Open Access

Genome sequence of Anoxybacillus ayderensis AB04T isolated from the Ayder hot spring in Turkey

Standards in Genomic Sciences201510:70

https://doi.org/10.1186/s40793-015-0065-2

Received: 18 March 2015

Accepted: 4 September 2015

Published: 26 September 2015

Abstract

Species of Anoxybacillus are thermophiles and, therefore, their enzymes are suitable for many biotechnological applications. Anoxybacillus ayderensis AB04T (= NCIMB 13972T = NCCB 100050T) was isolated from the Ayder hot spring in Rize, Turkey, and is one of the earliest described Anoxybacillus type strains. The present work reports the cellular features of A. ayderensis AB04T, together with a high-quality draft genome sequence and its annotation. The genome is 2,832,347 bp long (74 contigs) and contains 2,895 protein-coding sequences and 103 RNA genes including 14 rRNAs, 88 tRNAs, and 1 tmRNA. Based on the genome annotation of strain AB04T, we identified genes encoding various glycoside hydrolases that are important for carbohydrate-related industries, which we compared with those of other, sequenced Anoxybacillus spp. Insights into under-explored industrially applicable enzymes and the possible applications of strain AB04T were also described.

Keywords

Anoxybacillus Bacillaceae Bacillus Geobacillus Glycoside hydrolaseThermophile

Introduction

The family Bacillaceae [1, 2] is one of the largest bacterial families and currently consists of 57 genera [3]. The Bacillaceae are either rod-shaped (bacilli) or spherical (cocci) Gram-positive bacteria, the majority of which produce endospores [4]. Anoxybacillus [5, 6] is one of the genera within the Bacillaceae [1, 2], classified within the phylum Firmicutes [7], class Bacilli [8, 9], and order Bacillales [1, 10].

Anoxybacillus spp. are alkalo-thermophiles with optimum growth at temperatures between 50 °C and 65 °C and at pH 5.6–9.7 [4]. Most of the Anoxybacillus spp. are found in hot springs [4], but Anoxybacillus has also been found in animal manure [5], contaminated diary and meat products [4], animals (i.e., fish gut) [4], insects (i.e., glassy-winged sharpshooter and spiraling whitefly) [11], and plants (i.e., Indian mulberry) [11]. To date, a total of 22 species and two subspecies of Anoxybacillus have been described [4, 12, 13].

Almost all members of the Bacillaceae are excellent industrial enzyme producers [4, 14, 15]. Members of the genus Anoxybacillus exhibit the additional advantage of thermostability compared to the mesophilic Bacillaceae . It has been reported that enzymes from Anoxybacillus spp. can degrade various substrates such as starches, cellulose, fats, and proteins [4]. Many carbohydrase-encoding genes have been identified in Anoxybacillus spp. genomes, and some of the well-studied starch-degrading enzymes are α-amylase [16], pullulanase [17], amylopullulanase [18], CDase [19], and xylose-isomerase [20]. In addition, xylanolytic enzymes such as xylanase [21] and α-L-arabinofuranosidase [22] have been characterized from Anoxybacillus spp. Apart from their hydrolytic capabilities, Anoxybacillus spp. have been proposed as agents for bioremediation of Hg2+, Cr2+, Al3+, As3+ ions [4, 2325], and nitrogen oxide [26], and as possible candidates for biohydrogen production [4].

Among the members of the family Bacillaceae , intensive genome sequencing efforts have been undertaken for Geobacillus [27] (>80 projects) and Bacillus [1, 28] (>1,500 projects), which have been registered in the NCBI BioProject database. In contrast, genomic studies on Anoxybacillus are rather limited, with only 16 registered projects. At present, the genome of Anoxybacillus flavithermus WK1 is the only completely sequenced genome (BioProject accession number PRJNA59135) among the Anoxybacillus spp. [5, 29]. Draft genome sequences are available for Anoxybacillus ayderensis AB04T (PRJNA258494; this study) [30], Anoxybacillus sp. BCO1 (PRJNA261743) [31, 32], Anoxybacillus thermarum AF/04T (PRJNA260786) [3335], Anoxybacillus gonensis G2T (PRJNA264351) [36], Anoxybacillus sp. ATCC BAA-2555 (PRJNA260743), Anoxybacillus sp. KU2-6(11) (PRJNA258246), Anoxybacillus tepidamans PS2 (PRJNA214279) [37], A. flavithermus 25 (PRJNA258119) [5, 38], A. flavithermus AK1 (PRJNA190633) [5, 39], Anoxybacillus kamchatkensis G10 (PRJNA170961) [4042], A. flavithermus Kn10 (PRJDB1085) [5, 43], A. flavithermus TNO-09.006 (PRJNA169174) [5, 44], Anoxybacillus sp. SK3-4 (PRJNA174378) [45, 46], Anoxybacillus sp. DT3-1 (PRJNA182115) [45, 46], and A. flavithermus subsp. yunnanensis E13T (PRJNA213809) [35, 47, 48]. Therefore, the genomic study of Anoxybacillus spp. is essential not only to fully understand their biochemical networks, but also to discover their potential applicability in industrial processes.

In the present report, we describe the cellular features of A. ayderensis AB04T and we present a high-quality annotated draft genome of strain AB04T. Additionally, we provide a comparative analysis of the GHs of strain AB04T and other sequenced Anoxybacillus spp. In addition, we discuss the presence of other under-explored industrial enzymes and the potential applications of the bacterium.

Organism information

Classification and features

A. ayderensis AB04T (= NCIMB 13972T = NCCB 100050T ) was isolated from mud and water samples from the Ayder hot spring located in the province of Rize in Turkey [30]. Microscopic examination revealed that colonies of strain AB04T were cream-colored, regular in shape with round edges, and 1–2 mm in diameter.

Phenotypic analysis revealed that strain AB04T is a Gram-positive, rod-shaped, motile, and spore-forming bacterium [30]. It is a facultative anaerobe, moderate thermophile that grows well at 30–70 °C (optimum 50 °C) and at pH 6.0–11.0 (optimum pH 7.5–8.5) (Table 1). FESEM showed that cells of the strain AB04T were 0.7–0.8 × 3.5–5.0 μm in size (Fig. 1). The strain gave positive responses for catalase and oxidase activity, and was able to reduce nitrate to nitrite. Strain AB04T was capable of utilizing a wide range of carbon sources including starch, gelatin, d-glucose, d-raffinose, d-sucrose, d-xylose, d-fructose, l-arabinose, maltose, and d-mannose. The strain grew optimally in the presence of 1.5 % (w/v) NaCl, but it was able to grow in the absence of NaCl. Growth was inhibited in the presence of ampicillin (25 μg/ml), streptomycin sulphate (25 μg/ml), tetracycline (12.5 μg/ml), gentamicin (10 μg/ml), and kanamycin (10 μg/ml). The FAME profile showed that the major fatty acid in AB04T is C15:0iso (48.17 %), followed by C17:0 iso (20.62 %), C17:0 anteiso (9.22 %), C16:0 (9.10), C16:0 iso (7.47 %), C15:0 anteiso (3.58 %), C14:0 (1.02 %), and C15:0 (0.83 %) [30].
Table 1

Classification and general features of A. ayderensis AB04T [74]

MIGS ID

Property

Term

Evidence codea

 

Classification

Domain Bacteria

TAS [75]

 

Phylum Firmucutes

TAS [7]

 

Class Bacilli

TAS [8, 9]

 

Order Bacillales

TAS [1, 10]

 

Family Bacillaceae

TAS [1, 2]

 

Genus Anoxybacillus

TAS [5, 6]

 

Species Anoxybacillus ayderensis

TAS [30]

 

Type strain: AB04T (NCIMB 13972T, NCCB 100050T)

TAS [30]

 

Gram stain

Positive

TAS [30]

 

Cell shape

Rod

TAS [30]

 

Motility

Motile

TAS [30]

 

Sporulation

Terminal, spherical endospore

TAS [30]

 

Temperature range

30-70 °C

TAS [30]

 

Optimum temperature

50 °C

TAS [30]

 

pH range; Optimum

6.0-11.0; 7.5-8.5

TAS [30]

 

Carbon source

Carbohydrates

TAS [30]

MIGS-6

Habitat

Hot spring

TAS [30]

MIGS-6.3

Salinity

Optimum at 1.5 % NaCl (w/v)

TAS [30]

MIGS-22

Oxygen requirement

Facultative anaerobe

TAS [30]

MIGS-15

Biotic relationship

Free-living

TAS [30]

MIGS-14

Pathogenicity

Non-pathogenic

TAS [30]

MIGS-4

Geographic location

Ayder hot spring, Rize, Turkey

IDA

MIGS-5

Sample collection

January 1995

IDA

MIGS-4.1

Latitude

40°57’N

IDA

MIGS-4.2

Longitude

41°05’E

IDA

MIGS-4.4

Altitude

1350 m above sea level

IDA

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 the Gene Ontology project [76]

Fig. 1

FESEM micrograph of A. ayderensis AB04T. The micrograph was captured using FESEM (JEOL JSM-6701 F, Tokyo, Japan) operating at 5.0 kV at a magnification of 15,000 ×

The 16S rRNA-based phylogenetic tree constructed using MEGA6.0 [49] showed that strain AB04T clusters together with Anoxybacillus sp. SK3-4 [45, 46] and A. thermarum AF/04T [3335] (Fig. 2). Pairwise 16S rRNA sequence similarities among the strains were determined using the EzTaxon server [50], revealing that AB04T shares 99.6 % and 99.2 % similarity with Anoxybacillus sp. SK3-4 [45, 46] and A. thermarum AF/04T [3335], respectively.
Fig. 2

Phylogenetic tree based on 16S rRNA gene sequences showing the relationship between A. ayderensis AB04T and representative Anoxybacillus spp. The 16S rRNA accession number for each strain is shown in brackets. The 16S rRNA sequences were aligned using ClustalW and the tree was constructed using the ML method with 1000 bootstrap replicates embedded in the MEGA6.0 package [49]. The scale bar represents 0.01 nucleotide substitutions per position. Brevibacillus brevis NCIMB 9372T [77] was used as an out-group. Type strains are indicated with a superscript T. Published genomes are indicated in blue

Genome sequencing information

Genome project history

Genomic studies on the genus Anoxybacillus are relatively limited [45]. Hence, the findings of the genomic study on A. ayderensis AB04T presented in this study are important because they contribute to the body knowledge of the Anoxybacillus genomes. This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession number JXTG00000000. The NCBI BioProject accession number is PRJNA258494. The GOLD Project ID for strain AB04T is Gp0026071. Table 2 presents the project information and its association with MIGS version 2.0 compliance.
Table 2

Project information

MIGS ID

Property

Term

MIGS-31

Finishing quality

High-quality draft

MIGS-28

Libraries used

Illumina Paired-End library

MIGS-29

Sequencing platforms

Illumina MiSeq

MIGS-31.2

Fold coverage

239 ×

MIGS-30

Assemblers

IDBA-UD 1.0.9

MIGS-32

Gene calling method

Prodigal 2.60

 

Locus Tag

JV16

 

Genbank ID

JXTG00000000

 

Genome Data of Release

February 9, 2015

 

GOLD ID

Gp0026071

 

BIOPROJECT

PRJNA258494

MIGS-13

Source Material Identifier

NCIMB 13972T

 

Project relevance

Biotechnology

Growth conditions and genomic DNA preparation

A. ayderensis AB04T was plated on Nutrient Agar (pH 7.5) and incubated at 50 °C for 18 h. A single colony was transferred into Nutrient Broth (pH 7.5) and incubated at 50 °C with rotary shaking at 200 rpm for 18 h. The cells were harvested by centrifugation at 10,000 × g for 5 min using a Microfuge® 16 centrifuge (Beckman Coulter, Brea, CA, USA). Genomic DNA was extracted using a Qiagen DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. The purity, quality, and concentration of the genomic DNA were determined using a 6 % (w/v) agarose gel, NanoDrop 1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA), and Qubit 2.0 fluorometer (Invitrogen, Merelbeke, Belgium).

Genome sequencing and assembly

The genome of A. ayderensis AB04T was sequenced using the Illumina MiSeq sequencing platform (Illumina, San Diego, CA, USA) with 300-bp paired-end reads. The adapter sequences were removed and low quality regions and reads were filtered out using Trimmomatic [51] (Phred score = 25 (Q25), sliding window = 4 bp, leading and trailing qualities = 3, and minimum read length = 36 bp), Scythe (UC Davis Bioinformatics Core, Davis, DA, USA) (prior contamination rate = 0.3, minimum match length argument = 5, and minimum sequence to keep after trimming = 36 bp), and String Graph Assembler (SGA) [52] (k-mer threshold = 3, k-mer rounds = 10, and read error correction = 0.04). Next, the reads were subjected to de novo genome assembly using IDBA-UD 1.0.9 [53] (k min  = 35).

Genome annotation

Genes, tRNAs and tmRNAs, and rRNAs were predicted with Prodigal [54], ARAGORN [55], and RNAmmer [56], respectively. For functional annotation, the predicted coding sequences were translated and used to search for the closest matches in the NCBI non-redundant database and the UniProt [57], TIGRFAM [58], Pfam [59], CRISPRfinder [60], PRIAM [61], KEGG [62], COG [63], and InterProScan 5 [64] databases. The GHs were identified and verified using the dbCAN CAZy [65], NCBI BLASTp, and InterProScan 5 [64] databases. Genome comparison was done by the ANI function in the EzTaxon-e database [66].

Genome properties

The overall genome coverage was approximately 239-fold. The draft genome was assembled into 74 contigs with a total length of 2,832,347 bp and a G + C content of 41.8 % (Fig. 3 and Table 3). The longest and shortest contigs were 448,584 bp and 606 bp, respectively. The mean length of the contigs was 38,275 bp and the N50 contig length was 112,260 bp. We did not detect any additional DNA elements. The genome consisted of 2,998 predicted genes, of which 2,895 were protein-coding sequences and 103 were RNA genes including 14 rRNAs, 88 tRNAs, and 1 tmRNA. A total of 235 (8.1 %) genes were assigned a putative function. The remaining annotated genes (1023; 35.3 %) were hypothetical proteins. The properties and the statistics of the genome are summarized in Table 3. The distribution of genes into COGs and KEGG functional categories is presented in Table 4 and Fig. 3.
Fig. 3

A graphical circular map of the A. ayderensis AB04T genome. From outside to the center: genes on the forward strand (colored by COG categories), genes on forward strand (red), genes on reverse strand (blue) and genes on the reverse strand (colored by COG categories)

Table 3

Genome statistics

Attribute

Value

% of Totala

Genome size (bp)

2,832,347

100.00

DNA coding (bp)

2,517,744

88.89

DNA G + C (bp)

 

41.83

DNA scaffolds

74

100.00

Total genes

2,998

100.00

Protein coding genes

2,895

96.56

RNA genes

103

3.44

Pseudo genes

not determined

not determined

Genes in internal clusters

not determined

not determined

Genes with function prediction

1,637

54.60

Genes assigned to COGs

2,349

78.35

Genes with Pfam domains

2,158

71.98

Genes with signal peptides

103

3.44

Genes with transmembrane helices

674

23.28

Number of CRISPR candidates

3

 

Confirmed CRISPR(s)

1

 

Unconfirmed CRISPR(s)

2

 

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 4

Number of genes associated with general COG functional categories

Code

Value

% agea

Description

J

153

5.10

Translation, ribosomal structure and biogenesis

A

1

0.03

RNA processing and modification

K

169

5.64

Transcription

L

165

5.50

Replication, recombination and repair

B

1

0.03

Chromatin structure and dynamics

D

38

1.27

Cell cycle control, Cell division, chromosome partitioning

V

27

0.90

Defense mechanisms

T

162

5.40

Signal transduction mechanisms

M

117

3.90

Cell wall/membrane biogenesis

N

80

2.67

Cell motility

U

53

1.77

Intracellular trafficking and secretion

O

99

3.30

Posttranslational modification, protein turnover, chaperones

C

145

4.84

Energy production and conversion

G

169

5.64

Carbohydrate transport and metabolism

E

234

7.81

Amino acid transport and metabolism

F

71

2.37

Nucleotide transport and metabolism

H

120

4.00

Coenzyme transport and metabolism

I

81

2.70

Lipid transport and metabolism

P

140

4.67

Inorganic ion transport and metabolism

Q

29

0.97

Secondary metabolites biosynthesis, transport and catabolism

R

274

9.14

General function prediction only

S

261

8.71

Function unknown

-

409

13.64

Not in COGs

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

Insights from the genome sequence

Genome features of A. ayderensis AB04T and other Anoxybacillus spp

The genome sizes of the currently sequenced Anoxybacillus spp. are shown in Fig. 2. Most of the reported Anoxybacillus draft genome sizes are between 2.60 and 2.86 Mb [31, 33, 3840, 4345, 47], and the completely sequenced A. flavithermus WK1 genome has a size of 2.85 Mb [29]. The incomplete genome sequence of A. tepidamans PS2 has a size of 3.36 Mb (Fig. 2), which is the largest Anoxybacillus genome sequenced to date [37]. However, cumulative information on the Anoxybacillus genomes (Fig. 2) indicates that Anoxybacillus has a smaller genome size than the closest genus, Geobacillus (~3.50 Mb) [27, 45]. The genomes of other genera within Bacillaceae such as Bacillus [1, 28] and Lysinibacillus [67] are at least 40 % larger than that of Anoxybacillus [5, 6, 45]. The average G + C content of the Geobacillus spp. genomes (~50.0 %) [27, 45] is slightly higher than that of the A. ayderensis [30] genome (Fig. 2), while most Bacillus genomes have less than 40 % G + C content [1, 28, 45].

Table 5 summarizes the pairwise ANI values of Anoxybacillus spp. [66]. A. ayderensis AB04T showed the highest ANI of 97.6 % with Anoxybacillus sp. SK3-4 [46]. As this ANI value is greater than 95 % [68], Anoxybacillus sp. SK3-4 [45, 46] is likely to be a subspecies of A. ayderensis [30].
Table 5

Genomic comparison of A. ayderensis AB04T and 15 other sequenced Anoxybacillus spp. using ANI [66]

 

AB04T

WK1

E13T

SK3-4

DT3-1

TNO

G10

G2T

AF/04T

AK1

BCO1

KU2-6

Kn10

PS2

25

AB04T

100.00

87.9

87.3

97.6

94.5

87.3

94.3

94.3

94.7

85.7

97.5

89.6

88.2

72.4

97.6

WK1

87.9

100.00

88.4

88.0

88.1

91.8

88.2

88.2

87.8

84.8

87.7

89.8

95.0

72.5

87.5

E13T

87.3

88.3

100.00

87.3

87.2

88.3

86.9

87.1

86.9

85.2

87.1

89.9

89.1

72.3

87.1

SK3-4

97.5

88.1

87.2

100.00

94.0

87.5

93.7

93.9

94.2

85.8

96.9

89.5

88.3

72.5

96.9

DT3-1

94.6

88.0

87.2

94.1

100.00

87.0

98.5

98.6

94.1

85.3

94.4

89.8

88.0

72.4

94.1

TNO

87.5

91.8

88.4

87.7

87.1

100.00

87.1

87.0

87.3

87.5

87.4

88.6

92.5

72.5

87.3

G10

94.3

88.2

86.8

93.8

98.5

87.0

100.00

98.8

93.7

85.3

94.3

89.7

88.2

72.6

94.0

G2T

94.4

88.2

87.1

94.0

98.5

87.0

98.8

100.00

93.8

85.3

94.2

89.7

88.3

72.4

93.8

AF/04T

94.8

87.9

87.0

94.2

94.1

87.2

93.8

93.8

100.00

86.1

94.1

89.1

88.1

72.7

94.0

AK1

85.7

84.8

85.1

85.7

85.3

87.5

85.2

85.2

86.0

100.00

86.1

85.0

84.9

72.3

85.2

BCO1

97.6

87.6

87.1

97.0

94.4

87.2

94.3

94.1

94.2

86.1

100.00

89.4

87.9

72.4

97.1

KU2-6

89.5

89.8

90.0

89.5

89.7

88.6

89.5

89.6

89.0

85.0

89.4

100.00

90.8

72.5

89.3

Kn10

88.1

94.9

89.0

88.1

88.0

92.6

88.0

88.3

87.9

84.8

87.8

90.8

100.00

72.4

87.7

PS2

72.4

72.4

72.2

72.4

72.5

72.6

72.5

72.4

72.6

72.3

72.5

72.3

72.5

100.00

72.5

25

97.6

87.5

87.1

97.0

94.0

86.9

93.9

93.8

94.0

85.2

97.0

89.3

87.8

72.7

100.00

The ANI value (%) shared between genomes (above and below diagonal). AB04 T = A. ayderensis AB04T [30]; WK1 = A. flavithermus WK1 [5, 29]; E13T = A. flavithermus subsp. yunnanensis E13T [35, 47, 48]; SK3-4 = Anoxybacillus sp. SK3-4 [45, 46]; DT3-1 = Anoxybacillus sp. DT3-1 [45, 46]; TNO = A. flavithermus TNO-09.006 [5, 44]; G10 = A. kamchatkensis G10 [4042]; G2T = A. gonensis G2T [36]; AT T = A. thermarum AF/04T [3335]; AK1 = A. flavithermus AK1 [5, 39]; BCO1 = Anoxybacillus sp. BCO1 [31, 32]; KU2-6 = Anoxybacillus sp. KU2-6(11); Kn10 = A. flavithermus Kn10 [5, 43]; PS2 = A. tepidamans PS2 [37]; 25 = A. flavithermus 25 [5, 38]

Analysis of the GHs in A. ayderensis AB04T and other Anoxybacillus genomes

We detected 14 genes in the AB04T genome encoding GH enzymes belonging to GH families 1, 10, 13, 31, 32, 51, 52, and 67 (Table 6). On average, the AB04T GHs shared 93.9 % similarity with GHs identified in other Anoxybacillus spp. The GHs could be grouped into two types according to their predicted catalytic ability (Table 6). Nine GH enzymes were predicted to be active on α-chain polysaccharides whereas the remaining five GH enzymes were specific for β-linked polysaccharides (i.e., cellulose and xylan).
Table 6

List of several glycoside hydrolases (GHs) identified in various Anoxybacillus genomes

GH

Enzyme

Similarity within Anoxybacillus genomea

Number of studied enzymeb

AB04T

WK1

E13T

SK3-4

DT3-1

TNO

G10

G2T

AF/04T

AK1

BCO1

KU2-6

Kn10

PS2

1

β-glucosidase

91.2

100

90.7

39.5

92.4

-

-

-

-

-

-

91.6

92.0

-

-

10

Endo-1,4-β-xylanase

100.00

-

-

-

-

-

-

-

-

-

-

-

-

-

-

13

α-amylase (cell-bound)

98.6

100.00

98.4

97.4

96.2

97.2

96.2

98.2

98.6

94.5

76.6

98.8

99.8

84.3

2 [16]

13

α-amylase (extracellular)

77.6

-

 

100.00

-

-

95.0

-

-

-

54.2

-

-

-

-

13

Pullulanase

93.4

100.00

93.9

90.5

89.5

95.6

89.4

91.9

93.2

88.9

59.9

94.8

98.0

67.4

1 [17]

13

Amylopullulanase (>200 kDa)

-

100.00

90.2

88.8

-

-

99.1

-

-

59.6

-

-

89.6

-

1 [18]

13

Amylopullulanase (<200 kDa)

-

-

-

-

-

-

-

-

-

-

100

-

-

-

-

13

CDase

95.8

100.00

95.6

92.5

92.0

94.7

92.3

95.1

94.9

93.6

96.6

95.9

98.1

78.8

1 [19]

13

Oligo-1,6-glucosidase

98.2

100.00

61.7

96.0

96.0

98.1

96.0

53.9

97.5

97.0

53.6

96.5

98.6

90.9

-

13

Trehalose-6-phosphate hydrolase

95.6

100.00

-

94.2

93.7

-

93.9

-

96.2

94.9

-

95.8

99.1

-

-

13

1,4-α-glucan branching enzyme

93.4

100.00

-

93.2

92.8

94.1

92.4

-

93.9

 

94.9

-

-

-

-

31

α-glucosidase

92.1

100.00

92.9

91.0

89.2

88.1

89.2

91.5

91.7

88.5

71.1

93.4

96.9

67.4

-

32

Sucrase-6-phosphate hydrolase

94.7

100.00

-

91.3

91.8

91.0

91.3

-

93.5

92.5

-

93.9

93.3

-

-

36

α-galactosidase

-

-

91.2

-

-

-

79.2

100

-

90.5

72.3

93.7

-

79.5

-

51

α-L-arabinofuranosidase

93.6

100.00

-

-

-

-

-

-

-

99.4

-

-

-

-

1 [22]

52

β-xylosidase

91.5

-

90.4

-

-

-

100

99.6

-

 

-

-

-

89.5

-

65

Sugar hydrolase/phosphorylase

-

100.00

-

94.9

94.1

-

94.0

 

-

94.1

-

-

96.6

-

-

67

α-glucuronidase

100.00

-

-

-

-

-

-

-

-

-

-

-

-

-

-

aThe reference for the protein sequence alignment is denoted as 100 %; bThe numbers represent the respective cloned, purified, and characterized enzymes from Anoxybacillus species. AB04T = A. ayderensis AB04T [30]; WK1 = A. flavithermus WK1 [5, 29]; E13T = A. flavithermus subsp. yunnanensis E13T [35, 47, 48]; SK3-4 = Anoxybacillus sp. SK3-4 [45, 46]; DT3-1 = Anoxybacillus sp. DT3-1 [45, 46]; TNO = A. flavithermus TNO-09.006 [5, 44]; G10 = A. kamchatkensis G10 [4042]; G2T = A. gonensis G2T [36]; ATT = A. thermarum AF/04T [3335]; AK1 = A. flavithermus AK1 [5, 39]; BCO1 = Anoxybacillus sp. BCO1 [31, 32]; KU2-6 = Anoxybacillus sp. KU2-6(11); Kn10 = A. flavithermus Kn10 [5, 43]; PS2 = A. tepidamans PS2 [37]

Interestingly, we found two GH enzymes that were uniquely present in strain AB04T: endo-1,4-β-xylanase (NCBI locus ID: KIP21668) and α-glucuronidase (KIP21917) (Table 6). The closest homologs of endo-1,4-β-xylanase and α-glucuronidase were found in Geobacillus thermoglucosidans and Geobacillus stearothermophilus with 81.9 % and 87.1 % sequence similarity, respectively [27].

Genes coding for at least five of the aforementioned GHs including cell-bound α-amylase, pullulanase, CDase, oligo-1,6-glucosidase, and α-glucosidase were consistently found in the genomes of all Anoxybacillus spp. (Table 6). Therefore, these enzymes might play an important role in Anoxybacillus carbohydrate metabolism. A high molecular-mass amylopullulanase (>200 kDa) from Anoxybacillus sp. SK3-4 has been reported previously [18]. We detected this enzyme in other Anoxybacillus spp., for instance A. flavithermus WK1 [5, 29], A. flavithermus subsp. yunnanensis E13T [35, 47, 48], A. kamchatkensis G10 [4042], A. flavithermus AK1 [5, 39], and A. flavithermus Kn10 [5, 43]. From the current analysis, it can be concluded that amylopullulanase is the GH with greatest molecular-mass in Anoxybacillus (Table 6). Despite their widespread distribution in Anoxybacillus spp., only a limited number of GHs have been studied intensively. At present, only α-amylase [16], pullulanase [17], amylopullulanase [18], CDase [19], and α-L-arabinofuranosidase [22] have been cloned, purified, and biochemically characterized (Table 6). he number of underexplored GH enzymes such as β-glucosidase, endo-1,4-β-xylanase, α-L-arabinofuranosidase, α-glucuronidase, and β-xylosidase remains high; however, because of their interesting applications and their important roles in second-generation biofuel production [69], these enzymes are worthy of examination in the near future.

Other A. ayderensis AB04T enzymes with potential applications

Apart from the GHs, we found that A. ayderensis AB04T has genes coding for other industrially important enzymes such as xylose isomerase, esterase, and aldolase. Xylose isomerase (EC 5.3.1.5) catalyzes the isomerization of xylose to xylulose and of glucose to fructose, which is important in the industrial production of high-fructose corn syrup [20]. Earlier, a xylose isomerase from A. gonensis G2T was characterized and the enzyme displays 96.8 % amino acid sequence similarity to the one identified in strain AB04T (KIP21927) [20].

Previous studies have indicated that A. gonensis G2T, A. gonensis A4, and Anoxybacillus sp. PDF-1 produce esterase [7072]. We identified two esterases (KIP19922 and KIP21735) in the genome of strain AB04T, which shared 96.3 % and 96.0 % amino acid sequence similarity with the esterase from Anoxybacillus sp. PDF-1 [72] and A. gonensis G2T [70], respectively. In addition, a fructose-1,6-bisphosphate aldolase from A. gonensis G2T has been described [73]. Strain AB04T carries two aldolases, KIP21451 and KIP21450, which showed 95.9 % and 99.9 % amino acid similarity to aldolase from A. flavithermus WK1 [5, 29] and A. thermarum AF/04T [3335], respectively. We did not biochemically characterize these enzymes from strain AB04T in the current study.

Thermophilic bacteria are highly sought after for their potential use in bioremediation processes. Several Anoxybacillus spp. efficiently reduce metal ions such as Hg2+, Cr4+,Al3+, and As3+ [4, 2325]. The genome of strain AB04T contains at least six heavy metal resistance genes. Four genes are related to mercuric ion reduction; two of these are mercury resistance (mer) operons (KIP20706 and KIP20408) and the two other genes encode mercuric reductases, which catalyze the reduction of Hg2+ to Hg0 (KIP19952 and KIP20409). In addition, strain AB04T carries genes for an arsenate reductase (KIP20402) and an arsenic efflux pump protein (KIP20401). The function of these genes will be studied in the close future.

Conclusions

Knowledge on the genomics, industrial enzymes, and relevant applications of Anoxybacillus spp. are rather limited compared to that in their closest relatives, Geobacillus and Bacillus . In the present work we presented a whole-genome sequence of A. ayderensis AB04T and its annotation. Additionally, we provided insights into several GHs, under-explored enzymes, and putative applications of strain AB04T.

Abbreviations

ANI: 

Average Nucleotide Identity

CDase: 

Cyclomaltodextrinase

FAME: 

Fatty acid methyl esters

FESEM: 

Field emission scanning electron microscope

GH: 

Glycoside hydrolase

ML: 

Maximum-likelihood

Declarations

Acknowledgements

This work was supported by the University of Malaya via High Impact Research Grants (UM.C/625/1/HIR/MOHE/CHAN/01 [Grant No. A-000001-50001] and UM.C/625/1/HIR/MOHE/CHAN/14/1 [Grant No. H-50001-A000027]) awarded to K-GC. KMG appreciates the funding from Universiti Teknologi Malaysia (GUP Grants 06H31 and 09H98). ASY is grateful to UTM Zamalah for providing a graduate scholarship.

Open AccessThis 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)
Faculty of Sciences, Department of Biology, Karadeniz Technical University
(2)
Division of Genetics and Molecular Biology, Institute of Biological Sciences, Faculty of Science, University of Malaya
(3)
Faculty of Biosciences and Medical Engineering, Universiti Teknologi Malaysia

References

  1. Skerman VBD, McGowan V, Sneath PHA. Approved lists of bacterial names. Int J Syst Bacteriol. 1980;30:225–420.View ArticleGoogle Scholar
  2. Fischer A. Untersuchungen über bakterien. Jahrbücher für Wissenschaftliche Botanik. 1895;27:1–163.Google Scholar
  3. Taxon Abstract for the family Bacillaceae. NamesforLife, LLC. Retrieved June 13, 2015. http://dx.doi.org/10.1601/tx.4856.Google Scholar
  4. Goh KM, Kahar UM, Chai YY, Chong CS, Chai KP, Ranjani V, et al. Recent discoveries and applications of Anoxybacillus. Appl Microbiol Biotechnol. 2013;97:1475–88.View ArticlePubMedGoogle Scholar
  5. Pikuta E, Lysenko A, Chuvilskaya N, Mendrock U, Hippe H, Suzina N, et al. Anoxybacillus pushchinensis gen. nov., sp. nov., a novel anaerobic, alkaliphilic, moderately thermophilic bacterium from manure, and description of Anoxybacillus flavithermus comb. nov. Int J Syst Evol Microbiol. 2000;50:2109–17.View ArticlePubMedGoogle Scholar
  6. Pikuta E, Cleland D, Tang J. Aerobic growth of Anoxybacillus pushchinoensis K1T: emended descriptions of A. pushchinoensis and the genus Anoxybacillus. Int J Syst Evol Microbiol. 2003;53:1561–2.View ArticlePubMedGoogle Scholar
  7. Gibbons NE, Murray RGE. Proposals concerning the higher taxa of bacteria. Int J Syst Bacteriol. 1978;28:1–6.View ArticleGoogle Scholar
  8. Ludwig W, Schleifer K-H, Whitman WB, et al. Class I. Bacilli class nov. In: De Vos P, Garrity G, Jones D, Krieg NR, Ludwig W, Rainey FA, editors. Bergey's manual of systematic bacteriology, volume 3. 2nd ed. New York: Springer; 2009. p. 19–20.Google Scholar
  9. Validation List no. 132. List of new names and new combinations previously effectively, but not validly, published. Int J Syst Evol Microbiol. 2010;60:469–72.View ArticleGoogle Scholar
  10. Prévot AR. In: Hauderoy P, Ehringer G, Guillot G, Magrou J, Prévot AR, Rosset D, et al., editors. Dictionnaire des bactéries pathogènes. 2nd ed. Paris: Masson et Cie; 1953. p. 1–692.Google Scholar
  11. Rogers EE, Backus EA. Anterior foregut microbiota of the glassy-winged sharpshooter explored using deep 16S rRNA gene sequencing from individual insects. PLoS ONE. 2014;9:e106215.PubMed CentralView ArticlePubMedGoogle Scholar
  12. Cihan AC, Cokmus C, Koc M, Ozcan B. Anoxybacillus calidus sp. nov., a thermophilic bacterium isolated from soil near a thermal power plant. Int J Syst Evol Microbiol. 2014;64:211–9.View ArticlePubMedGoogle Scholar
  13. Zhang X-Q, Zhang Z-L, Wu N, Zhu X-F, Wu M. Anoxybacillus vitaminiphilus sp. nov., a strictly aerobic and moderately thermophilic bacterium isolated from a hot spring. Int J Syst Evol Microbiol. 2013;63:4064–71.View ArticlePubMedGoogle Scholar
  14. Joshi S, Satyanarayana T. In vitro engineering of microbial enzymes with multifarious applications: prospects and perspectives. Bioresour Technol. 2015;176:273–83.View ArticlePubMedGoogle Scholar
  15. Kananavičiūtė R, Čitavičius D. Genetic engineering of Geobacillus spp. J Microbiol Methods. 2015;111:31–9.View ArticlePubMedGoogle Scholar
  16. Chai YY, Rahman RNZRA, Illias RM, Goh KM. Cloning and characterization of two new thermostable and alkalitolerant α-amylases from the Anoxybacillus species that produce high levels of maltose. J Ind Microbiol Biotechnol. 2012;39:731–41.View ArticlePubMedGoogle Scholar
  17. Xu J, Ren F, Huang C-H, Zheng Y, Zhen J, Sun H, et al. Functional and structural studies of pullulanase from Anoxybacillus sp. LM18-11. Proteins: Struct, Funct, Bioinf. 2014;82:1685–93.View ArticleGoogle Scholar
  18. Kahar UM, Chan K-G, Salleh MM, Hii SM, Goh KM. A high molecular-mass Anoxybacillus sp. SK3-4 amylopullulanase: characterization and its relationship in carbohydrate utilization. Int J Mol Sci. 2013;14:11302–18.PubMed CentralView ArticlePubMedGoogle Scholar
  19. Turner P, Labes A, Fridjónsson ÓH, Hreggvidson GO, Schönheit P, Kristjánsson JK, et al. Two novel cyclodextrin-degrading enzymes isolated from thermophilic bacteria have similar domain structures but differ in oligomeric state and activity profile. J Biosci Bioeng. 2005;100:380–90.View ArticlePubMedGoogle Scholar
  20. Karaoglu H, Yanmis D, Sal FA, Celik A, Canakci S, Belduz AO. Biochemical characterization of a novel glucose isomerase from Anoxybacillus gonensis G2T that displays a high level of activity and thermal stability. J Mol Catal B Enzym. 2013;97:215–24.View ArticleGoogle Scholar
  21. Wang J, Bai Y, Yang P, Shi P, Luo H, Meng K, et al. A new xylanase from thermoalkaline Anoxybacillus sp. E2 with high activity and stability over a broad pH range. World J Microbiol Biotechnol. 2010;26:917–24.View ArticleGoogle Scholar
  22. Canakci S, Kacagan M, Inan K, Belduz AO, Saha BC. Cloning, purification, and characterization of a thermostable α-L-arabinofuranosidase from Anoxybacillus kestanbolensis AC26Sari. Appl Microbiol Biotechnol. 2008;81:61–8.View ArticlePubMedGoogle Scholar
  23. Beris FS, De Smet L, Karaoglu H, Canakci S, Van Beeumen J, Belduz AO. The ATPase activity of the G2alt gene encoding an aluminium tolerance protein from Anoxybacillus gonensis G2. J Microbiol. 2011;49:641–50.View ArticlePubMedGoogle Scholar
  24. Lim JC, Goh KM, Shamsir MS, Ibrahim Z, Chong CS. Characterization of aluminum resistant Anoxybacillus sp. SK3-4 isolated from a hot spring. J Basic Microbiol. 2014;55:514–9.View ArticlePubMedGoogle Scholar
  25. Jiang D, Li P, Jiang Z, Dai X, Zhang R, Wang Y, et al. Chemolithoautotrophic arsenite oxidation by a thermophilic Anoxybacillus flavithermus strain TCC9-4 from a hot spring in Tengchong of Yunnan, China. Front Microbiol. 2015;6:360.PubMed CentralView ArticlePubMedGoogle Scholar
  26. Chen J, Li Y, Hao H-H, Zheng J, Chen J-M. Fe(II)EDTA-NO reduction by a newly isolated thermophilic Anoxybacillus sp. HA from a rotating drum biofilter for NOx removal. J Microbiol Methods. 2015;109:129–33.View ArticlePubMedGoogle Scholar
  27. Nazina TN, Tourova TP, Poltaraus AB, Novikova EV, Grigoryan AA, Ivanova AE, et al. Taxonomic study of aerobic thermophilic bacilli: descriptions of Geobacillus subterraneus gen. nov., sp. nov. and Geobacillus uzenensis sp. nov. from petroleum reservoirs and transfer of Bacillus stearothermophilus, Bacillus thermocatenulatus, Bacillus thermoleovorans, Bacillus kaustophilus, Bacillus thermoglucosidasius and Bacillus thermodenitrificans to Geobacillus as the new combinations G. stearothermophilus, G. thermocatenulatus, G. thermoleovorans, G. kaustophilus, G. thermoglucosidasius and G. thermodenitrificans. Int J Syst Evol Microbiol. 2001;51:433–46.View ArticlePubMedGoogle Scholar
  28. Cohn F. Untersuchungen über bakterien. Beiträge zur Biologie der Pflanzen. 1872;1:127–224.Google Scholar
  29. Saw JH, Mountain BW, Feng L, Omelchenko MV, Hou S, Saito JA, et al. Encapsulated in silica: genome, proteome and physiology of the thermophilic bacterium Anoxybacillus flavithermus WK1. Genome Biol. 2008;9:1–16.View ArticleGoogle Scholar
  30. Dulger S, Demirbag Z, Belduz AO. Anoxybacillus ayderensis sp. nov. and Anoxybacillus kestanbolensis sp. nov. Int J Syst Evol Microbiol. 2004;54:1499–503.View ArticlePubMedGoogle Scholar
  31. Patel BKC. Draft genome sequence of Anoxybacillus strain BCO1, isolated from a thermophilic microbial mat colonizing the outflow of a bore well of the Great Artesian Basin of Australia. Genome Announc. 2015;3:e01547–14.PubMed CentralPubMedGoogle Scholar
  32. Ogg CD, Spanevello MD, Patel BKC. Exploring the ecology of thermophiles from Australia’s Great Artesian Basin during the genomic era. In: Satyanarayana T, Littlechild J, Kawarabayasi Y, editors. Thermophilic microbes in environmental and industrial biotechnology. New York: Springer; 2013. p. 61–97.View ArticleGoogle Scholar
  33. Poli A, Nicolaus B, Chan K-G, Kahar UM, Chan CS, Goh KM. Genome sequence of Anoxybacillus thermarum AF/04T, isolated from the Euganean Hot Springs in Abano Terme, Italy. Genome Announc. 2015;3:e00490–15.PubMed CentralView ArticlePubMedGoogle Scholar
  34. Poli A, Romano I, Cordella P, Orlando P, Nicolaus B, Berrini CC. Anoxybacillus thermarum sp. nov., a novel thermophilic bacterium isolated from thermal mud in Euganean hot springs, Abano Terme, Italy. Extremophiles. 2009;13:867–74.View ArticlePubMedGoogle Scholar
  35. Validation List no. 141. List of new names and new combinations previously effectively, but not validly, published. Int J Syst Evol Microbiol. 2011;61:2025–6.View ArticleGoogle Scholar
  36. Belduz AO, Dulger S, Demirbag Z. Anoxybacillus gonensis sp. nov., a moderately thermophilic, xylose-utilizing, endospore-forming bacterium. Int J Syst Evol Microbiol. 2003;53:1315–20.View ArticlePubMedGoogle Scholar
  37. Coorevits A, Dinsdale AE, Halket G, Lebbe L, De Vos P, Van Landschoot A, et al. Taxonomic revision of the genus Geobacillus: emendation of Geobacillus, G. stearothermophilus, G. jurassicus, G. toebii, G. thermodenitrificans and G. thermoglucosidans (nom. corrig., formerly ‘thermoglucosidasius’); transfer of Bacillus thermantarcticus to the genus as G. thermantarcticus comb. nov.; proposal of Caldibacillus debilis gen. nov., comb. nov.; transfer of G. tepidamans to Anoxybacillus as A. tepidamans comb. nov.; and proposal of Anoxybacillus caldiproteolyticus sp. nov. Int J Syst Evol Microbiol. 2011;62:1470–85.View ArticlePubMedGoogle Scholar
  38. Rozanov AS, Bryanskaya AV, Kotenko AV, Malup TK, Peltek SE. Draft genome sequence of Anoxybacillus flavithermus strain 25, isolated from the Garga hot spring in the Barguzin Valley, Baikal Region, Russian Federation. Genome Announc. 2014;2:e01258–14.PubMed CentralPubMedGoogle Scholar
  39. Khalil A, Sivakumar N, Qaraw S. Genome sequence of Anoxybacillus flavithermus strain AK1, a thermophile isolated from a hot spring in Saudi Arabia. Genome Announc. 2015;3:e00604–15.PubMed CentralView ArticlePubMedGoogle Scholar
  40. Lee S-J, Lee Y-J, Ryu N, Park S, Jeong H, Lee SJ, et al. Draft genome sequence of the thermophilic bacterium Anoxybacillus kamchatkensis G10. J Bacteriol. 2012;194:6684–5.PubMed CentralView ArticlePubMedGoogle Scholar
  41. Kevbrin VV, Zengler K, Lysenko AM, Wiegel J. Anoxybacillus kamchatkensis sp. nov., a novel thermophilic facultative aerobic bacterium with a broad pH optimum from the Geyser valley, Kamchatka. Extremophiles. 2005;9:391–8.View ArticlePubMedGoogle Scholar
  42. Validation List no. 109. List of new names and new combinations previously effectively, but not validly, published. Int J Syst Evol Microbiol. 2006;56:925–7.View ArticleGoogle Scholar
  43. Matsutani M, Shirakihara Y, Imada K, Yakushi T, Matsushita K. Draft genome sequence of a thermophilic member of the Bacillaceae, Anoxybacillus flavithermus strain Kn10, isolated from the Kan-nawa hot spring in Japan. Genome Announc. 2013;1:e00311–3.PubMed CentralPubMedGoogle Scholar
  44. Caspers MPM, Boekhorst J, Abee T, Siezen RJ, Kort R. Complete genome sequence of Anoxybacillus flavithermus TNO-09.006, a thermophilic sporeformer associated with a dairy-processing environment. Genome Announc. 2013;1:e00010–3.PubMed CentralView ArticlePubMedGoogle Scholar
  45. Goh KM, Gan HM, Chan K-G, Chan GF, Shahar S, Chong CS, et al. Analysis of Anoxybacillus genomes from the aspects of lifestyle adaptations, prophage diversity, and carbohydrate metabolism. PLoS ONE. 2014;9:e90549.PubMed CentralView ArticlePubMedGoogle Scholar
  46. Chai YY, Kahar UM, Salleh MM, Illias RM, Goh KM. Isolation and characterization of pullulan-degrading Anoxybacillus species isolated from Malaysian hot springs. Environ Technol. 2012;33:1231–8.View ArticlePubMedGoogle Scholar
  47. Wang Y, Zheng Y, Wang M, Gao Y, Xiao Y, Peng H. Non-contiguous finished genome sequence of Anoxybacillus flavithermus subsp. yunnanensis type strain (E13T), a strictly thermophilic and organic solvent-tolerant bacterium. Stand Genomic Sci. 2014;9:735–43.PubMed CentralView ArticlePubMedGoogle Scholar
  48. Dai J, Liu Y, Lei Y, Gao Y, Han F, Xiao Y, et al. A new subspecies of Anoxybacillus flavithermus ssp. yunnanensis ssp. nov. with very high ethanol tolerance. FEMS Microbiol Lett. 2011;320:72–8.View ArticlePubMedGoogle Scholar
  49. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol. 2013;30:2725–9.PubMed CentralView ArticlePubMedGoogle Scholar
  50. Kim O-S, Cho Y-J, Lee K, Yoon S-H, Kim M, Na H, et al. Introducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes thatrepresent uncultured species. Int J Syst Evol Microbiol. 2012;62:716–21.View ArticlePubMedGoogle Scholar
  51. Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30:2114–20.PubMed CentralView ArticlePubMedGoogle Scholar
  52. Simpson JT, Durbin R. Efficient construction of an assembly string graph using the FM-index. Bioinformatics. 2010;26:i367–i73.PubMed CentralView ArticlePubMedGoogle Scholar
  53. Peng Y, Leung HCM, Yiu SM, Chin FYL. IDBA-UD: a de novo assembler for single-cell and metagenomic sequencing data with highly uneven depth. Bioinformatics. 2012;28:1420–8.View ArticlePubMedGoogle Scholar
  54. Hyatt D, Chen G-L, LoCascio PF, Land ML, Larimer FW, Hauser LJ. Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics. 2010;11:119.PubMed CentralView ArticlePubMedGoogle Scholar
  55. Laslett D, Canback B. ARAGORN, a program to detect tRNA genes and tmRNA genes in nucleotide sequences. Nucleic Acids Res. 2004;32:11–6.PubMed CentralView ArticlePubMedGoogle Scholar
  56. Lagesen K, Hallin P, Rødland EA, Stærfeldt H-H, Rognes T, Ussery DW. RNAmmer: consistent and rapid annotation of ribosomal RNA genes. Nucleic Acids Res. 2007;35:3100–8.PubMed CentralView ArticlePubMedGoogle Scholar
  57. Magrane M, Consortium U. UniProt Knowledgebase: a hub of integrated protein data. Database. 2011;2011:bar009.PubMed CentralView ArticlePubMedGoogle Scholar
  58. Haft DH, Selengut JD, White O. The TIGRFAMs database of protein families. Nucleic Acids Res. 2003;31:371–3.PubMed CentralView ArticlePubMedGoogle Scholar
  59. Finn RD, Bateman A, Clements J, Coggill P, Eberhardt RY, Eddy SR, et al. Pfam: the protein families database. Nucleic Acids Res. 2014;42:D222–D30.PubMed CentralView ArticlePubMedGoogle Scholar
  60. Grissa I, Vergnaud G, Pourcel C. CRISPRFinder: a web tool to identify clustered regularly interspaced short palindromic repeats. Nucleic Acids Res. 2007;35:W52–W7.PubMed CentralView ArticlePubMedGoogle Scholar
  61. Claudel-Renard C, Chevalet C, Faraut T, Kahn D. Enzyme-specific profiles for genome annotation: PRIAM. Nucleic Acids Res. 2003;31:6633–9.PubMed CentralView ArticlePubMedGoogle Scholar
  62. Kanehisa M, Goto S, Sato Y, Kawashima M, Furumichi M, Tanabe M. Data, information, knowledge and principle: back to metabolism in KEGG. Nucleic Acids Res. 2014;42:D199–205.PubMed CentralView ArticlePubMedGoogle Scholar
  63. Tatusov RL, Galperin MY, Natale DA, Koonin EV. The COG database: a tool for genome-scale analysis of protein functions and evolution. Nucleic Acids Res. 2000;28:33–6.PubMed CentralView ArticlePubMedGoogle Scholar
  64. Jones P, Binns D, Chang H-Y, Fraser M, Li W, McAnulla C, et al. InterProScan 5: genome-scale protein function classification. Bioinformatics. 2014;30:1236–40.PubMed CentralView ArticlePubMedGoogle Scholar
  65. Yin Y, Mao X, Yang J, Chen X, Mao F, Xu Y. dbCAN: a web resource for automated carbohydrate-active enzyme annotation. Nucleic Acids Res. 2012;40:W445–W51.PubMed CentralView ArticlePubMedGoogle Scholar
  66. Goris J, Konstantinidis KT, Klappenbach JA, Coenye T, Vandamme P, Tiedje JM. DNA–DNA hybridization values and their relationship to whole-genome sequence similarities. Int J Syst Evol Microbiol. 2007;57:81–91.View ArticlePubMedGoogle Scholar
  67. Ahmed I, Yokota A, Yamazoe A, Fujiwara T. Proposal of Lysinibacillus boronitolerans gen. nov. sp. nov., and transfer of Bacillus fusiformis to Lysinibacillus fusiformis comb. nov. and Bacillus sphaericus to Lysinibacillus sphaericus comb. nov. Int J Syst Evol Microbiol. 2007;57:1117–25.View ArticlePubMedGoogle Scholar
  68. Richter M, Rosselló-Móra R. Shifting the genomic gold standard for the prokaryotic species definition. Proc Natl Acad Sci U S A. 2009;106:19126–31.PubMed CentralView ArticlePubMedGoogle Scholar
  69. Menon V, Rao M. Trends in bioconversion of lignocellulose: biofuels, platform chemicals and biorefinery concept. Prog Energy Combustion Sci. 2012;38:522–50.View ArticleGoogle Scholar
  70. Çolak A, Şişik D, Saglam N, Güner S, Çanakçi S, Beldüz AO. Characterization of a thermoalkalophilic esterase from a novel thermophilic bacterium, Anoxybacillus gonensis G2. Bioresour Technol. 2005;96:625–31.View ArticlePubMedGoogle Scholar
  71. Faiz Ö, Colak A, Saglam N, Çanakçi S, Beldüz AO. Determination and characterization of thermostable esterolytic activity from a novel thermophilic bacterium Anoxybacillus gonensis A4. J Biochem Mol Biol. 2007;40:588–94.View ArticlePubMedGoogle Scholar
  72. Ay F, Karaoglu H, Inan K, Canakci S, Belduz AO. Cloning, purification and characterization of a thermostable carboxylesterase from Anoxybacillus sp. PDF1. Protein Expr Purif. 2011;80:74–9.View ArticlePubMedGoogle Scholar
  73. Ertunga NS, Colak A, Belduz AO, Canakci S, Karaoglu H, Sandalli C. Cloning, expression, purification and characterization of fructose-1,6-bisphosphate aldolase from Anoxybacillus gonensis G2. J Biochem. 2007;141:817–25.View ArticlePubMedGoogle Scholar
  74. Field D, Garrity G, Gray T, Morrison N, Selengut J, Sterk P, et al. The minimum information about a genome sequence (MIGS) specification. Nat Biotechnol. 2008;26:541–7.PubMed CentralView ArticlePubMedGoogle Scholar
  75. Woese CR, Kandler O, Wheelis ML. Towards a natural system of organisms: proposal for the domains archaea, bacteria, and eucarya. Proc Natl Acad Sci U S A. 1990;87:4576–9.PubMed CentralView ArticlePubMedGoogle Scholar
  76. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, et al. Gene ontology: tool for the unification of biology. The gene ontology consortium. Nat Genet. 2000;25:25–9.PubMed CentralView ArticlePubMedGoogle Scholar
  77. Shida O, Takagi H, Kadowaki K, Komagata K. Proposal for two new genera, Brevibacillus gen. nov. and Aneurinibacillus gen. nov. Int J Syst Bacteriol. 1996;46:939–46.View ArticlePubMedGoogle Scholar

Copyright

© Belduz et al. 2015