High-coverage long-read sequencing of the Halobacterium salinarum type strain (91-R6) revealed a 2.17-Mb chromosome and two large plasmids (148 and 102 kb). Population heterogeneity and long repeats were observed. Strain 91-R6 and laboratory strain R1 showed 99.63% sequence identity in common chromosomal regions and only 38 strain-specific segments. This information resolves the previously uncertain relationship between type and laboratory strains

Dyall-Smith, M.

Habermann, B.

Marchfelder, A.

Pfeiffer, F.

International audienceHigh-coverage long-read sequencing of the Halobacterium salinarum type strain (91-R6) revealed a 2.17-Mb chromosome and two large plasmids (148 and 102 kb). Population heterogeneity and long repeats were observed. Strain 91-R6 and laboratory strain R1 showed 99.63% sequence identity in common chromosomal regions and only 38 strain-specific segments. This information resolves the previously uncertain relationship between type and laboratory strains

Pfeiffer, Friedhelm

Marchfelder, Anita

Habermann, Bianca

Dyall-Smith, Mike

HAL Descartes

The Genome Sequence of the Halobacterium salinarum Type Strain Is Closely Related to That of Laboratory Strains NRC-1 and R1

MPG.PuRe

The Genome Sequence of the Halobacterium salinarum TypeStrain Is Closely Related to That of Laboratory Strains NRC-1and R1Friedhelm Pfeiffer,a Anita Marchfelder,b Bianca Habermann,a,c Mike L. Dyall-Smitha,daComputational Biology Group, Max Planck Institute of Biochemistry, Martinsried, GermanybBiology II, Ulm University, Ulm, GermanycCNRS, IBDM UMR 7288, Computational Biology Group, Aix Marseille Université, Marseille, FrancedVeterinary Biosciences, Faculty of Veterinary and Agricultural Sciences, University of Melbourne, Parkville, AustraliaABSTRACT High-coverage long-read sequencing of the Halobacterium salinarumtype strain (91-R6) revealed a 2.17-Mb chromosome and two large plasmids (148and 102 kb). Population heterogeneity and long repeats were observed. Strain 91-R6and laboratory strain R1 showed 99.63% sequence identity in common chromosomalregions and only 38 strain-speciﬁc segments. This information resolves the previ-ously uncertain relationship between type and laboratory strains.Halobacterium salinarum is a well-studied model haloarchaeon ﬁrst isolated fromcured cod in 1922 (1). The source of this organism was found to be salt. Thisoriginal type strain was lost (2), and a neotype was assigned, H. salinarum isolate 91-R6(3) (NRC 34002  ATCC 33171  DSM 3754), which is referred to hereafter as strain91-R6. It was isolated in Canada from the red discoloration on a salted cow hide (3).Little experimental work has been reported for the type strain, but two laboratorystrains of H. salinarum have been sequenced, namely, strain NRC-1 (4) and strain R1 (5).Their relationship to the type strain has previously remained uncertain.A fresh culture of strain 91-R6 (DSM 3754T) was obtained from the DSMZ andinoculated directly into liquid medium, omitting any colony puriﬁcation. DNA from theresulting cells was used for genome sequencing using high-coverage PacBio long-readtechnology (5 single-molecule real-time [SMRT] cells; 253,044 reads; average length,5,400 bp; and 1.3 Gbp total, using kits from PacBio, including template preparation,MagBead loading, and sequencing). For assembly, we used the SMRTanalysis pipeline(RS_HGAP_assembly.2 v2.3.0, Paciﬁc Biosciences, with default parameters) which runsHGAP (DAGCON-based hierarchical genome assembly process) in three steps (6),namely, preassembly, de novo assembly with the Celera assembler, and ﬁnal polishingwith Quiver. Despite high coverage, the assembly gave 43 contigs. A supervisedgenome assembly was performed using Canu v1.7 (7) for assembly and Geneious v10.2(8) for integration and editing of contigs. Considerable population heterogeneity(transposon integrations and transposon-triggered genome inversions) was encoun-tered, which explained the failure of the automated assembly procedure. The repre-sentative genome sequence consists of 1 chromosome (2,178,608 bp, 67.1% GCcontent, and 400-fold coverage) and 2 large plasmids (pHSAL1, 148,406 bp and 60.6%GC content; pHSAL2, 102,666 bp and 56.5% GC content; 500-fold coverage forplasmids). The plasmids share a 39,230-bp duplication devoid of any sequence differ-ence.The chromosomes of strains 91-R6 and R1 were compared in detail by methods wepreviously described for strain comparisons of Haloquadratum walsbyi and Photorhab-dus laumondii (9, 10). They showed very high DNA sequence similarity (99.63% se-Citation Pfeiffer F, Marchfelder A, HabermannB, Dyall-Smith ML. 2019. The genomesequence of the Halobacterium salinarum typestrain is closely related to that of laboratorystrains NRC-1 and R1. Microbiol ResourAnnounc 8:e00429-19. https://doi.org/10.1128/MRA.00429-19.Editor Kenneth M. Stedman, Portland StateUniversityCopyright © 2019 Pfeiffer et al. This is anopen-access article distributed under the termsof the Creative Commons Attribution 4.0International license.Address correspondence to Friedhelm Pfeiffer,fpf@biochem.mpg.de.Received 22 April 2019Accepted 18 June 2019Published 11 July 2019GENOME SEQUENCEScrossmVolume 8 Issue 28 e00429-19 mra.asm.org 1 on August 20, 2019 at MAX PLANCK INSTITUTES Martinsriedhttp://mra.asm.org/Downloaded from quence identity covering 84.9% of 2.17 Mb in strain 91-R6 and 92.5% of 2.00 Mb instrain R1) and complete colinearity. Only 38 strain-speciﬁc regions were identiﬁed. Asthe chromosomes of strains R1 and NRC-1 are nearly identical, there is also very highsimilarity of the chromosomes from strains 91-R6 and NRC-1. The plasmids of strains91-R6 and R1 exhibited patches with very high interstrain similarity (107 kb, pHSAL1/pHS3; 42.5 kb, chromosome/pHS3; and 13.3 kb, pHSAL2/pHS1).Given the close genomic similarity of the strains, the annotation of strain R1 wasused as a reference for that of strain 91-R6. Strain 91-R6 codes for 2,451 regularproteins, of which 2,092 are shared with strain R1, with only a minority (73) having lessthan 98% protein sequence identity. Strain R1 is 1 of 12 haloarchaeal genomes whichhave been reliably annotated by our gold standard protein-based strategy (5, 11). Ourefforts also include regular systematic correlation with high-level databases (Swiss-Protand KEGG).During this project, we revised the annotation of the H. salinarum NRC-1 genomeand submitted it to NCBI as a third-party annotation (NCBI:TPA).Data availability. The genome sequence of strain 91-R6 has been deposited inGenBank under the accession numbers CP038631 (chromosome), CP038632 (pHSAL1),and CP038633 (pHSAL2). Raw reads have been deposited in the SRA archive underBioProject accession number PRJNA530823. The third-party annotation of the NRC-1genome has been deposited in the Third Party Annotation section of GenBank (acces-sion numbers BK010829, BK010830, and BK010831).ACKNOWLEDGMENTThis research received no speciﬁc grant from any funding agency in the public,commercial, or not-for-proﬁt sectors.REFERENCES1. Harrison FC, Kennedy ME. 1922. The red discolouration of cured codﬁsh.Proc Trans R Soc Can 16:101–152.2. Grant WD, Kamekura M, McGenity TJ, Ventosa A. 2001. Class III. Halo-bacteria class. nov., p 294–334. In Boone D, Castenholz R, Garrity G (ed),Bergey’s manual of systematic bacteriology, 2nd ed. Springer-Verlag,New York, NY.3. Lochhead AG. 1934. Bacteriological studies on the red discoloration ofsalted hides. Can J Res 10:275–286. https://doi.org/10.1139/cjr34-026.4. Ng WV, Kennedy SP, Mahairas GG, Berquist B, Pan M, Shukla HD, LaskySR, Baliga NS, Thorsson V, Sbrogna J, Swartzell S, Weir D, Hall J, Dahl TA,Welti R, Goo YA, Leithauser B, Keller K, Cruz R, Danson MJ, Hough DW,Maddocks DG, Jablonski PE, Krebs MP, Angevine CM, Dale H, IsenbargerTA, Peck RF, Pohlschroder M, Spudich JL, Jung KW, Alam M, Freitas T,Hou S, Daniels CJ, Dennis PP, Omer AD, Ebhardt H, Lowe TM, Liang P,Riley M, Hood L, DasSarma S. 2000. Genome sequence of Halobacteriumspecies NRC-1. Proc Natl Acad Sci U S A 97:12176–12181. https://doi.org/10.1073/pnas.190337797.5. Pfeiffer F, Schuster SC, Broicher A, Falb M, Palm P, Rodewald K, Ruepp A,Soppa J, Tittor J, Oesterhelt D. 2008. Evolution in the laboratory: thegenome of Halobacterium salinarum strain R1 compared to that of strainNRC-1. Genomics 91:335–346. https://doi.org/10.1016/j.ygeno.2008.01.001.6. Chin CS, Alexander DH, Marks P, Klammer AA, Drake J, Heiner C, Clum A,Copeland A, Huddleston J, Eichler EE, Turner SW, Korlach J. 2013. Non-hybrid, ﬁnished microbial genome assemblies from long-read SMRTsequencing data. Nat Methods 10:563–569. https://doi.org/10.1038/nmeth.2474.7. Koren S, Walenz BP, Berlin K, Miller JR, Bergman NH, Phillippy AM. 2017.Canu: scalable and accurate long-read assembly via adaptive k-merweighting and repeat separation. Genome Res 27:722–736. https://doi.org/10.1101/gr.215087.116.8. Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S,Buxton S, Cooper A, Markowitz S, Duran C, Thierer T, Ashton B, MeintjesP, Drummond A. 2012. Geneious Basic: an integrated and extendabledesktop software platform for the organization and analysis of sequencedata. Bioinformatics 28:1647–1649. https://doi.org/10.1093/bioinformatics/bts199.9. Dyall-Smith ML, Pfeiffer F, Klee K, Palm P, Gross K, Schuster SC, Rampp M,Oesterhelt D. 2011. Haloquadratum walsbyi: limited diversity in a globalpond. PLoS One 6:e20968. https://doi.org/10.1371/journal.pone.0020968.10. Zamora-Lagos MA, Eckstein S, Langer A, Gazanis A, Pfeiffer F, Haber-mann B, Heermann R. 2018. Phenotypic and genomic comparison ofPhotorhabdus luminescens subsp. laumondii TT01 and a widely usedrifampicin-resistant Photorhabdus luminescens laboratory strain. BMCGenomics 19:854. https://doi.org/10.1186/s12864-018-5121-z.11. Pfeiffer F, Oesterhelt D. 2015. A manual curation strategy to improvegenome annotation: application to a set of haloarchael genomes. Life(Basel) 5:1427–1444. https://doi.org/10.3390/life5021427.Pfeiffer et al.Volume 8 Issue 28 e00429-19 mra.asm.org 2 on August 20, 2019 at MAX PLANCK INSTITUTES Martinsriedhttp://mra.asm.org/Downloaded from 

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The Genome Sequence of the Halobacterium salinarum
Type Strain Is Closely Related to That of Laboratory
Strains NRC-1 and R1
Friedhelm Pfeiffer, Anita Marchfelder, Bianca Habermann, Mike Dyall-Smith
To cite this version:
Friedhelm Pfeiffer, Anita Marchfelder, Bianca Habermann, Mike Dyall-Smith. The Genome Sequence
of the Halobacterium salinarum Type Strain Is Closely Related to That of Laboratory Strains NRC-1
and R1. Microbiology Resource Announcements, American Society for Microbiology, 2019, 8 (28),
pp.e00429-19. ￿10.1128/MRA.00429-19￿. ￿hal-02368981￿
The Genome Sequence of the Halobacterium salinarum Type
Strain Is Closely Related to That of Laboratory Strains NRC-1
and R1
Friedhelm Pfeiffer,a Anita Marchfelder,b Bianca Habermann,a,c Mike L. Dyall-Smitha,d
aComputational Biology Group, Max Planck Institute of Biochemistry, Martinsried, Germany
bBiology II, Ulm University, Ulm, Germany
cCNRS, IBDM UMR 7288, Computational Biology Group, Aix Marseille Université, Marseille, France
dVeterinary Biosciences, Faculty of Veterinary and Agricultural Sciences, University of Melbourne, Parkville, Australia
ABSTRACT High-coverage long-read sequencing of the Halobacterium salinarum
type strain (91-R6) revealed a 2.17-Mb chromosome and two large plasmids (148
and 102 kb). Population heterogeneity and long repeats were observed. Strain 91-R6
and laboratory strain R1 showed 99.63% sequence identity in common chromosomal
regions and only 38 strain-speciﬁc segments. This information resolves the previ-
ously uncertain relationship between type and laboratory strains.
Halobacterium salinarum is a well-studied model haloarchaeon ﬁrst isolated fromcured cod in 1922 (1). The source of this organism was found to be salt. This
original type strain was lost (2), and a neotype was assigned, H. salinarum isolate 91-R6
(3) (NRC 34002  ATCC 33171  DSM 3754), which is referred to hereafter as strain
91-R6. It was isolated in Canada from the red discoloration on a salted cow hide (3).
Little experimental work has been reported for the type strain, but two laboratory
strains of H. salinarum have been sequenced, namely, strain NRC-1 (4) and strain R1 (5).
Their relationship to the type strain has previously remained uncertain.
A fresh culture of strain 91-R6 (DSM 3754T) was obtained from the DSMZ and
inoculated directly into liquid medium, omitting any colony puriﬁcation. DNA from the
resulting cells was used for genome sequencing using high-coverage PacBio long-read
technology (5 single-molecule real-time [SMRT] cells; 253,044 reads; average length,
5,400 bp; and 1.3 Gbp total, using kits from PacBio, including template preparation,
MagBead loading, and sequencing). For assembly, we used the SMRTanalysis pipeline
(RS_HGAP_assembly.2 v2.3.0, Paciﬁc Biosciences, with default parameters) which runs
HGAP (DAGCON-based hierarchical genome assembly process) in three steps (6),
namely, preassembly, de novo assembly with the Celera assembler, and ﬁnal polishing
with Quiver. Despite high coverage, the assembly gave 43 contigs. A supervised
genome assembly was performed using Canu v1.7 (7) for assembly and Geneious v10.2
(8) for integration and editing of contigs. Considerable population heterogeneity
(transposon integrations and transposon-triggered genome inversions) was encoun-
tered, which explained the failure of the automated assembly procedure. The repre-
sentative genome sequence consists of 1 chromosome (2,178,608 bp, 67.1% GC
content, and 400-fold coverage) and 2 large plasmids (pHSAL1, 148,406 bp and 60.6%
GC content; pHSAL2, 102,666 bp and 56.5% GC content; 500-fold coverage for
plasmids). The plasmids share a 39,230-bp duplication devoid of any sequence differ-
ence.
The chromosomes of strains 91-R6 and R1 were compared in detail by methods we
previously described for strain comparisons of Haloquadratum walsbyi and Photorhab-
dus laumondii (9, 10). They showed very high DNA sequence similarity (99.63% se-
Citation Pfeiffer F, Marchfelder A, Habermann
B, Dyall-Smith ML. 2019. The genome
sequence of the Halobacterium salinarum type
strain is closely related to that of laboratory
strains NRC-1 and R1. Microbiol Resour
Announc 8:e00429-19. https://doi.org/10.1128/
MRA.00429-19.
Editor Kenneth M. Stedman, Portland State
University
Copyright © 2019 Pfeiffer et al. This is an
open-access article distributed under the terms
of the Creative Commons Attribution 4.0
International license.
Address correspondence to Friedhelm Pfeiffer,
fpf@biochem.mpg.de.
Received 22 April 2019
Accepted 18 June 2019
Published 11 July 2019
GENOME SEQUENCES
crossm
Volume 8 Issue 28 e00429-19 mra.asm.org 1
quence identity covering 84.9% of 2.17 Mb in strain 91-R6 and 92.5% of 2.00 Mb in
strain R1) and complete colinearity. Only 38 strain-speciﬁc regions were identiﬁed. As
the chromosomes of strains R1 and NRC-1 are nearly identical, there is also very high
similarity of the chromosomes from strains 91-R6 and NRC-1. The plasmids of strains
91-R6 and R1 exhibited patches with very high interstrain similarity (107 kb, pHSAL1/
pHS3; 42.5 kb, chromosome/pHS3; and 13.3 kb, pHSAL2/pHS1).
Given the close genomic similarity of the strains, the annotation of strain R1 was
used as a reference for that of strain 91-R6. Strain 91-R6 codes for 2,451 regular
proteins, of which 2,092 are shared with strain R1, with only a minority (73) having less
than 98% protein sequence identity. Strain R1 is 1 of 12 haloarchaeal genomes which
have been reliably annotated by our gold standard protein-based strategy (5, 11). Our
efforts also include regular systematic correlation with high-level databases (Swiss-Prot
and KEGG).
During this project, we revised the annotation of the H. salinarum NRC-1 genome
and submitted it to NCBI as a third-party annotation (NCBI:TPA).
Data availability. The genome sequence of strain 91-R6 has been deposited in
GenBank under the accession numbers CP038631 (chromosome), CP038632 (pHSAL1),
and CP038633 (pHSAL2). Raw reads have been deposited in the SRA archive under
BioProject accession number PRJNA530823. The third-party annotation of the NRC-1
genome has been deposited in the Third Party Annotation section of GenBank (acces-
sion numbers BK010829, BK010830, and BK010831).
ACKNOWLEDGMENT
This research received no speciﬁc grant from any funding agency in the public,
commercial, or not-for-proﬁt sectors.
REFERENCES
1. Harrison FC, Kennedy ME. 1922. The red discolouration of cured codﬁsh.
Proc Trans R Soc Can 16:101–152.
2. Grant WD, Kamekura M, McGenity TJ, Ventosa A. 2001. Class III. Halo-
bacteria class. nov., p 294–334. In Boone D, Castenholz R, Garrity G (ed),
Bergey’s manual of systematic bacteriology, 2nd ed. Springer-Verlag,
New York, NY.
3. Lochhead AG. 1934. Bacteriological studies on the red discoloration of
salted hides. Can J Res 10:275–286. https://doi.org/10.1139/cjr34-026.
4. Ng WV, Kennedy SP, Mahairas GG, Berquist B, Pan M, Shukla HD, Lasky
SR, Baliga NS, Thorsson V, Sbrogna J, Swartzell S, Weir D, Hall J, Dahl TA,
Welti R, Goo YA, Leithauser B, Keller K, Cruz R, Danson MJ, Hough DW,
Maddocks DG, Jablonski PE, Krebs MP, Angevine CM, Dale H, Isenbarger
TA, Peck RF, Pohlschroder M, Spudich JL, Jung KW, Alam M, Freitas T,
Hou S, Daniels CJ, Dennis PP, Omer AD, Ebhardt H, Lowe TM, Liang P,
Riley M, Hood L, DasSarma S. 2000. Genome sequence of Halobacterium
species NRC-1. Proc Natl Acad Sci U S A 97:12176–12181. https://doi
.org/10.1073/pnas.190337797.
5. Pfeiffer F, Schuster SC, Broicher A, Falb M, Palm P, Rodewald K, Ruepp A,
Soppa J, Tittor J, Oesterhelt D. 2008. Evolution in the laboratory: the
genome of Halobacterium salinarum strain R1 compared to that of strain
NRC-1. Genomics 91:335–346. https://doi.org/10.1016/j.ygeno.2008.01
.001.
6. Chin CS, Alexander DH, Marks P, Klammer AA, Drake J, Heiner C, Clum A,
Copeland A, Huddleston J, Eichler EE, Turner SW, Korlach J. 2013. Non-
hybrid, ﬁnished microbial genome assemblies from long-read SMRT
sequencing data. Nat Methods 10:563–569. https://doi.org/10.1038/
nmeth.2474.
7. Koren S, Walenz BP, Berlin K, Miller JR, Bergman NH, Phillippy AM. 2017.
Canu: scalable and accurate long-read assembly via adaptive k-mer
weighting and repeat separation. Genome Res 27:722–736. https://doi
.org/10.1101/gr.215087.116.
8. Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S,
Buxton S, Cooper A, Markowitz S, Duran C, Thierer T, Ashton B, Meintjes
P, Drummond A. 2012. Geneious Basic: an integrated and extendable
desktop software platform for the organization and analysis of sequence
data. Bioinformatics 28:1647–1649. https://doi.org/10.1093/bioinformatics/
bts199.
9. Dyall-Smith ML, Pfeiffer F, Klee K, Palm P, Gross K, Schuster SC, Rampp M,
Oesterhelt D. 2011. Haloquadratum walsbyi: limited diversity in a global
pond. PLoS One 6:e20968. https://doi.org/10.1371/journal.pone.0020968.
10. Zamora-Lagos MA, Eckstein S, Langer A, Gazanis A, Pfeiffer F, Haber-
mann B, Heermann R. 2018. Phenotypic and genomic comparison of
Photorhabdus luminescens subsp. laumondii TT01 and a widely used
rifampicin-resistant Photorhabdus luminescens laboratory strain. BMC
Genomics 19:854. https://doi.org/10.1186/s12864-018-5121-z.
11. Pfeiffer F, Oesterhelt D. 2015. A manual curation strategy to improve
genome annotation: application to a set of haloarchael genomes. Life
(Basel) 5:1427–1444. https://doi.org/10.3390/life5021427.
Pfeiffer et al.
Volume 8 Issue 28 e00429-19 mra.asm.org 2


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The Genome Sequence of the Halobacterium salinarum Type Strain Is Closely Related to That of Laboratory Strains NRC-1 and R1

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