Open Access

Complete genome sequence of Geobacillus thermoglucosidasius C56-YS93, a novel biomass degrader isolated from obsidian hot spring in Yellowstone National Park

Standards in Genomic Sciences201510:73

https://doi.org/10.1186/s40793-015-0031-z

Received: 8 September 2014

Accepted: 29 June 2015

Published: 5 October 2015

Abstract

Geobacillus thermoglucosidasius C56-YS93 was one of several thermophilic organisms isolated from Obsidian Hot Spring, Yellowstone National Park, Montana, USA under permit from the National Park Service. Comparison of 16 S rRNA sequences confirmed the classification of the strain as a G. thermoglucosidasius species. The genome was sequenced, assembled, and annotated by the DOE Joint Genome Institute and deposited at the NCBI in December 2011 (CP002835). The genome of G. thermoglucosidasius C56-YS93 consists of one circular chromosome of 3,893,306 bp and two circular plasmids of 80,849 and 19,638 bp and an average G + C content of 43.93 %. G. thermoglucosidasius C56-YS93 possesses a xylan degradation cluster not found in the other G. thermoglucosidasius sequenced strains. This cluster appears to be related to the xylan degradation cluster found in G. stearothermophilus. G. thermoglucosidasius C56-YS93 possesses two plasmids not found in the other two strains. One plasmid contains a novel gene cluster coding for proteins involved in proline degradation and metabolism, the other contains a collection of mostly hypothetical proteins.

Keywords

Geobacillus thermoglucosidasius C56-YS93Hot springsBiomassXylanProphage

Introduction

Identification of new organisms that produce biomass-degrading enzymes is of considerable interest. Commercial uses for these enzymes include paper manufacturing, brewing, biomass deconstruction and the production of animal feeds [13]. Hot springs, especially those at Yellowstone National Park, have been a source of many new organisms including Thermus aquaticus [4, 5], Thermus brockianus [6], and Acidothermus cellulolyticus [7] that possess enzymes with significant potential in biotechnological applications [8]. As part of a project in conjunction with the Great Lakes Bioenergy Research Center, Dept. of Energy, C5-6 Technologies and Lucigen Corp. isolated, characterized, and sequenced a number of new enzyme-producing aerobic organisms from Yellowstone hot springs.

Geobacillus species were the most common aerobic organisms isolated during the cultivation of most hot springs samples. Geobacillus species were originally classified as members of the genus Bacillus , but were reclassified as a separate genus based on 16S rRNA gene sequence analysis, lipid and fatty acid analysis, phenotypic characterization, and DNA–DNA hybridization experiments [9]. Geobacillus species have been isolated from a number of hostile environments including high-temperature oilfields [10], a corroded pipeline in an extremely deep well [11], African [12] and Russian [13] hot springs, marine vents [14], and the Mariana Trench [15], yet they can also be found in garden soils [16] and hay composts [17]. In many cases though, it is unclear if these isolations of Geobacillus species represent vegetative cells growing in these environments or merely spores spread from other locations [18]. The ability of Geobacillus species to thrive in varied and often hostile environments suggests that these species possess enzymes suitable for applications in hostile industrial environments. We therefor sequenced a number of these Geobacillus isolates including G. thermoglucosidasius C56-YS93 to identify new enzymes suitable for use in biomass conversion into fuels and chemicals.

Organism Information

Classification and Features

G. thermoglucosidasius C56-YS93 is one of a number of novel thermophilic species isolated from Obsidian Hot Spring, Yellowstone National Park, Montana, USA (44.6100594° latitude and −110.4388217° longitude) under a sampling permit from the National Park Service. The hot spring possesses a pH of 6.37 and a temperature range of 42–90 °C. The organism was isolated from a sample of hot spring water by enrichment and plating on YTP-2 medium (YTP-2 media contains (per liter) 2.0 g yeast extract, 2.0 g tryptone, 2.0 g sodium pyruvate, 1.0 g KCl, 2.0 g KNO3, 2.0 g Na2HPO4.7H2O, 0.1 g MgSO4, 0.03 g CaCl2, and 2.0 ml clarified tomato juice) at 70 °C. The culture is freely available from the Bacillus Genetic Stock Center (BGSC). Cultures are routinely grown on tryptic soy broth without glucose (TSB) (Difco) media and maintained on TSB agar plates. C5-6 Technologies, Lucigen, the National Park Service, and the Joint Genome Institute have placed no restrictions on the use of the culture or sequence data. G. thermoglucosidasius C56-YS93, is a gram-positive, rod-shaped facultative anaerobe, (Table 1, Additional file 1: Table S1), with optimum growth temperature of 65 °C and maximum growth temperature of 75 °C. This is similar to the optimum growth temperature reported for G. thermoglucosidasius TNO-09.020 [19], but significantly higher than reported for previously isolated strains (<60 °C) [9]. G. thermoglucosidasius C56-YS93 appears to grow as a mixture of single cells and large clumps in liquid culture (Fig. 1).
Table 1

Classification and general features of Geobacillus thermoglucosidasius C56-YS93 [48]

MIGS ID

Property

Term

Evidence codea

 

Classification

Domain Bacteria

TAS [49]

Phylum Firmicutes

TAS [9]

Class Bacilli

TAS [9]

Order Bacillales

TAS [9]

Family Bacillaceae

TAS [9]

Genus Geobacillus

TAS [9]

Species Geobacillus thermoglucosidasius

TAS [9]

Strain C56-YS93

Gram stain

Positive

IDA

Cell shape

Rods and chains of rods

IDA

Motility

Motile

IDA

Sporulation

Subterminal spores

IDA

Temperature range

55 to 75 °C

IDA

Optimum temperature

65 °C

IDA

pH range; Optimum

5.8–8.0; 7.5

IDA

Carbon source

Carbohydrate or protein

IDA

GS-6

Habitat

Hot spring

 

MIGS-6.3

Salinity

Not reported

IDA

MIGS-22

Oxygen requirement

Facultative anaerobe

IDA

MIGS-15

Biotic relationship

Free-living

IDA

MIGS-14

Pathogenicity

Non-pathogen

IDA

MIGS-4

Geographic location

Obsidian Spring, Yellowstone National Park, USA

IDA

MIGS-5

Sample collection

September 2003

IDA

MIGS-4.1

Latitude

44.6603028

TAS [50]

MIGS-4.2

Longitude

−110.865194

TAS [50]

MIGS-4.4

Altitude

2416 m

TAS [50]

aEvidence codes - IDA inferred from direct assay, TAS traceable author statement (i.e., a direct report exists in the literature), NAS non-traceable author statement (i.e., not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). These evidence codes are from http://www.geneontology.org/GO.evidence.shtml of the Gene Ontology project [51]

Fig. 1

Micrograph of Geobacillus thermoglucosidasius C56-YS93 cells showing individual cells and clumps of cells. Cells were grown in TSB plus 0.4 % glucose for 18 h. at 70 °C. A 1.0 ml aliquot was removed, centrifuged, re-suspended in 0.2 ml of sterile water, and stained using a 50 μM solution of SYTO® 9 fluorescent stain in sterile water (Molecular Probes). Dark field fluorescence microscopy was performed using a Nikon Eclipse TE2000-S epifluorescence microscope at 2000× magnification using a high-pressure Hg light source and a 500 nm emission filter

Fig. 2

Molecular phylogenetic analysis by Maximum Likelihood method as detailed in the Material and Methods section. The tree with the highest log likelihood (−3014.19) is shown. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The novel sequenced Geobacillus strains are indicated in bold. The type strains of all validly described species are included (NCBI accession numbers): G. caldoxylolyticus ATCC700356T (AF067651), G. galactosidasius CF1BT (AM408559), G. jurassicus DS1T (FN428697), G. kaustophilus NCIMB8547T (X60618), G. lituanicus N-3T (AY044055), G. stearothermophilus R-35646T (FN428694), G. subterraneus 34 T (AF276306), G. thermantarcticus DSM9572T(FR749957), G. thermocatenulatus BGSC93A1T (AY608935), G. thermodenitrificans R-35647T (FN538993), G. thermoglucosidasius BGSC95A1T (FN428685), G. thermoleovoransDSM5366T (Z26923), G. toebii BK-1T (FN428690), G. uzenensis UT (AF276304) and G. vulcani 3S-1T (AJ293805). The 16S rRNA sequence of Paenibacillus lautusJCM9073T (AB073188) was used to root the tree

A phylogenetic tree was constructed to identify the relationship of G. thermoglucosidasius C56-YS93 to other members of the Geobacillus family. The phylogeny of G. thermoglucosidasius C56-YS93 was determined using its 16S rRNA gene sequence, as well as those of the type strains of all validly described Geobacillus spp. The 16S rRNA gene sequences were aligned using MUSCLE [20], pairwise distances were estimated using the Maximum Composite Likelihood (MCL) approach, and initial trees for heuristic search were obtained automatically by applying the Neighbour-Joining method in MEGA 5 [21]. The alignment and heuristic trees were then used to infer the phylogeny using the Maximum Likelihood method based on Tamura-Nei [22]. The phylogenetic tree confirms the identification of G. thermoglucosidasius C56-YS93 as a G. thermoglucosidasius sp. (Fig. 2).

Genome sequencing information

Genome project history

G. thermoglucosidasius C56-YS93 was selected for sequencing on the basis of its biotechnological potential as part of the U.S. Department of Energy’s Genomic Science program (formerly Genomics:GTL). The genome sequence is deposited in the Genomes On Line Database [23, 24] (GOLD ID = Gc01858), and in GenBank (NCBI Reference Sequence = CP002835.1). Sequencing, finishing and annotation were performed by the DOE Joint Genome Institute (JGI). A summary of the project information and its association with MIGS identifiers is shown in Table 2.
Table 2

Project information

MIGS ID

Property

Term

MIGS 31

Finishing quality

Finished

MIGS 28

Libraries used

6 and 24 kb

MIGS 29

Sequencing platforms

454 Titanium, Illumina GAii

MIGS 31.2

Fold coverage

5.8

MIGS 30

Assemblers

Phred/Phrap/Consed

MIGS 32

Gene calling method

Prodigal, GenePRIMP

Locus tag

GEOTH

Genbank ID

CP002835.1

Genbank date of release

Dec. 1, 2011

GOLD ID

Gc01858

BIOPROJECT

PRJNA40781

MIGS 13

Project relevance

Biotechnological

 

Source material identifier

Genome

Growth conditions and genomic DNA preparation

For preparation of genomic DNA, 1 l cultures of G. thermoglucosidasius C56-YS93 were grown from a single colony in YTP-2 medium at 70 °C in flasks agitated at 200 rpm and collected by centrifugation. Culture stocks were maintained on YTP-2 agar plates grown at 70 °C. The cell concentrate was lysed using a combination of SDS and proteinase K, and genomic DNA was isolated using a phenol/chloroform extraction [25]. The genomic DNA was precipitated, and treated with RNase to remove residual contaminating RNA.

Genome sequencing and assembly

The genome of G. thermoglucosidasius C56-YS93 was sequenced at the JGI using a combination of Illumina and 454 technologies [26]. An Illumina GAii shotgun library with reads of 878 Mb, a 454 Titanium draft library with average read length of 510–525 bp bases, and a paired end 454 library with average insert size of 13 Kb were generated for this genome. All general aspects of library construction and sequencing performed at the JGI [27]. Illumina sequencing data was assembled with VELVET [28], and the consensus sequences were shredded into 1.5 kb overlapped fake reads and assembled together with the 454 data. Draft assemblies were based on 197.18 MB 454 draft data, and all of the 454 paired end data. Newbler parameters are consed -a 50–1 350 –g –m –ml 20. The initial Newbler assembly contained 54 contigs in 2 scaffolds. We converted the initial 454 assembly into a phrap assembly by making fake reads from the consensus and collecting the read pairs in the 454 paired end library. The Phred/Phrap/Consed software package was used for sequence assembly and quality assessment ([2931] in the following finishing process. Illumina data was used to correct potential base errors and increase consensus quality using a software Polisher developed at JGI (Alla Lapidus, unpublished). After the shotgun stage, reads were assembled with parallel phrap (High Performance Software, LLC). Possible mis-assemblies were corrected with gapResolution (Cliff Han, unpublished), Dupfinisher (Han, 2006), or sequencing cloned bridging PCR fragments with subcloning. Gaps between contigs were closed by editing in Consed, by PCR and by Bubble PCR primer walks. A total of 301 additional reactions and 7 shatter libraries were necessary to close gaps and to raise the quality of the finished sequence. The total number of reads used in final assembly was 190,696. The overall average error rate of the final assembly was 0.02 errors/10 kb.

Genome annotation

Genes were identified using Prodigal [32] as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline [33]. The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) nonredundant database, UniProt, TIGRFam, Pfam, PRIAM, KEGG, COG, and InterPro databases. These data sources were combined to assert a product description for each predicted protein. Non-coding genes and miscellaneous features were predicted using tRNAscan-SE [33], RNAMMer [34], Rfam [35], TMHMM [36], and signalP [36].

Genome properties

The genome of G. thermoglucosidasius C56-YS93 consists of one circular chromosome (Table 3 and Fig. 3) of 3,893,306 bp and two circular plasmids of 80,849 and 19,638 bp and an average G + C content of 43.93 % (Table 4). There are 90 tRNA genes, 27 rRNA genes and 4 “other” identified RNA genes. There are 4014 predicted protein-coding regions and 255 pseudogenes in the genome. A total of 2757 genes (66.7 %) have been assigned a predicted function while the rest have been designated as hypothetical proteins (Table 4). The numbers of genes assigned to each COG functional category are listed in Table 5. About 37 % of the annotated genes were not assigned to a COG or have an unknown function.
Table 3

Summary of genome: one chromosome and two plasmids

Label

Size (Mb)

Topology

INSDC identifier

RefSeq ID

Chromosome

3.65

Circular

CP002050.1

NC_14206.1

Plasmid 1

0.081

Circular

CP002836.1

NC_015665

Plasmid 2

0.020

Circular

CP002837.1

NC_015661

Fig. 3

Graphical circular map of the chromosome. From outside to the center: Genes on forward strand (color by COG categories) Genes on reverse strand (color by COG categories) RNA genes (tRNAs green, rRNAs red, other RNAs black) GC content, GC skew

Table 4

Genome statistics

Attribute

Value

% of Totala

Genome size (bp)

3,993,793

100

DNA coding (bp)

3,437,131

86

DNA G+C (bp)

1,754,637

44

DNA Scaffolds

3

100

Total genes

4,135

100

Protein-coding genes

4,014

97

RNA genes

121

3

Pseudo genes

255

6

Genes in internal clusters

1,984

48

Genes with function prediction

1,257

30

Genes assigned to COGs

2,607

63

Genes with Pfam domains

3,278

79

Genes with signal peptides

161

4

Genes with transmembrane helices

948

23

CRISPR repeats

6

 

aThe total is based on either the size of the genome in base pairs or the total number of protein coding genes in the annotated genome

Table 5

Number of genes associated with general COG functional categories

Code

Value

Percent

Description

J

156

5.4

Translation, ribosomal structure and biogenesis

A

0

0

RNA processing and modification

K

195

6.8

Transcription

L

208

7.2

Replication, recombination and repair

B

1

0.03

Chromatin structure and dynamics

D

30

1.0

Cell cycle control, cell division, chromosome partitioning

V

44

1.5

Defense mechanisms

T

116

4.0

Signal transduction mechanisms

M

103

3.6

Cell wall/membrane/envelope biogenesis

N

61

2.1

Cell motility

U

48

1.7

Intracellular trafficking, secretion, and vesicular transport

O

100

3.5

Posttranslational modification, protein turnover, chaperones

C

207

7.2

Energy production and conversion

G

194

6.7

Carbohydrate transport and metabolism

E

285

9.9

Amino acid transport and metabolism

F

72

2.5

Nucleotide transport and metabolism

H

132

4.6

Coenzyme transport and metabolism

I

105

3.6

Lipid transport and metabolism

P

160

5.6

Inorganic ion transport and metabolism

Q

71

2.5

Secondary metabolites biosynthesis, transport and catabolism

R

330

11.5

General function prediction only

S

261

9.1

Function unknown

 

1528

37.0

Not in COGs

The total is based on the total number of protein coding genes in the annotated genome

Insights from the genome sequence

To better understand the unique features of G. thermoglucosidasius C56-YS93, whole genome comparisons were carried out between G. thermoglucosidasius C56-YS93 and G. thermoglucosidasius M10EXG (M10EXG) and G. thermoglucosidasius TNO-09.020 (TNO-09.020) (accession number: NZ_CM001483.1) using RAST genome annotation [37] and SEED curation into subsystems [38]. (The genome sequence of M10EXG is available from the Integrated Microbial Genomes (IMG) database [27, 39].) Basic features of the three genomes are shown in Table 6. Genome comparisons revealed that C56-YS93 possessed a number of unique features.
Table 6

G. thermoglucosidasius strains used in comparisons

G. thermoglucosidasius

C56-YS93

M10EXG

TNO-09.020

Chromosome size

3,993,793

3,773,252

3,740,238

Plasmids

2

0

0

Protein coding genes

4326

4228

4164

Isolation source

Hot Spring, YNP, United States

Sydney, New South Wales, Australia

Dairy production, Netherlands

Xylan degradation cluster in G. thermoglucosidasius C56-YS93

The most significant unique feature of G. thermoglucosidasius C56-YS93 is a 26-gene cluster coding for xylan utilization not found in any G. thermoglucosidasius genome. Included in the cluster are regulatory elements, transporters, intracellular and extracellular xylanases, and enzymes involved in xylose metabolism (Table 7). Manual curation of the cluster indicates that the genes and organization of the G. thermoglucosidasius C56-YS93 xylan utilization cluster are essentially identical to those of the G. stearothermophilus cluster (Bst T-6) [40]. This identity suggests the cluster may be highly conserved within the xylanolytic geobacilli. No homologs of the corresponding G. stearothermophilus arabinan utilization [41] cluster genes are present in G. thermoglucosidasius C56-YS93, indicating G. thermoglucosidasius C56-YS93 is unable to utilize arabinan.
Table 7

Xylan degradation cluster of Geobacillus thermoglucosidasius C56-YS93

 

Annotated protein function

C56-YS93

Bst T-6

1

Integral membrane sensor signal transduction histidine kinase

Geoth_2272

xynD

2

AraC family transcriptional regulator

Geoth_2271

xynC

3

Family 1 extracellular solute-binding protein

Geoth_2270

xynE

4

Binding-protein-dependent transporters inner membrane component

Geoth_2269

xynF

5

Binding-protein-dependent transporters inner membrane component

Geoth_2268

xynG

6

Aldose 1-epimerase

Geoth_2267

araK

7

Polysaccharide deacetylase

Geoth_2266

axe1

8

Xylan 1,4-beta-xylosidase

Geoth_2265

xynB2

9

Endo-1,4-beta-xylanase

Geoth_2264

xynA2

10

Family 1 extracellular solute-binding protein

Geoth_2262

aguE

11

Binding-protein-dependent transporters inner membrane component

Geoth_2261

aguF

12

Binding-protein-dependent transporters inner membrane component

Geoth_2260

aguG

13

Alpha-glucuronidase

Geoth_2259

aguA

14

Xylan 1,4-beta-xylosidase

Geoth_2258

xynB

15

PfkB domain-containing protein

Geoth_2257

kdgK

16

2-dehydro-3-deoxyphosphogluconate aldolase/4-hydroxy-2-oxoglutarate aldolase

Geoth_2256

kgdA

17

GntR family transcriptional regulator

Geoth_2255

uxuR

18

Uronate isomerase

Geoth_2254

uxaC

19

Mannonate dehydratase

Geoth_2253

uxuA

20

Short-chain dehydrogenase/reductase SDR

Geoth_2252

uxuB

21

Hypothetical protein

Geoth_2251

orfA

22

Endo-1,4-beta-xylanase

Geoth_2250

xynA1

23

Hypothetical protein

Geoth_2247

xynX

24

G-D-S-L family lipolytic protein

Geoth_2246

axe2

25

Xylose isomerase

Geoth_2243

xylA

26

Xylulokinase

Geoth_2242

xylB

Nitrogen clusters in G. thermoglucosidasius C56-YS93

G. thermoglucosidasius C56-YS93 has a number of nitrogen utilization systems. The absence of an arabinan cluster in G. thermoglucosidasius C56-YS93 is the result of an 11-gene insert (Geoth_2276 through Geoth_2288) coding for a peptide utilization cluster that replaces part of the arabinan cluster. The cluster does not code for a secreted protease or peptidase, but contains an annotated five-gene ABC peptide transporter system and two intracellular peptidases. Downstream from the peptide utilization cluster is a 12-gene urea uptake and utilization cluster (Geoth_2301 through Geoth_2312). The organism contains clusters for reduction of nitrate to nitrite (Geoth_2197 through Geoth_2200) and reduction of nitrite to dintrogen (Geoth_3084 through Geoth_3090).

Presence of plasmids in G. thermoglucosidasius C56-YS93

While the genomes of strains TNO-09.020 and M10EXG contain no plasmids, the genome of G. thermoglucosidasius C56-YS93 includes two plasmids, one of approximately 81 Kb and one of approximately 20 Kb. The 20 Kb plasmid contains genes coding for a number of small hypothetical proteins with no identifiable function. Among the annotated proteins, the 20Kb plasmid contains an annotated P4 phage/plasmid primase with no close homologs in other Geobacillus strains (Geoth_0020) and an annotated ArpU family phage transcriptional regulator (Geoth_0016). The 20 Kb plasmid contains an annotated transcriptional modulator of MazE/toxin, MazF (Geoth_0007) that may function in maintaining the plasmid. The 80 Kb plasmid contains a gene cluster that may function for proline and hydroxyproline capture, transport and metabolism. The cluster includes two peptidases (Geoth_3970 and Geoth_3979), a transport system and hydroxyglutarate oxidase cluster (Geoth_4004 and Geoth_3999), four annotated oxoprolinases (Geoth_3972, Geoth_3973, Geoth_3984, and Geoth_3987), and a hydantoin racemase (Geoth_3976) The plasmid also contains genes coding for proteins that metabolize proline to glutamate via proline dehydrogenase. (Geoth_3968 and Geoth_3969). BLAST analysis indicates that these two proteins are not common to Geobacillus species, but appear to have been acquired from an Anoxybacillus species. In addition, the 80 kb plasmid contains genes coding for eight proteins annotated as integrase or transposon-related and annotated death-on-curing and addiction module antidote proteins (Geoth_4023 and Geoth_4024) that may function in maintaining the plasmid.

Prophage insert in G. thermoglucosidasius C56-YS93

Prophage analysis of the G. thermoglucosidasius C56-YS93 genome was performed using PHAST genome search software [42]. PHAST identified a 56 KB insert containing an intact prophage between 735,196 and 780,775 bp. The insert contains 75 genes, of which 51 are annotated as having a phage origin, 20 are annotated as hypothetical proteins and four are annotated as bacterial (Fig. 4). BLAST analysis indicates the phage proteins in the insert most closely match those of Geobacillus virus E2 (Accession: NC_009552.2) with 26 protein hits.
Fig. 4

Prophage genes identified in G. thermoglucosidasius C56-YS93 using PHAST genome search software

Conclusions

G. thermoglucosidasius species were first isolated by Suzuki and given the name Bacillus thermoglucosidasius [43]. The organisms were reclassified as Geobacillus and their name corrected to thermoglucosidasius [9]. G. thermoglucosidasius C56-YS93 is the first G. thermoglucosidasius strain from a hot spring environment for which a whole genome sequence is available. While it is possible that G. thermoglucosidasius C56-YS93 was present only as wind-blown spores in the hot spring [18], there are a number of strong arguments for the growth of this and other Geobacillus species in hot springs. The first and most compelling argument is that, in our lab, boiled samples of Obsidian hot spring water resulted in isolation of no viable organisms, either in liquid culture or by plating. If Geobacillus spores were present in a significant quantity, a significant number of isolates would be expected. Secondly, we have been able to isolate Geobacillus species only from alkaline or neutral hot springs with temperatures between 60 and 80 °C, essentially the environment in which Geobacillus species can grow. No Geobacillus species were isolated from acidic hot springs located close to the alkaline and neutral springs. The isolation of wind-borne spore cultures would predict equal numbers of Geobacillus species isolated from acidic and alkaline springs. Thirdly, in our work, Geobacillus species and Thermus species were the predominant organisms isolated from Yellowstone hot springs under aerobic conditions. Thermus species share temperature and pH optima with Geobacillus species. Thermus species do not sporulate, so the presence of Thermus species cannot be attributed to wind-blown spores, but indicates the organism is growing in the hot spring. If these hot springs support growth of Thermus species, it would be difficult to argue that the hot springs can support growth of Thermus species but cannot support growth of with Geobacillus species. Finally, Geobacillus species have been isolated from microbial mats from other hot springs in Yellowstone [44].

G. thermoglucosidasius , C56-YS93, appears to have a number of unique features as a result of its growth in the hot spring environment. The organism possesses a large xylan degradation cluster that increases the substrate range of this strain relative to the other two sequenced strains. A number of other biomass-degrading organisms have been identified in Obsidian Hot Spring [45], but this is the first reported biomass-degrading Geobacillus species from the hot spring. The organization of this cluster appears to match the glucuronic acid utilization cluster described for G. stearothermophilus [40], suggesting this cluster may be conserved in other Geobacillus species. G. thermoglucosidasius C56-YS93 possesses both chromosomal and plasmid-borne peptide utilization clusters that may allow the organism to scavenge proteins and peptides from the medium. G. thermoglucosidasius C56-YS93 also possesses the ability to reduce nitrate to dinitrogen, possibly utilizing nitrate as an alternate electron acceptor in the oxygen-poor high temperature environment. Genetic exchange with other Geobacillus species in the hot spring may be facilitated by the presence of the two plasmids not found in the other two strains. Further work is needed to identify the function of the genes present on these two plasmids and clarify the role they play in survival in the hot spring. Metagenomic analysis of samples from two other hot springs in Yellowstone National Park, Bear Paw and Octopus, shows the presence of active archaeal and bacterial phage populations [46, 47]. The prophage identified in G. thermoglucosidasius C56-YS93 (43.9 % G + C) is unrelated to the prophages identified in Geobacillus species Y412MC52 and Y412MC61 (52.3 % G + C), isolated from the same hot spring. This suggests that the identified prophage identified in G. thermoglucosidasius C56-YS93 may be specific to G. thermoglucosidasius , or to the lower G + C species. Additional work is needed to understand the relationship between Geobacillus species and the phages that infect them.

Abbreviations

IMG: 

Integrate microbial genomes database

JGI: 

Joint Genome Institute

Declarations

Acknowledgements

This work was funded by the DOE Great Lakes Bioenergy Research Center (DOE Office of Science BER DE-FC02-07ER64494). Sequencing work was performed under the auspices of the US Department of Energy’s Office of Science, Biological and Environmental Research Program, and by the University of California, Lawrence Berkeley National Laboratory under contract No. DE-AC02-05CH11231, Lawrence Livermore National Laboratory under Contract No. DE-AC52-07NA27344, and Los Alamos National Laboratory under contract No. DE-AC02-06NA25396.

Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Lucigen Corporation
(2)
Oak Ridge National Laboratory
(3)
Great Lakes Bioenergy Research Center, University of Wisconsin

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