Open Access

Complete genome sequence of Halorhodospira halophila SL1

  • Jean F. Challacombe
  • , Sophia Majid
  • , Ratnakar Deole,
  • , Thomas S. Brettin,
  • , David Bruce
  • , Susana F. Delano,
  • , John C. Detter
  • , Cheryl D. Gleasner
  • , Cliff S. Han
  • , Monica Misra
  • , Krista G. Reitenga
  • , Natalia Mikhailova,
  • , Tanja Woyke
  • , Sam Pitluck
  • , Matt Nolan
  • , Miriam L. Land
  • , Elizabeth Saunders
  • , Roxanne Tapia
  • , Alla Lapidus,
  • , Natalia Ivanova
  • and Wouter D. Hoff

DOI: 10.4056/sigs.3677284

Received: 15 April 2013

Accepted: 15 April 2013

Published: 15 June 2013


Halorhodospira halophila is among the most halophilic organisms known. It is an obligately photosynthetic and anaerobic purple sulfur bacterium that exhibits autotrophic growth up to saturated NaCl concentrations. The type strain H. halophila SL1 was isolated from a hypersaline lake in Oregon. Here we report the determination of its entire genome in a single contig. This is the first genome of a phototrophic extreme halophile. The genome consists of 2,678,452 bp, encoding 2,493 predicted genes as determined by automated genome annotation. Of the 2,407 predicted proteins, 1,905 were assigned to a putative function. Future detailed analysis of this genome promises to yield insights into the halophilic adaptations of this organism, its ability for photoautotrophic growth under extreme conditions, and its characteristic sulfur metabolism.


halophilesaturated saltsulfur metabolismpurple sulfur bacteriumphototrophic


Halorhodospira halophila is an anoxygenic photosynthetic halophile that was isolated from salt-encrusted mud along the shore of Summer Lake in Oregon [1], and from the hypersaline Wadi Natrun lakes in Egypt [2]. The original name of this organism, Ectothiorhodospira halophila, was modified to Halorhodospira halophila when the genus Ectothiorhodospira was divided into two genera (Ectothiorhodospira and Halorhodospira), and E. halophila was reclassified as a member of the genus Halorhodospira, serving as the type species of the new genus [3]. Over the last decade, the genomes of a number of extremely halophilic Archaea have been sequenced and analyzed, including Halobacterium salinarum [4,5], Haloarcula marismortui [6], Natronomonas pharaonis [7], and Haloquadratum walsbyi [8]. In addition, the genomes of three halophilic Bacteria have become available: Salinibacter ruber [9], Halothermothrix orenii [10], and ‘Halanaerobium hydrogenoformans’ [11]. All of these organisms are obligate chemotrophs. Thus, H. halophila is the first phototrophic extreme halophile to have its genome sequence determined and analyzed. In contrast to other extreme halophiles that grow well in saturated salt concentrations, H. halophila has a high flexibility with respect to the salt concentrations that it tolerates, and grows optimally at all NaCl concentrations from 15% to 35%, with growth down to 3.5% NaCl [12]. In contrast, the above extremely halophilic archaea and S. ruber require 15% NaCl for growth.

H. halophila is of significant interest because it is an obligately anaerobic purple sulfur bacterium, and among the most halophilic organisms known [13]. To date, genome sequences are available for two phototrophic purple sulfur bacteria, Allochromatium vinosum DSM 180 and the H. halophila SL1 genome reported here. H. halophila has very few growth requirements. However, it does need reduced sulfur compounds for growth, as does A. vinosum [14]. Its pathways for both photosynthetic electron transfer [15-17] and nitrogen fixation [18] have attracted attention. In addition, H. halophila contains photoactive yellow protein [19,20]. This is the first member of a novel class of blue light receptors, and triggers a negative phototaxis response in H. halophila [21]. The photoactive yellow protein (PYP) from H. halophila has been studied extensively for its biophysical characteristics [22-24].

The sulfur metabolism of H. halophila is unusual, resulting in the transient accumulation of extracellular sulfur globules via metabolic pathways that are not yet fully resolved [14]. While purple non-sulfur phototrophs such as Rhodobacter sphaeroides and Rhodospirillum rubrum use organic compounds like malate as electron donors, H. halophila obtains electrons from reduced sulfur compounds. The genome sequence of H. halophila promises to reveal insights into its adaptations to hypersaline environments, and to allow a better understanding of its unique combination of metabolic capabilities, combining properties from extreme halophiles, anoxygenic phototrophs, and purple sulfur bacteria.

Classification and features

H. halophila belongs to the Gammaproteobacteria [3] (Table 1). The 16S rRNA gene sequence of H. halophila SL1 reveals closer relationships with H. halochloris and Alkalilimnicola ehrlichii, the other representatives of the Ectothiorhodospiraceae (Figure 1), than with A. vinosum, a purple sulfur bacterium in the Chromatiaceae family, and the haloalkaliphilic chemolithoautotrophic Thioalkalivibrio strains.

Table 1

Classification and general features of H. halophila SL1 according to the MIGS recommendations [25].




   Evidence codea

    Current classification

     Domain Bacteria

   TAS [26]

     Phylum Proteobacteria

   TAS [27]

     Class Gammaproteobacteria

   TAS [28,29]

     Order Chromatiales

   TAS [28,30]

     Family Ectothiorhodospiraceae

   TAS [31]

     Genus Halorhodospira

   TAS [32-34]

     Species Halorhodospira halophila

   TAS [32,33]


    Subspecific genetic lineage

     DSM 244T

    Gram stain



    Cell shape


   TAS [1]



   TAS [1]




    Temperature range



    Optimum temperature


   TAS [1]

    Carbon source

     CO2, succinate, acetate

   TAS [35]

    Energy source


   TAS [1]



     salt lake mud

   TAS [1]



     Extreme halophile

   TAS [1]




   TAS [1]


    Biotic relationship

     free living







    Geographic location

     Summer Lake, Lake County, OR

   TAS [1]


    Sample collection time

     about 1967

   TAS [1]



     not reported



     not reported



     not reported



     not reported

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 [36]. If the evidence code is IDA, then the property should have been directly observed, for the purpose of this specific publication, for a live isolate by one of the authors, or an expert or reputable institution mentioned in the acknowledgements.

Figure 1

Phylogram representation of a phylogenetic tree highlighting the position of Halorhodospira halophila strain SL1 relative to other organisms of interest, including members of the Ectothiorhodospiraceae, as well as additional strains that were included for comparison purposes, based on environmental and functional considerations. The strains (type=T) and their corresponding GenBank accession numbers (and coordinates) for 16S rRNA genes are: H. halophila strain SL1T, CP00544:380025-381562; Alkalilimnicola ehrlichii strain MLHE-1, CP00453:369818-369894; Thioalkalivibrio sp. HL-EbGR7, CP001339:2548250-2549775; Thioalkalivibrio sp. K90mix, CP001905:423231-424758; Allochromatium vinosum DSM 180T, CP001896:112452-113967; Ectothiorhodospira halochloris M59152; Burkholderia phytofirmans PsJN, CP001052:1541578-1543101; Desulfovibrio vulgaris subsp. vulgaris strain Hildenborough, AE017285:105921-107426; Rhodobacter sphaeroides 2.4.1, CP000143:1-1464; Rhodospirillum rubrum ATCC 11170, CP000230: 192528-194004; Escherichia coli B strain REL606, CP000819: 226609-228150. The 16S rRNA sequences were aligned by MUSCLE [37]. The tree was determined by the maximum likelihood model of PhyML [38] and rendered with TreeDyn [39], using the “one click” pipeline of the web resource [40].

Genome sequencing and annotation

Genome project history

This organism was selected for sequencing to better understand its halophilic adaptations, its unusual sulfur metabolism, its photosynthetic pathways, and to provide a framework for better understanding signaling pathways for photoactive yellow protein. The complete genome sequence has been deposited in GenBank. Sequencing, finishing and annotation were performed by the DOE Joint Genome Institute (JGI). Table 2 presents the project information and its association with MIGS version 2.0 compliance [25].

Table 2

Project information





     Finishing quality



     Libraries used

    40kb, 8kb, 3kb


     Sequencing platforms



     Fold coverage






     Gene calling method


     Genbank ID


     Genbank Date of Release

    January 12, 2012

     GOLD ID


     Project relevance


Growth conditions and DNA isolation

H. halophila SL1 strain DSM 44T was obtained from Deutsche Sammlung vor Mikroorganismen und Zellkulturen (DSMZ), Braunschweig, Germany, and were grown in DSMZ 253 medium. The cells were grown anaerobically and photosynthetically by placing them in 20 ml glass culture tubes completely filled with growth medium and sealed with screw caps. The tubes were kept at 42ºC in a water bath and illuminated with 70 W tungsten light bulbs. Chromosomal DNA was purified from the resulting cell cultures using the CTAB procedure.

Genome sequencing and assembly

The random shotgun method was used in sequencing the genome of H. halophila SL1. Large (40 kb), median (8 kb) and small (3 kb) insert random sequencing libraries were sequenced for this genome project with an average success rate of 88% and average high-quality read lengths of 750 nucleotides. After the shotgun stage, reads were assembled with parallel phrap (High Performance Software, LLC). Possible mis-assemblies were corrected with Dupfinisher (unpublished, C. Han) or by transposon bombing of bridging clones (EZ-Tn5 <P6Kyori/KAN-2> Tnp Transposome kit, Epicentre Biotechnologies). Gaps between contigs were closed by editing, custom primer walks or PCR amplification. The completed genome sequence of H. halophila SL1 contains 36,035 reads, achieving an average of 12-fold sequence coverage per base with error rate less than 1 in 100,000.

Genome annotation

Identification of putative protein-encoding genes and initial automated annotation of the genome was performed by the Oak Ridge National Laboratory genome annotation pipeline. Additional gene prediction analysis and functional annotation was performed within the IMG platform [41].

Genome properties

The genome is 2,678,452 bp long and comprises one circular chromosome with 67% GC content (Figure 2). For the main chromosome, 2,493 genes were predicted, 2,407 of which are protein-coding genes. A total of 1,905 of protein coding genes were assigned to a putative function, with the remaining annotated as hypothetical proteins. In addition, 31 pseudo genes were identified. The properties and the statistics of the genome are summarized in Tables 3-4.

Figure 2

Graphical circular map of the genome. From outside to the center: Circle 1, genes on forward strand (colored by COG categories); Circle 2, genes on reverse strand (colored by COG categories); Circle 3, RNA genes (tRNAs green, rRNAs red, other RNAs black); Circle 4, mobile element genes; Circle 5, CRISPR-associated protein genes; Circle 6, GC content; Circle 7, GC skew.

Table 3

Nucleotide content and gene count levels of the genome



    % of total

Genome size (bp)



DNA coding region (bp)



DNA G+C content (bp)



Total genes


RNA genes



rRNA operons


Protein-coding genes



Pseudo genes



Genes in paralog clusters



Genes assigned to COGs



Genes with signal peptides



Genes with transmembrane helices



Table 4

Number of genes associated with the 25 general COG functional categories












     RNA processing and modification








     Replication, recombination and repair




     Chromatin structure and dynamics




     Cell cycle control, mitosis and meiosis




     Nuclear structure




     Defense mechanisms




     Signal transduction mechanisms




     Cell wall/membrane biogenesis




     Cell motility








     Extracellular structures




     Intracellular trafficking and secretion




     Posttranslational modification, protein turnover, chaperones




     Energy production and conversion




     Carbohydrate transport and metabolism




     Amino acid transport and metabolism




     Nucleotide transport and metabolism




     Coenzyme transport and metabolism




     Lipid transport and metabolism




     Inorganic ion transport and metabolism




     Secondary metabolites biosynthesis, transport and catabolism




     General function prediction only




     Function unknown




     Not in COGs

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


H. halophila is among the most halophilic eubacteria known. Further analysis and characterization of its genome will provide insights into the mechanisms it uses to adapt to hypersaline environments.



This work was performed under the auspices of the US Department of Energy Office of Science, Biological and Environmental Research Program, and by the University of California, Lawrence Berkeley National Laboratory under contract No. DE-AC02-05CH11231, Lawrence Livermore National Laboratory under Contract No. DE-AC52-07NA27344, and Los Alamos National Laboratory under contract No. DE-AC02-06NA25396, UT-Battelle, and Oak Ridge National Laboratory under contract DE-AC05-00OR22725.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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