DNA
310
G+C content (mol %) 51.7.
311
The type strain IG-V01T (=KCTC 42159T =MCC 2532T) and one additional strain IG-V01b were
312
isolated from the gut of red-flour beetles Tribolium castaneum.
313
Acknowledgements
314
This work was supported by the Department of Biotechnology (DBT), Government of India
315
under the project ‘‘Establishment of Microbial Culture Collection’’ (grant no.
316
BT/PR/0054/NDB/52/94/2007). We acknowledge Dr. Neetha Joseph and Prachi Koradi for
317
FAME analysis and assistance in biochemical tests respectively. Dr. Praveen Rahi is
318
acknowledged for ribosomal protein profiling by MALDI-TOF (MS). Mr. Dhananjay P. Patil is
319
acknowledged for his assistance during initial isolation. We thank Dr. Bernhand Schink for
14
320
etymology of the novel genus and species. Dr. Tapan Chakrabarti is acknowledged for valuable
321
suggestions in the discussion section and etymology.
322
323
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324
325
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451
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452
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453
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Figure Legends
457
458
Fig. 1. A neighbor-joining phylogenetic tree based on 16S rRNA gene sequences depicting the
459
phylogenetic relatedness of strains IG-V01T and IG-V01b and other species of the
460
phylogenetically related genera of the family Enterobacteriaceae. Bootstrap values (>50 %)
461
obtained from 1000 replicates are expressed as percentages at branching nodes. Morganella
462
morganii is included as an out group. Bar, 0.005 substitutions per site
463
464
Fig. S1. A neighbor-joining phylogenetic tree based on 16S rRNA gene sequences of the species
465
of the family Enterobacteriaceae. Bootstrap values (>50 %) obtained from 1000 replicates are
19
466
expressed as percentages at branching nodes. Thorsellia anophelis is included as an out group.
467
Bar, 0.01 substitutions per site.
468
469
Fig. S2. A neighbor-joining phylogenetic tree based on rpoB gene sequences showing the
470
relatedness of IG-V01T and IG-V01b with most of the species of the family Enterobacteriaceae.
471
Bootstrap values (>50 %) obtained from 1000 replicates are expressed as percentages at
472
branching nodes. Thorsellia anophelis is included as outgroup. Bar, 0.05 substitutions per site.
473
474
Fig. S3. A neighbor-joining phylogenetic tree based on gyrB gene sequences showing the
475
relatedness of IG-V01T and IG-V01b with most of the species of the family Enterobacteriaceae.
476
Bootstrap values (>50 %) obtained from 1000 replicates are expressed as percentages at
477
branching nodes. Thorsellia anophelis is included as outgroup. Bar, 0.05 substitutions per site.
478
479
Fig. S4. A neighbor-joining phylogenetic tree based on 16S rRNA gene sequences depicting the
480
phylogenetic affiliation of “Flavobacterium acidificum” (which is wrongly classified under the
481
family Enterobacteriaceae), and novel strains IG-V01T, IG-V01b within the family
482
Enterobacteriaceae. Bootstrap values (>50 %) obtained from 1000 replicates are expressed as
483
percentages at branching nodes. Bar, 0.05 substitutions per site.
484
485
Fig. S5. Phase-contrast microphotograph of cells of strains IG-V01T (IG-V01b) showing cell
486
morphology. Bar, 5 µm.
487
20
488
Fig. S6. Two-dimensional thin-layer chromatogram of whole cell lipid extracts of strains IG-
489
V01T (IG-V01b). The first dimension was developed in CHCl3:CH3OH:H2O (65:25:4 by volume)
490
and the second in CHCl3:CH3OH:CH3COOH:H2O (80:12:15:4 by volume). PE,
491
phosphatidylethanolamine; DPG, diphosphatidylglycerol; UAL, unidentified aminolipid.
492
493
Fig. S7 (a). Ribosomal protein profiles of IG-V01T, IG-V01b and other phylogenetically close
494
members of the family Enterobacteriaceae generated by MALDI-TOF (MS).
495
496
Fig. S7 (b). PCA (principal component analysis) dendrogram generated from the ribosomal
497
protein profiles (by MALDI-TOF-MS) showing the relatedness of strains IG-V01T and IG-V01b
498
with other closely related members of different genera of the family Enterobacteriaceae.
499
500
Supplementary Table 1. Pairwise similarity of 16S rRNA gene sequences of all the type strains
501
used for phylogenetic analysis in Fig. S1. The table was made by using an alignment file of 16S
502
rRNA gene sequences.
503
504
Supplementary Table 2. Pairwise similarity of rpoB gene sequences of all the type strains used
505
for phylogenetic analysis in Fig. S2. The table was made by using an alignment file of rpoB gene
506
sequences.
507
508
21
509
Supplementary Table 3. Pairwise similarity of gyrB gene sequences of all the type strains used
510
for phylogenetic analysis in Fig. S3. The table was made by using an alignment file of gyrB gene
511
sequences.
512
513
514
515
22
516
Table 1. Phenotypic characteristics differentiating Enterobacillus tribolii IG-V01T (n=2) from its
517
closest phylogenetic relatives within the family Enterobacteriaceae
518
α
519
DZ0503SBS1T; 3, Serratia marcescens DSM 30121T; 4, Cronobacter sakazakii ATCC 29544T; 5,
520
Pantoea agglomerans DSM 3493T; 6, Raoultella planticola DSM 3069T; 7, Klebsiella pneumoniae DSM
521
30104T; 8, Enterobacter cloacae DSM 30054T; 9, Citrobacter freundii DSM 30039T; 10, Leclercia
522
adecarboxylata DSM 30081T; 11, Escherichia coli DSM 30083T; 12, Tatumella ptyseos LMG 7888T; 13,
523
Rosenbergiella necterea DSM 24150T.
524
Data of type strain from the present study (column 1, 7, 9 and 11); Zhang et al. 2009 (2); Iversen et al.
525
2008 (4); Kageyama et al. 1992 (5); Gavini et al. 1989 and Drancourt et al. 2001 (6); Werkman and Gillen
526
1932, Kampfer et al. 2005, Hormaeche and Edwards 1960 (8); Tamura et al. 1986 (10); Hollis et al. 1981
527
and Brady et al. 2010 (12); Grimont and Grimont 2006 (3); Brenner and Farmer 2005 (4 -12); Malka et al.
528
2013 (3-13).
1, Enterobacillus tribolii IG-V01T (n=2), (Data from present study); 2, Serratia nematodiphila
529
530
α
531
strains compared unless otherwise mentioned. ¥red pigment; #At 22 ˚C; *at 25 ˚C (Hollis et al. 1981);
532
+, positive; -, negative; v, variable (reaction for the type strain in parenthesis); ++, > 90 % positive
533
reactions; --, <10 % positive reactions; [+], week positive; x, not determined. All the strains produce acids
534
from D-mannose and D-glucose.
All the phenotypic characterizations were carried out under the same conditions used for all
535
536
537
538
539
23
§
1
-
2
+¥
3
+¥
4
+
5
+
6
--
7
-
8
+
9
-
10
+
11
-
12
+
+
+
v[+]
+
+
+
+
+
-x
-x
x
++
++
-++
x
+
--++
++
++
-++
++
+
+
+
+
+
+
+
+
+
+
+
+
+
v(-)
v(-)
+
+
+
x
+
+
+
v
+
+
+
+
+
+
+
v(-)
+
+
x
+
+
+
+
+
+
v(+)
v(+)
v(+)
++
++
v
++
++
++
++
++
++
++
++
++
+
+
+
v
x
+
v
+
+
+
+
+
v
+
+
+
v
v
v
+
+
+
+
+
v
+
+
+
++
v
+
-+
+
+
+
v
+
+
+
+
v
++
v
++
++
++
v
v
v
++
+
v(+)
v(+)
+
+
+
x
x
+
[+]
-
Nitrate reduction
Gelatinase
Urease
Tryptophanase
MethylRed
Acetoin production
(Voges-Proskauer )
H2S production
+
-
+
+
+
+
+
x
v
+
+
v
+
+
+
+
v(+)
-#
v(+)
-v(+)
x
v(+)
v
-+
v
+
-
v(-)
+
+
-
+
+
-
+
x
+
-
-
-
-
-
-
-
-
+
-
-
-
-
Arginine dihydrolase
Citritase
Utilization of carbohydrates
-
+
+
+
+
+
+
+
v(+)
v(+)
+
v(-)
-
-
v(-)
+*
+
v(-)
Characteristics
Yellow pigment production
Acid from
D-arabinose
Inositol
Lactose
Cellobiose
D-arabitol
Dulcitol
D-xylose
Glycerol
L-arabinose
Maltose
Mannitol
Melibiose
Raffinose
Salicine
Trehalose
Enzymes
24
13
+
Adonitol
Citrate
Dulcitol
Glucose
Erythritol
Lactose
L-Arabinose
L-Rhamnose
Melezitose
Melibiose
Growth at
5˚C
10˚C
45˚C
50˚C
G+C content (mol %)
+
[+]
+
-
+
+
x
+
+
+
x
x
+
+
+
+
+
v
-_
+
v
+
x
+
+
+
+
+
+
+
+
-
+
+
x
+
+
+
+
+
+
x
x
+
+
+
+
x
v
+
+
x
+
v
v
+
x
v
+
+
x
+
+
+
+
+
+
+
+
-v
+
x
+
+
v (+)
x
v
+
x
+
x
x
+
x
x
+
x
+
x
x
+
+
+
+
+
-
x
x
x
+
+
-
+
+
-
+
+
-
+
x
x
x
x
x
x
x
x
x
+
x
-
+
+
-
x
x
-
+
+
-
51.7
59.5
57.5-60
57
55.6
53.9
57
54.7
51.0
50.8
53
46.8
540
541
542
543
544
545
25
52.454.8
546
Table 2. Cellular fatty acid profile of Enterobacillus tribolii IG-V01T and type species of phylogenetically closest genera.
547
1, Enterobacillus tribolii IG-V01T; 2, Serratia nematodiphila DZ0503SBS1T; 3, Serratia marcescens DSM 30121T; 4, Cronobacter sakazakii
548
ATCC 29544T; 5, Pantoea agglomerans DSM 3493T; 6, Raoultella planticola DSM 3069T; 7, Klebsiella pneumoniae DSM 30104T; 8,
549
Enterobacter cloacae DSM 30054T; 9, Citrobacter freundii DSM 30039T; 10, Leclercia adecarboxylata DSM 30081T; 11, Escherichia coli DSM
550
30083T; 12, Tatumella ptyseos LMG 7888T; 13, Rosenbergiella necterea DSM 24150T.
551
Data from present study (1); Zhang et al. 2009 (2); Kimura et al. 2014 (6); Gu et al. 2014 (10); Malka et al. 2013 (13); http://www.ccug.se/ (3-
552
5,7-9,11 and 12). €iso-C 16:1 and/or C14:0 3-OH; γC16:1 ω7c / C16:1 ω6c; £C18:1 ω6c / C18:1 ω7c; §fatty acids >10% are given. ^Fatty acids
553
of type strain is given in the table.
γ
1^
1.1
11.2
8.1
38.3
2.7
2
2.4
8.4
1
7.5
34.7
< 1.0
3
1.9
7.9
2.2
8.8
31.8
27
4
1.8
9.9
8
28.9
20.9
5
3.9
6
8.4
33.1
29.3
6
3
5.5
3
7.8
21.5
2.4
21.4
7
2.3
10.4
1.7
0.7
7.9
27.2
1.1
13.9
8
2.7
6.3
2.9
0.8
6
28.8
2.3
19.1
9
3.6
6.8
6.8
26
0.8
29.9
10
4.3
6.6
6.9
29.2
3.0
7.6
11
2.7
8.4
2.3
6.2
35.8
1.8
7.8
12
4.4
7.9
0.8
7
30.1
0.8
30
13
33.4
17.1
£
20.4
7.1
20
1.6
2.7
15.1
1.5
28.1
9.7
9.6
6.7
24.8
11.6
19.9
8
19.9
2.5
18.1
20.8
18.1
13.8
17.6
4.8
12.6
14
13.8
8.7
17.2
1.9
0.4
1.8
3.9
3
2.4
2.7
-
0.8
0.5
3.6
0.8
-
Fatty acids
C12:0
C14:0
C15:0
C14:0 2-OH
€
Sum In Feature 2
C16:0
C17:0
Sum In Feature 3
C17:0 cyclo
Sum In Feature 8
C18:0
C19:0 cyclo ω8c
554
555
26
§
556
27
557
Fig. 1
60
558
T
Klebsiella pneumoniae DSM 30104 (X87276)
T
Enterobacter cloacae ATCC 13047 (AJ251469)
T
Leclercia adecarboxylata GTC 1267 (AB273740)
T
50
Citrobacter freundii DSM 30039 (AJ233408)
T
Raoultella planticola DSM 3069 (X93215)
T
62
Serratia marcescens DSM 30121 (AJ233431)
100 Serratia nematodiphila DZ0503SBS1T (EU036987)
T
Tatumella ptyseos LMG7888 (EU344770)
92
50
T
Pantoea agglomerans strain DSM 3493 (AJ233423)
T
100 Enterobacillus tribolii IG-V01 (HG972968)
Enterobacillus tribolii IG-V01b (LK934679)
T
Cronobacter sakazakii ATCC 29544 (EF088379)
54
T
Escherichia coli ATCC 11775 (X80725)
T
Rosenbergiella nectarea strain 8N4 (HQ284827)
T
Morganella morganii CIPA231 (AJ301681)
0.005
28