Complete genome sequence of the halophilic and highly halotolerant Chromohalobacter salexigens type strain (1H11(T)).

Chromohalobacter salexigens is one of nine currently known species of the genus Chromohalobacter in the family Halomonadaceae. It is the most halotolerant of the so-called 'moderately halophilic bacteria' currently known and, due to its strong euryhaline phenotype, it is an established model organism for prokaryotic osmoadaptation. C. salexigens strain 1H11(T) and Halomonas elongata are the first and the second members of the family Halomonadaceae with a completely sequenced genome. The 3,696,649 bp long chromosome with a total of 3,319 protein-coding and 93 RNA genes was sequenced as part of the DOE Joint Genome Institute Program DOEM 2004.

Introduction

Strain 1H11T (= DSM 3043 = ATCC BAA-138 = CECT 5384) is the type strain of the species Chromohalobacter salexigens [1], which is one of currently nine species in the genus Chromohalobacter [1,2]. The genus name was derived from the Greek words chroma, color, hals halos, salt, and the Neo-Latin bacter, rod, meaning the colored salt rod. The species epithet originated from the Latin words sal salis, salt, and exigo, to demand; salt-demanding [3]. Strain 1H11T was originally isolated in 1974 in Bonair, Netherlands Antilles, from salterns containing 18.6% salt, and was initially published as a strain belonging to the species Halomonas elongata [4]. In 2001, Arahal et al. transferred the strain to the genus Chromohalobacter [2] as the type strain of the then novel species C. salexigens [1] following detailed phenotypic, genotypic, and phylogenetic analyses. C. salexigens is known for its very broad salinity range [1] and for its role as a model organism for prokaryotic osmosadaptation [5-7], e.g. the synthesis of ectoines (ectoine and hydroxyectoine) for cell stress protection [8,9]. Here we present a summary classification and characteristics of C. salexigens 1H11T, together with the description of the complete genomic sequencing and annotation.

Classification and features

The sequences of the five identical 16S rRNA genes of strain 1H11T were compared using NCBI BLAST [10] under default settings (e.g., considering only the high-scoring segment pairs (HSPs) from the best 250 hits) with the most recent release of the Greengenes database [11] and the relative frequencies of taxa and keywords (reduced to their stem [12]) were determined and weighted by BLAST scores. The most frequently occurring genera were Halomonas (50.7%), Chromohalobacter (46.3%), 'Haererehalobacter' (1.7%), Bacillus (0.8%) and Pseudomonas (0.5%) (214 hits in total). For 16 hits to sequences from members of the C. salexigens species, the average identity within HSPs was 99.9% and the average coverage by HSPs was 97.9%. For 22 hits to sequences from other members of the genus Chromohalobacter, the average identity within HSPs was 98.2% and the average coverage by HSPs was 98.6%. Among all other species, the one yielding the highest score was Chromohalobacter marismortui (X87222), which corresponded to an identity of 99.9% and an HSP coverage of 100.0%. (Note that the Greengenes database uses the INSDC (= EMBL/NCBI/DDBJ) annotation, which is not an authoritative source for nomenclature or classification.) The highest-scoring environmental sequence was EU799899 ('It's all ranking aquatic Newport Harbor RI clone 1C227569'), which showed an identity of 100.0% and an HSP coverage of 100.0%. The most frequently occurring keywords within the labels of environmental samples which yielded hits were 'soil' (12.1%), 'lake' (3.6%), 'salin' (3.0%), 'agricultur' (2.9%) and 'alkalin, chang, flood, former, mexico, texcoco' (2.6%) (36 hits in total). The most frequently occurring keyword within the labels of environmental samples which yielded hits of a higher score than the highest scoring species was 'aquat, harbour, newport, rank' (25.0%) (2 hits in total). These keywords fit reasonably well with the ecological and physiological properties reported for strain 1H11T in the original description [1].

Figure 1 shows the phylogenetic neighborhood of C. salexigens in a 16S rRNA based tree. The sequences of the five identical 16S rRNA gene copies in the genome differ by two nucleotides from the previously published 16S rRNA sequence (AJ295146), which contains three ambiguous base calls.

Figure 1

Phylogenetic tree highlighting the position of C. salexigens relative to the type strains of the other species within the genus and the type species of the other genera within the family Halomonadaceae. The tree was inferred from 1,440 aligned characters [13,14] of the 16S rRNA gene sequence under the maximum likelihood (ML) criterion [15]. Rooting was done initially using the midpoint method [16] and then checked for its agreement with the current classification (Table 1). The branches are scaled in terms of the expected number of substitutions per site. Numbers adjacent to the branches are support values from 1,000 ML bootstrap replicates [17] (left) and from 1,000 maximum parsimony bootstrap replicates [18] (right) if larger than 60%. Lineages with type strain genome sequencing projects registered in GOLD [19] are labeled with one asterisk, those also listed as 'Complete and Published' with two asterisks [20].

Table 1

Classification and general features of C. salexigens according to the MIGS recommendations [21].

MIGS ID	Property	Term	Evidence code
	Current classification	Domain Bacteria	TAS [22]
		Phylum Proteobacteria	TAS [23]
		Class Gammaproteobacteria	TAS [24,25]
		Order Oceanospirillales	TAS [24,26]
		Family Halomonadaceae	TAS [27-31]
		Genus Chromohalobacter	TAS [2,32]
		Species Chromohalobacter salexigens	TAS [1]
		Type strain 1H11	TAS [1,4]
	Gram stain	negative	TAS [1]
	Cell shape	rod-shaped	TAS [1]
	Motility	motile	TAS [1]
	Sporulation	none	TAS [1]
	Temperature range	mesophilic, 15–45°C	TAS [1]
	Optimum temperature	37°C	TAS [1]
	Salinity	halophilic and halotolerant. Salinity range from 0.9 to 32% (w/v) NaCl in rich media,     2.9% to 19% (0.5 M to 3.75M) NaCl in minimal media;    halotolerance increased by osmoprotectants;    halotolerance decreases at high temperature.	TAS [1,4,6,33]
MIGS-22	Oxygen requirement	uses O2 and NO3- as electron acceptors; does not grow fermentatively	TAS [4]
	Carbon source	various organic acids, alcohols, sugars, and aromatic compounds	TAS [1]
	Energy metabolism	chemoorganotrophic	NAS
MIGS-6	Habitat	saltern, fresh water	TAS [4]
MIGS-15	Biotic relationship	free living	TAS [1]
MIGS-14	Pathogenicity	none	NAS
	Biosafety level	1	TAS [34]
	Isolation	solar salt facility, concentration more than 10% NaCl	TAS [1]
MIGS-4	Geographic location	Bonaire, Netherlands Antilles	TAS [1]
MIGS-5	Sample collection time	June 1974	TAS [4]
MIGS-4.1	Latitude	12.25	NAS
MIGS-4.2	Longitude	-68.26	NAS
MIGS-4.3	Depth	surface	NAS
MIGS-4.4	Altitude	sea level	NAS

Evidence codes - 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 of the Gene Ontology project [35].

Cells of C. salexigens strain 1H11T are straight or slightly curved rods, 0.7 to 1.0 by 2 to 3 µm in size (Figure 2) with squared ends and occur singly or in pairs [1,4]. Cells of strain 1H11T stain Gram-negative, are motile with polar flagella, strictly aerobic, and are non-spore-forming [1,4]. Carbon and nitrogen source utilization and biochemistry of the strain were reported by Arahal et al. [1]. A partial characterization of the carbon-source utilization by the organism has also been presented by Csonka et al. [36], who reported that the strain can degrade a number of aromatic compounds, including benzoate, protocatechuate, 4-hydroxybenzoate, and toluene.

Figure 2

Light microscopic image of C. salexigens 1H11T

C. salexigens 1H11T is a halophile, which according to the classification proposed by Kushner [37], is on the borderline between "moderate" halophiles (those growing optimally between 2.9 -14.5% NaCl) and "extreme" halophiles (those growing optimally between 8.7 -23.2% NaCl). In addition, it displays extraordinarily high halotolerance (considered as the ability to live and survive under high salt concentrations), and is able to grow at salt concentrations over 17.4% and 32% in defined and complex media, respectively. However, both the minimum NaCl requirement and the upper limit of NaCl tolerance are dependent on growth medium and temperature. The organism can tolerate higher NaCl concentrations in LB or in other complex media than in defined media. In defined media, halotolerance is enhanced by osmoprotectants, such as glycine betaine or its precursor, choline [4,6,33]. In the complex medium SW (‘sea water’), which is routinely used for growing this type of microorganism, strain 1H11T grows optimally at 7.5 to 10% (w/v) NaCl, with growth occurring over the range of 0.9% to ­25% NaCl [1]. In casein medium, which was initially used for strain isolation, growth occurs in the presence of 32% solar salts [4]. In SW medium containing 10% (w/v) total salts, C. salexigens 1H11T can grow at a pH range from 5 to 10, with an optimum at pH 7.5 [1]. In the same medium, the temperature range for growth is 15 – 45°C, with an optimum at 37°C [1]. In the standard defined medium M63, supplemented with glucose as the sole carbon source, growth is optimal at 8.7 to 11.6% NaCl but occurs over the range of 2.9% NaCl or a maximum of 19% NaCl [6]. Interestingly, C. salexigens 1H11T exhibits maximal growth rate in glucose-M63 with only 1.8% (0.3M) NaCl in the presence of high concentrations of salts of other inorganic ions, including K+, Rb+, NH4+, Br-, NO3-, or SO4- [38]. However, it is an open question whether this strain is unique among halophiles in being able to use other inorganic ions in addition to Na+ and Cl- for maximal growth rate.

Chemotaxonomy

Data on the structure of the cell wall, fatty acids lipid composition, quinones and polar lipids are not available.

Genome sequencing and annotation

Genome project history

This organism was selected for sequencing on the basis of the DOE Joint Genome Institute Program DOEM 2004. The genome project is deposited in the Genomes On Line Database [19] and the complete genome sequence is deposited in GenBank. Sequencing, finishing and annotation were performed by the DOE Joint Genome Institute (JGI). A summary of the project information is shown in Table 2.

Table 2

Genome sequencing project information

MIGS ID	Property	Term
MIGS-31	Finishing quality	Finished
MIGS-28	Libraries used	Three genomic Sanger libraries: 4 kb pUC, 8kb pMCL200 and fosmid pcc1Fos libraries.
MIGS-29	Sequencing platforms	ABI3730
MIGS-31.2	Sequencing coverage	11.5 × Sanger
MIGS-30	Assemblers	Phrap
MIGS-32	Gene calling method	Critica complemented with the output of Glimmer
	INSDC ID	CP000285
	GenBank Date of Release	April 16, 2006
	GOLD ID	Gc00371
	NCBI project ID	12636
	Database: IMG	637000075
MIGS-13	Source material identifier	DSM 3043
	Project relevance	Bioremediation, Biotechnology, Environmental

Strain history

The history of strain 1H11T begins with R.H. Vreeland, who deposited the organism in the DSMZ open collection, where cultures of the strain are maintained freeze dried as well as in liquid nitrogen (since 1984). The strain used for the project was provided by the Carmen Vargas – Joaquín Nieto lab in Seville (Spain), who acquired it from the DSMZ.

Growth conditions and DNA isolation

The culture of strain 1H11T, DSM 3043, used to prepare genomic DNA (gDNA) for sequencing was grown in LB medium with 1 M NaCl. DNA was extracted as described by O’Connor and Zusman [39]. The purity, quality and size of the bulk gDNA preparation were assessed by JGI according to DOE-JGI guidelines.

Genome sequencing and assembly

The genome was sequenced using a combination of 4 kb, 8 kb and fosmid DNA libraries. All general aspects of library construction and sequencing can be found at the JGI website [40]. Draft assemblies were based on 44,750 total reads. The Phred/Phrap/Consed software package was used for sequence assembly and quality assessment [41]. After the shotgun stage, reads were assembled with parallel phrap (High Performance Software, LLC). Possible mis-assemblies were corrected with Dupfinisher or transposon bombing of bridging clones (Epicentre Biotechnologies, Madison, WI) [42]. Gaps between contigs were closed by editing in Consed, custom priming, or PCR amplification (Roche Applied Science, Indianapolis, IN). A total of 920 additional reactions, 14 shatter and 18 transposon bomb libraries were needed to close gaps and to raise the quality of the finished sequence. The error rate of the completed genome sequence is less than 1 in 100,000. Together all libraries provided 11.5 × coverage of the genome.

Genome annotation

Genes were identified using two gene modeling programs, Glimmer [43] and Critica [44] as part of the Oak Ridge National Laboratory genome annotation pipeline. The two sets of gene calls were combined using Critica as the preferred start call for genes with the same stop codon. Genes specifying fewer than 80 amino acids that were predicted by only one of the gene callers and had no Blast hit in the KEGG database at ≤1e-05, were deleted. Automated annotation was followed by a round of manual curation to eliminate obvious overlaps. The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) non-redundant database, UniProt, TIGRFam, Pfam, PRIAM, KEGG, COG, and InterPro databases. These data sources were combined to assert a product description for each predicted protein. Non-coding genes and miscellaneous features were predicted using tRNAscan-SE [45], TMHMM [46], and signalP [47].

Genome properties

The genome consists of a 3,696,649 bp long chromosome with a 63.9% G+C content (Table 3 and Figure 3). Of the 3,412 putative genes, 3,319 are protein-coding, and 93 specify RNAs; 21 pseudogenes were also identified. The majority of the protein-coding genes (76.8%) were assigned a putative function while the remaining ones were annotated as encoding hypothetical proteins. The distribution of genes into COGs functional categories is presented in Table 4.

Table 3

Genome Statistics

Attribute	Value	% of Total
Genome size (bp)	3,696,649	100.00%
DNA coding region (bp)	3,333,410	90.17%
DNA G+C content (bp)	2,362,597	63.91%
Number of replicons	1
Extrachromosomal elements	0
Total genes	3,412	100.00%
RNA genes	93	2.73%
rRNA operons	5
Protein-coding genes	3,319	97.27%
Pseudogenes	21	0.62%
Genes with function prediction	2,621	76.82%
Genes in paralog clusters	402	11.78%
Genes assigned to COGs	2,842	83.29%
Genes assigned Pfam domains	2,928	85.81%
Genes with signal peptides	689	20.19%
Genes with transmembrane helices	828	24.27%
CRISPR repeats	5

Figure 3

Graphical circular map of the genome. From outside to the center: Genes on forward strand (color by COG categories), Genes on reverse strand (color by COG categories), RNA genes (tRNAs green, rRNAs red, other RNAs black), GC content, GC skew.

Table 4

Number of genes associated with the general COG functional categories

Code	value	%age	Description
J	166	5.2	Translation, ribosomal structure and biogenesis
A	1	0.0	RNA processing and modification
K	251	7.8	Transcription
L	114	3.5	Replication, recombination and repair
B	1	0.0	Chromatin structure and dynamics
D	34	1.1	Cell cycle control, cell division, chromosome partitioning
Y	0	0.0	Nuclear structure
V	33	1.0	Defense mechanisms
T	152	4.7	Signal transduction mechanisms
M	184	5.7	Cell wall/membrane biogenesis
N	81	2.5	Cell motility
Z	0	0.0	Cytoskeleton
W	0	0.0	Extracellular structures
U	77	2.4	Intracellular trafficking and secretion, and vesicular transport
O	122	3.8	Posttranslational modification, protein turnover, chaperones
C	207	6.4	Energy production and conversion
G	227	7.1	Carbohydrate transport and metabolism
E	324	10.1	Amino acid transport and metabolism
F	81	2.5	Nucleotide transport and metabolism
H	152	4.7	Coenzyme transport and metabolism
I	110	3.4	Lipid transport and metabolism
P	175	5.4	Inorganic ion transport and metabolism
Q	76	2.4	Secondary metabolites biosynthesis, transport and catabolism
R	385	12.0	General function prediction only
S	269	8.4	Function unknown
-	570	16.7	Not in COGs

Insights into the genome

The publication of genome sequence strain 1H11T is preceded by some publications that were based on draft versions of the sequence or on publicly available genome sequence and annotation. Oren et al. [48] found that the predicted isoelectric points of periplasmic proteins of C. salexigens 1H11T are significantly more acidic than those of orthologous proteins in mesophilic bacteria, and they suggested that this feature may contribute to the halophilic characteristics of 1H11T. Analysis of the genomic sequence indicted that the organism has all of the enzymes of the Embden-Meyerhof glycolytic pathway, hexose monophosphate shunt, and TCA cycle but seemed to lack the standard fructose-1,6-bisphosphate phosphatase of the gluconeogenetic pathway [36]. Krejcík et al. predicted the isethionate formation from taurine based on the genome sequence [49]. Ates et al. recently presented a genome-scale reconstruction of a metabolic network for strain 1H11T focusing on the uptake and accumulation of industrially important organic osmolytes such as ectoine and betaine [5].