Open Access
Vishnuvardhan Reddy Sultanpuram and Thirumala Mothe
Microbial Ecology Laboratory, Department of Applied Biosciences, Mahatma Gandhi University, Anneparthy, Yellareddygudem (PO), Nalgonda – 508 254, Telangana, India.
J Pure Appl Microbiol. 2017;11(2):879-884
https://doi.org/10.22207/JPAM.11.2.26 | © The Author(s). 2017
Received: 03/02/2017 | Accepted: 30/04/2017 | Published: 30/06/2017
Abstract

Six novel Gram-stain-positive, rod shaped strains of  bacteria designated as 10nlgT, 25nlgT, SPT, S5T, S7T and S9T which were isolated from marine ecosystems and soda lake from India, represents  novel members of the family Bacillaceae. Apart from 10nlgT, 25nlgT and SPT all the other three strains represented members of novel genus from the family. Here we report the draft genome of the strains 10nlgT, 25nlgT, SPT, S5T, S7T and S9T. Strains 10nlgT, 25nlgT  and SPT genomes comprised ~ 2.93 Mb,  ~ 2.58 Mb and ~ 5.87 Mb with the G + C content of 47.13%, 43.26% 44.85%, respectively. A total of 2772 protein-coding genes in strain 10nlgT, 2581 protein-coding genes in strain 25nlgT and 5199 protein-coding genes in strain SPT were reported. Strain S5T genome comprised ~ 2.52 Mb with the G + C content of 37.08 % and a total of 2407 protein-coding genes. Strains S7T and S9T genomes comprised ~ 3.61 Mb, ~ 4.43 Mb with the G + C content of 47.65 %, 42.42% and a total of 3662, 4232 protein-coding genes, respectively.

Keywords

Compatible solute producing, Firmicutes, Whole genome sequencing

Introduction

The strain Salisediminibacterium haloalkalitolerans 10nlg was recently described as a novel species of the genus Salisedimini bacterium. The cells are Gram-stain-positive, rod shaped, non-motile, orange pigmented and non-endospore forming. The species showed catalase positive and oxidase negative activities (Sultanpuram et al., 2015a).  The two novel Bacillus species described Bacillus lonarensis 25nlgT and Bacillus caseinilyticus SPT were Gram-stain-positive, terminal endospore forming, motile rods (Sultanpuram et al., 2015b; Sultanpuram et al., 2015c). Strains Pelagirhabdus alkalitolerans S5, Salibacterium halotolerans S7 and Salipaludibacillus aurantiacus S9 were three novel recently described members representing novel genus from the family Bacillaceae. These strains were Gram-stain-positive, rod shaped, non-motile and non-endospore forming, except Salipaludibacillus aurantiacus S9 (Sultanpuram et al., 2016; Sultanpuram et al., 2015d; Sultanpuram and Mothe 2016). Here we report the genome sequencing of these six strains.

Materials and Methods

Library construction, genome sequencing and data analysis
Sequencing of the type strains was part of Genomic Encyclopedia of Type Strains, Phase III: the (KMG-III) project (Whitman et al., 2015). Project information is deposited in the Genomes on Line Database (GOLD) (Reddy et al., 2015) and the high quality draft genome sequence is deposited in GenBank and in the Integrated Microbial Genomes database (IMG) (Markowitz et al., 2014). Draft sequencing, initial gap closure and annotation were performed by the DOE Joint Genome Institute (JGI) using state-of-the-art sequencing technology (Mavromatis et al., 2012).

An Illumina standard shotgun library was constructed and sequenced using the Illumina HiSeq 2500-1TB. Illumina paired-end reads of average 250-bp length and the clean data were assembled using Velvet (version 1.1.04) (Zerbino and Birney 2008), simulated paired end reads were created from Velvet contigs using wgsim and simulated read pairs were reassembled using Allpaths-LG (version r42328) (MacCallum et al., 2009). Protein-coding genes were identified using Prodigal (Hyatt et al., 2010), as part of the DOE-JGI genome annotation pipeline (Huntemann et al., 2015). Additional gene prediction analysis and manual functional annotation were performed within the Integrated Microbial Genomes (IMG) platform, which provides tools for analyzing and reviewing the structural and functional annotations of genomes in a comparative context (Markowitz et al., 2014). Genome annotation procedures are detailed in Markowitz et al.  and references therein. Briefly, the predicted CDSs were translated and used to search the NCBI nonredundant database, UNIProt, TIGRFam, Pfam, KEGG, COG and InterPro databases. Transfer RNA genes were identified using the tRNAScan-SE tool and other non-coding RNAs were found using INFERNAL. Ribosomal RNA genes were predicted using hmmsearch against the custom models generated for each type of rRNA.

RESULTS AND DISCUSSION

Genome features
The Table 1 describes the classification, general features and genome sequencing projects information for the strains according to the MIGS recommendations (Field et al., 2008). The assembled draft genome of Salisediminibacterium haloalkalitolerans 10nlg contained 2,93,7914 bps with 47.13% G + C content. Further, the genome analysis predicted 2,282 proteins coding sequences (CDS) of which 80.35% were assigned to recognized functional genes. The genome analysis revealed 57 tRNA and 13 rRNA genes. The genome of Bacillus lonarensis 25nlgT contained 2,58,2209 bps with 43.26 % G + C content. Further, the genome analysis predicted 2,581 proteins coding sequences (CDS) of which 96.34% were assigned to recognized functional genes with 67 tRNA and 16 rRNA genes. The assembled genome of Bacillus caseinilyticus SPT contained 5,87,2627 bps with 44.85% GC content. Further, the genome analysis predicted 5,199 proteins coding sequences (CDS) of which 97.41 % were assigned to recognized functional genes. The genome analysis revealed 101 tRNA and 12 rRNA genes. The genome of Pelagirhabdus alkalitolerans S5 contained 2,52,7601 bps with 88.99% DNA coding bases and 37.08 % GC content. The genome revealed 2,033 protein coding sequences which were about 81.61 % of the total genome. Around 15.01% CDS present weren’t assigned to any of the known functions. Further, it revealed 56 tRNA and 13 rRNA genes. Strain Salibacterium halotolerans S7 showed 3, 61, 4063 bps with 47.65% G + C mol%. Around 75.89% of the identified CDS were protein sequences with known functions and about 807 protein coding genes had some unknown functions. Sixty one tRNA and 13 rRNA genes were revealed in the analysis of strain Salibacterium halotolerans S7 genome. Salipaludibacillus aurantiacus S9 draft genome revealed 4,43,5185 bps genome size with 42.42% G + C mol%. The protein functions of 3349 CDS were known, whereas, 883 CDS protein functions were unknown. Further, it revealed 73 tRNA and 16 rRNA genes.

Table (1):
Classification, general features and genome sequencing projects information for the strains according to the MIGS recommendations (Field et al., 2008)

MIGS ID
Property Current  classification
Domain Bacteria
Domain Bacteria
Domain Bacteria
Domain Bacteria
Domain Bacteria
Domain Bacteria
Phylum Firmicutes
Phylum Firmicutes
Phylum Firmicutes
Phylum Firmicutes
Phylum Firmicutes
Phylum Firmicutes
Class Bacilli
Class Bacilli
Class Bacilli
Class Bacilli
Class Bacilli
Class Bacilli
Order Bacillales
Order Bacillales
Order Bacillales
Order Bacillales
Order Bacillales
Order Bacillales
Family Bacillaceae
Family Bacillaceae
Family Bacillaceae
Family Bacillaceae
Family Bacillaceae
Family Bacillaceae
Genus Salisediminibacterium
Genus Bacillus
Genus Bacillus
Genus Pelagirhabdus
Genus Salibacterium
Genus Salipaludibacillus
Species haloalkalitolerans
Species lonarensis
Species caseinilyticus
Species alkalitolerans
Species halotolerans
Species aurantiacus
Strain 10nlg
Strain 25nlg
Strain SP
Strain S5
Strain S7
Strain S9
Gram stain
Positive
Positive
Positive
Positive
Positive
Positive
Cell shape
Rod
Rod
Rod
Rod
Rod
Rod
Motility
Non-motile
Motile
Motile
Non-motile
Non-Motile
Non-motile
Sporulation
Non-endospore former
Terminal endospore former
Terminal endospore former
Non-Endospore-former
Non-Endospore-former
Endospore-former
Temperature range
20-50°C
20-50°C
15-60°C
20-55°C
25-45°C
20-45°C
Optimum temperature
 37°C
35- 37°C
 37°C
37°C
37°C
37°C
Carbon source
Varied
Varied
Varied
Varied
Varied
Varied
Energy source
Heterotrophic
Heterotrophic
Heterotrophic
Heterotrophic
Heterotrophic
Heterotrophic
MIGS-6
Habitat
Soda lake
Soda lake
Soda lake
Marine
Marine salt pan
Marine salt marsh
MIGS-6.3
Salinity
NaCl
NaCl
NaCl
    NaCl
NaCl
NaCl
MIGS-22
Oxygen
Aerobic/Facultative anaerobe
Aerobic/Facultative anaerobe
Aerobic/Facultative anaerobe
Aerobic/Facultative anaerobe
Aerobic/Facultative anaerobe
Aerobic/Facultative anaerobe
MIGS-15
Biotic relationship
Free living
Free living
Free living
Free living
Free living
Free living
MIGS-14
Pathogenicity
None
None
None
None
None
None
MIGS-4
Geographic location
Lonar soda lake, India
Lonar soda lake, India
Lonar soda lake, India
Pingaleshwar beach, India
Khavda, India
Narayan sarovar, India
MIGS-5
Sample collection time
    05/08/2013
    05/08/2013
    05/08/2013
    12/06/2015
    18/09/2014
   21/09/2014
MIGS-4.3
Depth
1 m
1 m
1 m
1 m
1 m
1 m
MIGS-4.4
Altitude
Not recorded
Not recorded
Not recorded
Not recorded
Not recorded
Not recorded
MIGS-31
Finishing quality
Permanent draft
Permanent draft
Permanent draft
Permanent draft
Permanent draft
Permanent draft
MIGS-28
Libraries used
Shotgun
Shotgun
Shotgun
Shotgun
Shotgun
Shotgun
MIGS-29
Sequencing platforms
Illumina HiSeq 2500-1TB
Illumina HiSeq 2500-1TB
Illumina HiSeq 2500-1TB
Illumina HiSeq 2500-1TB
Illumina HiSeq 2500-1TB
Illumina HiSeq 2500-1TB
MIGS-30
Assemblers
Newbler v.2.8
Newbler v.2.8
Newbler v.2.8
Newbler v.2.8
Newbler v.2.8
Newbler v.2.8
MIGS-32
Gene calling method
Glimmer 3.02
Glimmer 3.02
Glimmer 3.02
Glimmer 3.02
Glimmer 3.02
Glimmer 3.02
NCBI project ID
    335132
    329819
    329820
343906
335130
335131
NCBI Bio-Project Accession
    PRJNA335132
    PRJNA329819
    PRJNA329820
PRJNA330706
PRJNA335130
PRJNA335131
Project relevance
Environmental and biotechnological
Environmental and biotechnological
Environmental and biotechnological
Environmental and biotechnological
Environmental and biotechnological
Environmental and biotechnological

The genomes of all the above described six strains harboured a cluster of genes coding for compatible solute production, thus explaining their osmotolerance. These genes from strain Salisediminibacterium haloalkalitolerans 10nlg include, L- ectoine synthase (130 aa; locus tag SAMN05444126_10550; accession number FOGV01000005.1) and choline dehydrogenase (561 aa, locug tag SAMN05444126_10967; accession number FOGV01000009.1). The strain, Bacillus lonarensis 25nlgT contains ectoine synthase (127 aa; locus tag SAMN05421737_10167; accession number FMYM01000001), glycerol-3-phosphate dehydrogenase (NAD(P)+ (339 aa; locus tag SAMN05421737_105223; accession number FMYM01000005.1), 1-acyl-sn-glycerol-3-phosphate acyltransferase (192 aa; locus tag SAMN05421737_105218; accession number FMYM01000005.1), choline dehyrogenase 402 aa; locus tag SAMN05421737_10199; accession number FMYM01000001.1).The strain, Bacillus caseinilyticus SPT contains L-ectoine synthase (128 aa; locus tag SAMN05421736_104185; accession number FNPI01000004.1), glycerol-3-phosphate dehydrogenase (NAD(P)+ (338 aa; locus tag SAMN05421736_10427; accession number FNPI01000004.1), glycerol-1-phosphate dehydrogenase (351 aa; locus tag SAMN05421736_10511; accession number FNPI01000005.1), glycerol-1-phosphate dehydrogenase (561 aa; locus tag SAMN05421736_10785; accession number FNPI01000007.1), 1-acyl-sn-glycerol-3-phosphate acyltransferase (196 aa; locus tag SAMN05421736_10420; accession number FNPI01000004.1), choline dehyrogenase (569 aa; locus tag SAMN05421736_101228; accession number FNPI01000001.1). Pelagirhabdus alkalitolerans S5 contains ectoine synthase (129 aa; locus tag SAMN05421734_10588; accession number FMYI01000005.1), glycerol-3-phosphate dehydrogenase (NAD(P)+ (347 aa; locus tag SAMN05421734_102431; accession number FMYI01000002.1), 1-acyl-sn-glycerol-3-phosphate acyltransferase (243 aa; locus tag SAMN05421734_102368; accession number FMYI01000002.1/ 193 aa; locus tag SAMN05518683_10435; accession number FMYI01000002.1/ 237 aa; locus tag SAMN05518683_10446; accession number FMYI01000004.1). Whereas, strain Salibacterium halotolerans S7 codes for L- ectoine synthase (128 aa; locus tag SAMN05518683_10726; accession number FOXD01000007.1), ectoine hydroxylase (314 aa; locus tag SAMN05518683_10644; accession number FOXD01000006.1) and choline dehydrogenase (569 aa; locus tag SAMN05518683_10452; accession number FOXD01000004.1 / 265 aa; locus tag SAMN05518683_10464; accession number FOXD01000004.1 / 402 aa; locus tag SAMN05518683_12711; accession number FOXD01000027.1). Salipaludibacillus aurantiacus S9 codes for L- ectoine synthase (131 aa; locus tag SAMN05518684_11244; accession number FOGT01000012.1), ectoine hydroxylase (310 aa; locus tag SAMN05518684_10667; accession number FOGT01000006.1), choline dehydrogenase (563 aa; locus tag SAMN05518684_106165; accession number FOGT01000006.1) and betaine aldehydyde dehydrogenase (490 aa; locus tag SAMN05518684_106162; accession number FOGT01000006.1). These draft genome sequences will further help in understanding the genetic potential of the above described six strains belonging to Firmicutes for osmotolerance, especially in relation to ectoine and hydroxyectoine production which are very commercially viable products.

Nucleotide sequence accession numbers
The draft sequence of Salisedimini bacterium haloalkalitolerans 10nlg, Bacillus lonarensis 25nlgT, Bacillus caseinilyticus SPT, Pelagirhabdus alkalitolerans S5, Salibacterium halotolerans S7 and Salipaludibacillus aurantiacus S9 obtained in this Whole Genome Shotgun project has been deposited at GenBank under the accession numbers FOGV00000000, FMYM00000000, FNPI00000000,  FOGV00000000, FOXD00000000 and FOGT00000000, respectively. The Genomes on Line Database (GOLD) IDs of the strains Salisediminibacterium haloalkalitolerans 10nlg, Bacillus lonarensis 25nlgT, Bacillus caseinilyticus SPT, Pelagirhabdus alkalitolerans S5, Salibacterium halotolerans S7 and Salipaludibacillus aurantiacus S9 are PRJEB16955, PRJEB15470, PRJEB16536, PRJEB15473, PRJEB17326 and PRJEB16964, respectively.

Declarations

ACKNOWLEDGMENTS
We are thankful to Prof. William Whitman for proposing the genomic sequencing of these organisms under KMG-III. These organisms were isolated and described in Department of Science & Technology, Government of India, FTSYS project grants (SB/FT/LS-115/2012-13 and SB/FT/LS-320/2012-13, respectively).

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