IJSEM Papers in Press. Published August 20, 2014 as doi:10.1099/ijs.0.068205-0

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Genome-based reclassification of Bacillus cibi as a latter heterotypic synonym of

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Bacillus indicus and emended description of Bacillus indicus.

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Running Title: Reclassification of Bacillus cibi as Bacillus indicus.

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Contents category: New Taxa (Firmicutes)

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Samantha J. Stropko†, Shannon E. Pipes† and Jeffrey D. Newman

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Correspondence: Jeffrey D. Newman, [email protected] ,

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phone: 570-321-4386, fax: 570-321-4073 Biology Department, Lycoming College, Williamsport PA 17701 USA

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The Whole Genome Shotgun projects of Bacillus indicus LMG 22858T and B. cibi DSM

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16189T have been deposited at DDBJ/EMBL/GenBank under the accessions

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JGVU00000000 and JNVC00000000, respectively. The versions described in this paper

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are versions JGVU02000000 and JNVC02000000.

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The online version of this paper includes the following supplemental data: Fig. S1. Dot

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plot of B.indicus LMG 22858T vs B.cibi DSM 16189T. Table S1. B. indicus LMG 22058T

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genes not present in B. cibi DSM 16189T. Table S2. B. cibi DSM 16189T genes not

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present in B. indicus LMG 22058T. Fig. S2. Pigmentation of B.indicus and B.cibi.

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These authors contributed equally to this work.

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Abstract While characterizing a related strain, it was noted that there was little difference

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between the 16S rRNA sequences of Bacillus indicus LMG 22858T and Bacillus cibi

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DSM 16189T. Phenotypic characterization revealed differences only in the utilization of

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mannose and galactose and slight variation in pigmentation. Whole genome shotgun

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sequencing and comparative genomics were used to calculate established

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phylogenomic metrics and explain phenotypic differences. The full, genome-derived

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16S rRNA sequences were 99.74% similar. The average nucleotide identity (ANI) of the

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two strains was 98.0%, the average amino acid identity (AAI) was 98.3%, and the

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estimated DNA-DNA hybridization (DDH) determined by the genome-genome distance

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calculator (GGDC) was 80.3%. These values are higher than the species thresholds for

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these metrics, which are 95%, 95%, and 70% respectively, suggesting that these two

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strains should be classified as members of the same species.

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Bacillus indicus Sd/3T was isolated from arsenic-contaminated aquifer sand and

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described by Suresh et al. (2004). Bacillus cibi JG-30T was isolated from the Korean

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fermented seafood dish jeotgal, and published in 2005 (Yoon et al.). While Yoon et al.

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used information about reference strains and cited Suresh et al., 2004, a comparison to

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the B. indicus Sd/3T 16S rRNA was not reported, and B. indicus Sd/3T was not used as

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a reference strain despite tight clustering of the original DNA sequences on the

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neighbor-joining tree (Fig. 1).

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While investigating environmental unknown organisms isolated from a freshwater

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creek in the undergraduate microbiology course at Lycoming College (Newman, 2000;

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Strahan et al., 2011; Kirk et al., 2013), strain SJS was found to be closely related to B.

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indicus and B. cibi. The type strains B. indicus LMG 22858T and B. cibi DSM 16189T

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were obtained from BCCM and DSMZ, respectively, characterized phenotypically and

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their genomes were sequenced.

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Genomic DNA was isolated from B. indicus LMG 22858T and B. cibi DSM 16189T

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using the Qiagen DNeasy Blood and Tissue Kit according to the manufacturer’s

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instructions for Gram positive bacteria. Libraries were prepared and then sequenced on

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an Illumina MiSeq (V3 2x300 base) by the Indiana University Center for Genome

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Studies as part of a Genome Consortium for Active Teaching NextGen Sequencing

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Group (GCAT-SEEK) shared run (Buonaccorsi et al., 2011, 2014). Sequencing reads

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were filtered (median phred score>20), trimmed (phred score >16), and assembled

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using the paired-end de novo assembly option in NextGENe V2.3.4.2 (SoftGenetics).

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The assembled genomes were uploaded to the Rapid Annotation with Subsystem

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Technology web service (Aziz et al., 2008; Overbeek et al., 2014) for analysis, guided

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contig reordering, and assembly improvement.

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Approximately 0.9 million reads from the B. indicus LMG 22858T genome were

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assembled into 22 contigs with a total genome length of 4,129,127 bp, an average

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coverage of 52x and a G+C content of 44.4 mol%, significantly higher than the 41.2%

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reported by Suresh et al., (2004). Automated annotation identified 4285 potential protein

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coding sequences and 83 RNAs.

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Approximately 1.4 million B. cibi DSM 16189T reads were assembled into 24 contigs with a total genome length of 4,072,175 bp, an average coverage of 71x and a

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G+C content of 44.4 mol%, in good agreement with the 45% reported by Yoon et al.,

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2005). Automated annotation identified 4253 potential protein coding sequences and 85

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RNAs. Contigs were ordered and oriented to maximize synteny of the 3964 shared

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coding sequences (Supplementary Figure S1).

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The 16S rRNA sequences obtained from the sequenced genomes were

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compared to the partial sequences reported at the time of initial publication using the

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pairwise alignment (Myers and Miller, 1988) feature implemented on the EzTaxon-e

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web-based service (Kim et al., 2012). The Bacillus indicus LMG 22858T genome-

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derived 16S rRNA sequence was identical to the Bacillus indicus Sd/3T 16s rRNA partial

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sequence (AJ583158) (Suresh et al., 2004) except for 2 differences and an insertion

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within the first 14 bases of the partial rRNA sequence, which apparently did not have

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the amplification primer sequences trimmed. Because the genome-derived sequence is

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present within a large contig as part of a complete rRNA operon with the 23S and 5S

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rRNA genes, it is likely to be the correct sequence. There was one difference between

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the B. cibi DSM 16189T genome-derived 16S rRNA sequence and the B. cibi JG-30T

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partial sequence (AY550276) (Yoon et al., 2005). The two genome-derived sequences

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differed at four of 1533 bases for a similarity of 99.74%. The four differences

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corresponded to two pairs of substitutions that maintained base-pairing within stems.

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Several phylogenomic metrics have been developed to measure genome

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similarity for taxonomic determinations. The traditional metric of DNA-DNA hybridization

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(DDH) (Tindall et al., 2010) has been criticized due to technical difficulty, lack of

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reproducibility, and inability to incorporate into databases. Meier-Kolthoff et al. (2013)

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developed the Genome-Genome Distance Calculator (GGDC) to estimate the DNA-

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DNA hybridization value based on high scoring segment pairs (HSPs) from genome

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sequence comparisons. The estimated DDH between the B. cibi DSM 16189T and B.

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indicus LMG 22858T genomes was 80.3% (95% confidence interval 78.3-82.3%), which

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exceeds the 70% DDH species boundary.

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Average Nucleotide Identity (ANI) reflects the similarity of 1 kb sequence

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fragments using the algorithm described by Goris et al., (2007). The implementation on

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the ExGenome Web service (http://www.ezbiocloud.net/ezgenome/ani) (Kim et al.,

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2012) yielded a value of 97.8%, while the Kostas Lab implementation (http://enve-

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omics.ce.gatech.edu/ani/ ) calculated the value as 98.24%. In both cases, this is higher

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than the 94-96% threshold that correlates to the demarcation of separate species

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(Konstantinidis & Tiedje, 2005a, Richter & Rosselló-Móra, 2009).

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Average Amino Acid Identity (AAI) reflects the similarity of orthologous proteins.

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Because amino acid sequences change more slowly than nucleotide sequences and

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have greater complexity, AAI values are more sensitive over greater evolutionary

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distances, yet can still be used to distinguish organisms at the species level as well

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(Konstantinidis & Tiedje 2005b). Based on the bidirectional best hits (3964 cds)

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identified by the sequence-based comparison tool of the SEED viewer (Overbeek et al.,

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2005), AAI was calculated as 98.33% with the web-based NewmanLab AAI calculator

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(http://lycofs01.lycoming.edu/~newman/aai/). Again, this is greater than the 95%

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threshold that correlates to separate species.

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During phenotypic characterization as described by Kirk et al. (2013), several

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discrepancies with published results were noted. Motility during wet mounts was

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observed for both strains, which each have more than 80 genes for motility and

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chemotaxis, however, B. indicus was originally described as non-motile (Suresh et al.,

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2004). Both organisms grew at 4%, 6%, and 8% NaCl on TSA plates and at 4% and 8%

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NaCl in Biolog GenIII plates, though B. indicus was originally reported as negative for

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growth under these moderately high NaCl concentrations. Growth at 8% NaCl and

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motility were also observed for both strains by Khaneja et al. (2010). Neither organism

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grew at 44oC, in contrast to the positive growth result reported by Yoon et al. (2005) at

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this temperature. Khaneja et al. (2010) also observed that both strains show resistance

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to arsenic and arsenate, though B. cibi DSM 16189T exhibited slightly lower maximum

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tolerated concentrations.

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While examining the utilization of carbon sources on the Biolog GenIII plates,

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both organisms yielded a strong positive response in the negative control well when

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using Biolog Inoculating Fluid A, suggesting that they contained stored carbon sources.

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Consistent with this hypothesis, both organisms contained a glycogen synthesis and

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utilization operon. A weaker response in the negative control well was observed with

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inoculating fluid B which allowed the assessment of exogenous carbohydrate utilization

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abilities. The primary nutritional differences between the two strains were that B. indicus

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LMG 22858T utilized mannose, but not galactose, while B. cibi DSM 16189T utilized

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galactose but not mannose. Examination of the genome sequences revealed the

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existence of a 55 kb region with a mannose utilization operon that was present in B.

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indicus LMG 22858T but absent in B. cibi DSM 16189T(Supplemental Table S1). A likely

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explanation for the lack of galactose utilization by B. indicus LMG 22858T is a 1 base

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deletion near the 3’ end of the Galactose-1-phosphate uridylyltransferase gene that

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results in an apparently non-functional in-frame fusion with the adjacent aldose-1-

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epimerase gene. While these organisms shared approximately 93% of their genomes,

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B. indicus LMG 22858T contained 321 unique coding sequences, including a 34 kb

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prophage (Table S1). B. cibi DSM 16189T contained 282 unique coding sequences

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(Table S2).

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All other standard phenotypes tested and listed in the emended species

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description below were identical to each other and to previously reported results except

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for β-glucosidase activity on the API ZYM test strip (Biomerieux) which was present in

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B. indicus LMG 22858T but not in B. cibi DSM 16189T.

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Fatty acid profiles were generated by Gas-Liquid Chromatography (MIDI

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Sherlock version 6.1, method RTSBA6) using cells grown on tryptic soy broth agar

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(TSBA) for 24 hr at 30oC and extracted using the standard method (Sasser, 1990). The

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fatty acid composition (Table 1) was essentially identical and the most abundant fatty

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acids were iso-C15:0 , and anteiso-C15:0.

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While colonies of B. indicus LMG 22858T and B. cibi DSM 16189T grown on

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TSBA at 30oC for 24 hr appear similar, the pigmentation of B. cibi DSM 16189T colonies

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is darker by 72 hr (Fig S2a). To extract the pigments, 5-10 mg of cell mass was

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scraped from a plate, spread around the inside of a 1.5 mL microcentrifuge tube, which

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was incubated in the shaker-incubator at 250 rpm and 28oC with 1 mL acetone for 1 hr.

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The acetone extract was decanted into a 2 mL sample vial and analyzed on an Agilent

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1200 HPLC with an Agilent Zorbax Eclipse XDB C18 column (4.6 mm x 250 mm) at

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40oC and a diode array detector. Separation was achieved using 50 mM NaH2PO4 (pH

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4.5) as Solvent A; methanol containing 0.l% v/v glacial acetic acid as Solvent B; a flow

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rate of 1 ml min-1; and the following gradient: 0-5 minutes at 0%; increase to 75%

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solvent B at 10 min; increase to 100% solvent B at 30 min; remain at 100% solvent B

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until 45 min. The elution profiles revealed a variety of compounds with different spectra

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that absorbed light in the visible range (Fig. S2b-c). The optimal wavelength to detect all

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of the peaks was 452 nm (Fig. 2a). There were three clusters of peaks that could be

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distinguished based on absorbance spectrum (Fig. 2b); the 29-32 min peaks had

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spectra consistent with 4,4’-diaponeurosporene, 36-40 min peaks had spectra

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consistent with 4,4’-diaponeurosporenoate, and the 33-36 min peaks had spectra

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consistent with Staphyloxanthin. Analysis of the two strains’ genomes revealed several

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operons with homologs of the Staphylococcus aureus crtOPQMN genes that are

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responsible for the synthesis of the golden pigment staphyloxanthin (Pelz et al., 2005).

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Some of the different peaks in each region are likely to be different geometric isomers

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(Melendez-Martinez et al., 2013). Both organisms contain all of the genes necessary to

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produce staphyloxanthin, but B. indicus LMG 22858T contains less of this pigment (Fig.

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2b), which is apparent in the more yellow, less golden color (Fig. S2a).

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Based on the minor phenotypic and genomic differences, it is proposed that B.

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cibi Yoon et al., 2005 should be considered as a later heterotypic synonym of B. indicus

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Suresh et al. 2004.

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Emended description of Bacillus indicus Suresh et al., 2004)

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The description is the same as that given by Suresh et al. (2004) except for the

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following traits. Cells are motile. Growth is observed on R2A and tryptic soy agars, but

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not on phenylethanol or mannitol-salt agars. Yellow-golden non-diffusible carotenoid-

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type pigments are produced that are consistent with staphyloxanthin and biosynthetic

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precursors. The pH range for growth is 6–9, and cells grow at up to 8% NaCl. Cells

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exhibit oxidase, catalase, amylase, and caseinase activities, but do not liquefy gelatin.

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On the API ZYM panel, positive for C4 esterase, C8 esterase lipase, leucine

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arylamidase (weak), α-chymotrypsin, acid phosphatase (weak), naphthol-AS-BI-

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phosphohydrolase, β-galactosidase, and α-glucosidase, and negative for alkaline

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phosphatase, C14 lipase, valine and cystine arylamidase, trypsin, α-galactosidase, β-

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glucuronidase, and N-acetyl-β-glucosaminidase, α-mannosidase, and α-fucosidase, but

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β-glucosidase varies.

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When using inoculating fluid B and the Biolog GenIII plate (27oC), positive for utilization

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of dextrin, D-maltose, D-trehalose, D-cellobiose, gentiobiose, sucrose, D-turanose,

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stachyose, D-raffinose, D-melibiose, β-methyl-D-glucoside, D-salicin

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N-acetyl-D-glucosamine, α-D-glucose, D-fructose, glycerol, gelatin, glycyl-L-proline, L-

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alanine, L-aspartic acid, L-glutamic acid, L-histidine, L-pyroglutamic acid, L-serine,

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pectin, D-gluconic acid, D-glucuronic acid, methyl pyruvate, D-lactic acid methyl ester,

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L-lactic acid, α-keto-glutaric acid, L-malic acid, Tween-40, β-hydroxy-D,L-butyric acid

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acetoacetic acid, and acetic acid; utilization of D-mannose and galactose varies;

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negative for the remaining utilization tests. Also on the GenIII plate, resistant to pH 6,

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NaCl at 1, 4 and 8%, 1% Na-lactate, LiCl, K-tellurite, aztreonam, and Na-butyrate;

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sensitive to the remaining inhibitory conditions. The DNA G+C content is 44.4 mol%.

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The Type strain is Sd/3T (=MTCC 4374T=DSM 15820T), the former Type strain of B.cibi,

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JG-30T (=KCTC 3880T=DSM 16189T), is a second strain of B. indicus.

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Acknowledgements

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This work has been funded by a Lycoming College Professional Development Grant to

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JDN and a Summer Student Research Grant to SJS. GCAT-SEEK has been supported

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by NSF Award # DBI-1248096: RCN-UBE - GCAT-SEEK: The Genome Consortium for

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Active Undergraduate Research and Teaching Using Next-Generation Sequencing.

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Table 1. Fatty acid composition (%) of B. indicus LMG 22858T and B. cibi DSM

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16189T grown on TSBA at 30oC for 24 hr.

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Strains: 1, B. indicus LMG 22858T; 2, B. cibi DSM 16189T. Fatty acids amounting to

Genome-based reclassification of Bacillus cibi as a later heterotypic synonym of Bacillus indicus and emended description of Bacillus indicus.

While characterizing a related strain, it was noted that there was little difference between the 16S rRNA gene sequences of Bacillus indicus LMG 22858...
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