Vishnuvardhan Reddy Sultanpuram* and Thirumala Mothe

Microbial Ecology Laboratory, Department of Applied Biosciences, Mahatma Gandhi University, Anneparthy, Yellareddygudem (PO), Nalgonda – 508 254, Telangana, India.

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 Salisediminibacteriumhaloalkalitolerans10nlg was recently described as a novel species of the genus Salisediminibacterium. 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 Pelagirhabdusalkalitolerans S5, Salibacteriumhalotolerans S7 and Salipaludibacillusaurantiacus S9 were threenovel 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 Salipaludibacillusaurantiacus S9 (Sultanpuram et al., 2016; Sultanpuram et al., 2015d; Sultanpuram and Mothe 2016). Here we report the genome sequencing of these six strains.

 

Material 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 IlluminaHiSeq 2500-1TB. Illumina paired-endreadsofaverage250-bplengthandthe cleandatawere 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 discussions

Genome features

The Table 1 describes the classification,generalfeaturesandgenomesequencingprojectsinformationforthe strainsaccordingtotheMIGSrecommendations(Fieldetal.,2008).Theassembleddraftgenomeof Salisediminibacteriumhaloalkalitolerans10nlgcontained2,93,7914 bps with 47.13 %GCcontent.Further, the genome analysispredicted 2,282proteins coding sequences (CDS) of which 80.35 % were assigned to recognized functional genes.The genome analysis revealed 57 tRNA and 13 rRNA genes. Thegenomeof Bacillus lonarensis 25nlgTcontained2,58,2209 bps with 43.26 %GCcontent.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.Theassembledgenomeof Bacillus caseinilyticus SPTcontained5,87,2627 bps with 44.85 %GCcontent.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 101tRNA and 12 rRNA genes. The genome of Pelagirhabdusalkalitolerans S5 contained 2,52,7601bps 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 Salibacteriumhalotolerans 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 onetRNA and 13 rRNA genes were revealed in the analysis of strain Salibacteriumhalotolerans S7 genome. Salipaludibacillusaurantiacus 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.

 

 

Table1: Classification,generalfeaturesandgenomesequencingprojectsinformationforthe strainsaccordingtotheMIGSrecommendations(Fieldetal.,2008).

 

MIGS ID

 

Property
Current  classification
DomainBacteria
DomainBacteria
DomainBacteria
DomainBacteria
DomainBacteria
DomainBacteria
PhylumFirmicutes
PhylumFirmicutes
PhylumFirmicutes
PhylumFirmicutes
PhylumFirmicutes
PhylumFirmicutes
ClassBacilli
ClassBacilli
ClassBacilli
ClassBacilli
ClassBacilli
ClassBacilli
OrderBacillales
OrderBacillales
OrderBacillales
OrderBacillales
OrderBacillales
OrderBacillales
FamilyBacillaceae
FamilyBacillaceae
FamilyBacillaceae
FamilyBacillaceae
FamilyBacillaceae
FamilyBacillaceae
GenusSalisediminibacterium
Genus

Bacillus

Genus

Bacillus

GenusPelagirhabdus
GenusSalibacterium
GenusSalipaludibacillus
    Specieshaloalkalitolerans
    Species

lonarensis

    Speciescaseinilyticus
Speciesalkalitolerans
Specieshalotolerans
Species

aurantiacus

Strain10nlg
Strain25nlg
StrainSP
StrainS5
StrainS7
StrainS9
Gramstain
Positive
Positive
Positive
Positive
Positive
Positive
Cellshape
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
Temperaturerange
    20-50°C
    20-50°C
15-60°C
    20-55°C
    25-45°C
    20-45°C
Optimumtemperature
 37°C
35- 37°C
 37°C
37°C
37°C
37°C
Carbonsource
Varied
Varied
Varied
Varied
Varied
Varied
Energysource
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
Bioticrelationship
Freeliving
Freeliving
Freeliving
Freeliving
Freeliving
Freeliving
MIGS-14
Pathogenicity
None
None
None
None
None
None
MIGS-4
Geographiclocation
Lonar soda lake, India
Lonar soda lake, India
Lonar soda lake, India
Pingaleshwar beach, India
Khavda, India
Narayan sarovar, India
MIGS-5
Samplecollectiontime
    05/08/2013
    05/08/2013
    05/08/2013
    12/06/2015
    18/09/2014
   21/09/2014
MIGS-4.3
Depth
1m
1m
1m
1m
1m
1m
MIGS-4.4
Altitude
Notrecorded
Notrecorded
Notrecorded
Notrecorded
Notrecorded
Notrecorded
MIGS-31
Finishingquality
Permanent draft
Permanent draft
Permanent draft
Permanent draft
Permanent draft
Permanent draft
MIGS-28
Librariesused
Shotgun
Shotgun
Shotgun
Shotgun
Shotgun
Shotgun
MIGS-29
Sequencingplatforms
IlluminaHiSeq 2500-1TB
IlluminaHiSeq 2500-1TB
IlluminaHiSeq 2500-1TB
IlluminaHiSeq 2500-1TB
IlluminaHiSeq 2500-1TB
IlluminaHiSeq 2500-1TB
MIGS-30
Assemblers
Newblerv.2.8
Newblerv.2.8
Newblerv.2.8
Newblerv.2.8
Newblerv.2.8
Newblerv.2.8
MIGS-32
Genecallingmethod
Glimmer3.02
Glimmer3.02
Glimmer3.02
Glimmer3.02
Glimmer3.02
Glimmer3.02
NCBIprojectID
    335132
    329819
    329820
343906
335130
335131
NCBI Bio-Project Accession
    PRJNA335132
    PRJNA329819
    PRJNA329820
PRJNA330706
PRJNA335130
PRJNA335131
Projectrelevance
Environmentalandbiotechnological
Environmentalandbiotechnological
Environmentalandbiotechnological
Environmentalandbiotechnological
Environmentalandbiotechnological
Environmentalandbiotechnological

 

 

The genomes of all the above described six strains harboured a cluster of genes coding for compatible solute production, thus explaining theirosmotolerance. These genes from strain Salisediminibacteriumhaloalkalitolerans10nlginclude, L- ectoine synthase (130 aa; locus tag SAMN05444126_10550; accession number FOGV01000005.1) and choline dehydrogenase (561aa, 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 dehyrogenase402 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 (196aa; locus tag SAMN05421736_10420; accession number FNPI01000004.1), choline dehyrogenase(569aa; locus tag SAMN05421736_101228; accession number FNPI01000001.1).Pelagirhabdusalkalitolerans 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, strainSalibacteriumhalotolerans 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). Salipaludibacillusaurantiacus 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) andbetainealdehydyde 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 four strains belonging to Firmicutesfor osmotolerance, especially in relation to ectoine and hydroxyectoine production which are very commercially viable products.

 

 

Nucleotide sequence accession numbers

The draft sequence of Salisediminibacteriumhaloalkalitolerans10nlg,Bacillus lonarensis 25nlgT, Bacillus caseinilyticus SPT, Pelagirhabdusalkalitolerans S5, Salibacteriumhalotolerans S7 and Salipaludibacillusaurantiacus S9obtained in this Whole Genome Shotgun project has been deposited at GenBank under the accession numbers FOGV00000000, FMYM00000000,FNPI00000000,  FOGV00000000, FOXD00000000 and FOGT00000000, respectively. TheGenomes on Line Database (GOLD) IDs of the strainsSalisediminibacteriumhaloalkalitolerans10nlg,Bacillus lonarensis 25nlgT, Bacillus caseinilyticus SPT,Pelagirhabdusalkalitolerans S5, Salibacteriumhalotolerans S7 and Salipaludibacillusaurantiacus S9are PRJEB16955, PRJEB15470, PRJEB16536,PRJEB15473, PRJEB17326 and PRJEB16964, respectively.

 

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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