S anda ds in Genomic Sciences (2011) 5:379-388 DOI:10.4056/sigs.2285059
The Genomic S anda ds Conso ium
Comple e genome sequence o he halophilic and highly
halo ole an Ch omohalobac e salexigens ype s ain
(1H11T)
Alex Copeland1, Ka hleen O’Conno 2, Susan Lucas1, Alla Lapidus1, Ke ie W. Be y1, John C.
De e 1,3, Tijana Gla ina Del Rio1, Nancy Hammon1, Eileen Dalin1, Hope Tice1, Sam Pi -
luck1, Da id B uce1,3, Lynne Goodwin1,3, Cli Han1,3, Roxanne Tapia1,3, Elizabe h Saund-
e s1,3, Je emy Schmu z3, Thomas B e in1,4 F ank La ime 1,4, Mi iam Land1,4, Lo en Hause 1,4,
Ca men Va gas5, Joaquin J. Nie o5, Nikos C. Ky pides1, Na alia I ano a1, Ma kus Göke 6,
Hans-Pe e Klenk6*, Laszlo N. Csonka2*, and Tanja Woyke1
1 DOE Join Genome Ins i u e, Walnu C eek, Cali o nia, USA
2 Depa men o Biological Sciences, Pu due Uni e si y, Wes La aye e, Indiana, USA
3 Los Alamos Na ional Labo a o y, Bioscience Di ision, Los Alamos, New Mexico, USA
4 Oak Ridge Na ional Labo a o y, Oak Ridge, Tennessee, USA
5 Depa men o Mic obiology and Pa asi ology, Uni e si y o Se ille, Spain
6 Leibniz Ins i u e DSMZ – Ge man Collec ion o Mic oo ganisms and Cell Cul u es,
B aunschweig, Ge many
*Co esponding au ho s: [email protected], [email protected] due.edu
Keywo ds: ae obic, chemoo gano ophic, G am-nega i e, mo ile, mode a ely halophilic, halo
ole an , ec oine syn hesis, Halomonadaceae, Gammap o eobac e ia, DOEM 2004
Ch omohalobac e salexigens is one o nine cu en ly known species o he genus Ch omoha-
lobac e in he amily Halomonadaceae. I is he mos halo ole an o he so-called ‘mod-
e a ely halophilic bac e ia’ cu en ly known and, due o i s s ong eu yhaline pheno ype, i is
an es ablished model o ganism o p oka yo ic osmoadap a ion. C. salexigens s ain 1H11T
and Halomonas elonga a a e he i s and he second membe s o he amily Halomonada-
ceae wi h a comple ely sequenced genome. The 3,696,649 bp long ch omosome wi h a o al
o 3,319 p o ein-coding and 93 RNA genes was sequenced as pa o he DOE Join Genome
Ins i u e P og am DOEM 2004.
In oduc ion
S ain 1H11T (= DSM 3043 = ATCC BAA-138 =
CECT 5384) is he ype s ain o he species Ch o-
mohalobac e salexigens [1], which is one o cu -
en ly nine species in he genus Ch omohalobac e
[1,2]. The genus name was de i ed om he G eek
wo ds ch oma, colo , hals halos, sal , and he Neo-
La in bac e , od, meaning he colo ed sal od.
The species epi he o igina ed om he La in
wo ds sal salis, sal , and exigo, o demand; sal -
demanding [3]. S ain 1H11T was o iginally iso-
la ed in 1974 in Bonai , Ne he lands An illes, om
sal e ns con aining 18.6% sal , and was ini ially
published as a s ain belonging o he species Ha-
lomonas elonga a [4]. In 2001, A ahal e al. ans-
e ed he s ain o he genus Ch omohalobac e
[2] as he ype s ain o he hen no el species C.
salexigens [1] ollowing de ailed pheno ypic, geno-
ypic, and phylogene ic analyses. C. salexigens is
known o i s e y b oad salini y ange [1] and o
i s ole as a model o ganism o p oka yo ic os-
mosadap a ion [5-7], e.g. he syn hesis o ec oines
(ec oine and hyd oxyec oine) o cell s ess p o-
ec ion [8,9]. He e we p esen a summa y classi i-
ca ion and cha ac e is ics o C. salexigens 1H11T,
oge he wi h he desc ip ion o he comple e ge-
nomic sequencing and anno a ion.
Classi ica ion and ea u es
The sequences o he i e iden ical 16S RNA genes
o s ain 1H11T we e compa ed using NCBI BLAST
[10] unde de aul se ings (e.g., conside ing only he
high-sco ing segmen pai s (HSPs) om he bes
250 hi s) wi h he mos ecen elease o he G een-
genes da abase [11] and he ela i e equencies o
axa and keywo ds ( educed o hei s em [12])
we e de e mined and weigh ed by BLAST sco es.
Ch omohalobac e salexigens ype s ain (1H11T)
380 S anda ds in Genomic Sciences
The mos equen ly occu ing gene a we e Halo-
monas (50.7%), Ch omohalobac e (46.3%),
'Hae e ehalobac e ' (1.7%), Bacillus (0.8%) and
Pseudomonas (0.5%) (214 hi s in o al). Fo 16 hi s
o sequences om membe s o he C. salexigens spe-
cies, he a e age iden i y wi hin HSPs was 99.9%
and he a e age co e age by HSPs was 97.9%. Fo
22 hi s o sequences om o he membe s o he ge-
nus Ch omohalobac e , he a e age iden i y wi hin
HSPs was 98.2% and he a e age co e age by HSPs
was 98.6%. Among all o he species, he one yielding
he highes sco e was Ch omohalobac e ma ismo -
ui (X87222), which co esponded o an iden i y o
99.9% and an HSP co e age o 100.0%. (No e ha
he G eengenes da abase uses he INSDC (=
EMBL/NCBI/DDBJ) anno a ion, which is no an au-
ho i a i e sou ce o nomencla u e o classi ica-
ion.) The highes -sco ing en i onmen al sequence
was EU799899 ('I 's all anking aqua ic Newpo
Ha bo RI clone 1C227569'), which showed an
iden i y o 100.0% and an HSP co e age o 100.0%.
The mos equen ly occu ing keywo ds wi hin he
labels o en i onmen al samples which yielded hi s
we e 'soil' (12.1%), 'lake' (3.6%), 'salin' (3.0%),
'ag icul u ' (2.9%) and 'alkalin, chang, lood, o me ,
mexico, excoco' (2.6%) (36 hi s in o al). The mos
equen ly occu ing keywo d wi hin he labels o
en i onmen al samples which yielded hi s o a high-
e sco e han he highes sco ing species was 'aqua ,
ha bou , newpo , ank' (25.0%) (2 hi s in o al).
These keywo ds i easonably well wi h he ecolog-
ical and physiological p ope ies epo ed o s ain
1H11T in he o iginal desc ip ion [1].
Figu e 1 shows he phylogene ic neighbo hood o C.
salexigens in a 16S RNA based ee. The sequences
o he i e iden ical 16S RNA gene copies in he ge-
nome di e by wo nucleo ides om he p e iously
published 16S RNA sequence (AJ295146), which
con ains h ee ambiguous base calls.
Figu e 1. Phylogene ic ee highligh ing he posi ion o C. salexigens ela i e o he ype s ains o he o h-
e species wi hin he genus and he ype species o he o he gene a wi hin he amily Halomonadaceae.
The ee was in e ed om 1,440 aligned cha ac e s [13,14] o he 16S RNA gene sequence unde he
maximum likelihood (ML) c i e ion [15]. Roo ing was done ini ially using he midpoin me hod [16] and
hen checked o i s ag eemen wi h he cu en classi ica ion (Table 1). The b anches a e scaled in e ms
o he expec ed numbe o subs i u ions pe si e. Numbe s adjacen o he b anches a e suppo alues
om 1,000 ML boo s ap eplica es [17] (le ) and om 1,000 maximum pa simony boo s ap eplica es
[18] ( igh ) i la ge han 60%. Lineages wi h ype s ain genome sequencing p ojec s egis e ed in GOLD
[19] a e labeled wi h one as e isk, hose also lis ed as 'Comple e and Published' wi h wo as e isks [20].
Copeland e al.
h p://s anda dsingenomics.o g 381
Cells o C. salexigens s ain 1H11T a e s aigh o
sligh ly cu ed ods, 0.7 o 1.0 by 2 o 3 µm in size
(Figu e 2) wi h squa ed ends and occu singly o in
pai s [1,4]. Cells o s ain 1H11T s ain G am-
nega i e, a e mo ile wi h pola lagella, s ic ly
ae obic, and a e non-spo e- o ming [1,4]. Ca bon
and ni ogen sou ce u iliza ion and biochemis y o
he s ain we e epo ed by A ahal e al. [1]. A pa -
ial cha ac e iza ion o he ca bon-sou ce u iliza-
ion by he o ganism has also been p esen ed by
Csonka e al. [36], who epo ed ha he s ain can
deg ade a numbe o a oma ic compounds, includ-
ing benzoa e, p o oca echua e, 4-hyd oxybenzoa e,
and oluene.
C. salexigens 1H11T is a halophile, which acco ding
o he classi ica ion p oposed by Kushne [37], is
on he bo de line be ween "mode a e" halophiles
( hose g owing op imally be ween 2.9 -14.5%
NaCl) and "ex eme" halophiles ( hose g owing
op imally be ween 8.7 -23.2% NaCl). In addi ion, i
displays ex ao dina ily high halo ole ance (con-
side ed as he abili y o li e and su i e unde
high sal concen a ions), and is able o g ow a
sal concen a ions o e 17.4% and 32% in de-
ined and complex media, espec i ely. Howe e ,
bo h he minimum NaCl equi emen and he up-
pe limi o NaCl ole ance a e dependen on
g ow h medium and empe a u e. The o ganism
can ole a e highe NaCl concen a ions in LB o in
o he complex media han in de ined media. In
de ined media, halo ole ance is enhanced by os-
mop o ec an s, such as glycine be aine o i s p e-
cu so , choline [4,6,33]. In he complex medium
SW (‘sea wa e ’), which is ou inely used o g ow-
ing his ype o mic oo ganism, s ain 1H11T
g ows op imally a 7.5 o 10% (w/ ) NaCl, wi h
g ow h occu ing o e he ange o 0.9% o 25%
NaCl [1]. In casein medium, which was ini ially
used o s ain isola ion, g ow h occu s in he
p esence o 32% sola sal s [4]. In SW medium
con aining 10% (w/ ) o al sal s, C. salexigens
1H11T can g ow a a pH ange om 5 o 10, wi h
an op imum a pH 7.5 [1]. In he same medium,
he empe a u e ange o g ow h is 15 – 45°C,
wi h an op imum a 37°C [1]. In he s anda d de-
ined medium M63, supplemen ed wi h glucose as
he sole ca bon sou ce, g ow h is op imal a 8.7 o
11.6% NaCl bu occu s o e he ange o 2.9%
NaCl o a maximum o 19% NaCl [6]. In e es ingly,
C. salexigens 1H11T exhibi s maximal g ow h a e
in glucose-M63 wi h only 1.8% (0.3M) NaCl in he
p esence o high concen a ions o sal s o o he
ino ganic ions, including K+, Rb+, NH4+, B -, NO3-, o
SO4- [38]. Howe e , i is an open ques ion whe he
his s ain is unique among halophiles in being
able o use o he ino ganic ions in addi ion o Na+
and Cl- o maximal g ow h a e.
Chemo axonomy
Da a on he s uc u e o he cell wall, a y acids
lipid composi ion, quinones and pola lipids a e
no a ailable.
Figu e 2. Ligh mic oscopic image o C. salexigens 1H11T
Ch omohalobac e salexigens ype s ain (1H11T)
382 S anda ds in Genomic Sciences
Table 1. Classi ica ion and gene al ea u es o C. salexigens acco ding o he MIGS ecommenda ions [21].
MIGS ID P ope y Te m E idence code
Cu en classi ica ion
Domain Bac e ia
TAS [22]
Phylum P o eobac e ia
TAS [23]
Class Gammap o eobac e ia
TAS [24,25]
O de Oceanospi illales
TAS [24,26]
Family Halomonadaceae TAS [27-31]
Genus Ch omohalobac e
TAS [2,32]
Species Ch omohalobac e salexigens
TAS [1]
Type s ain 1H11
TAS [1,4]
G am s ain nega i e TAS [1]
Cell shape od-shaped TAS [1]
Mo ili y mo ile TAS [1]
Spo ula ion none TAS [1]
Tempe a u e ange
mesophilic, 15–45°C
TAS [1]
Op imum empe a u e 37°C TAS [1]
Salini y
halophilic and halo ole an . Salini y ange om 0.9 o 32%
(w/ ) NaCl in ich media, 2.9% o 19% (0.5 M o 3.75M)
NaCl in minimal media; halo ole ance inc eased by osmo-
p o ec an s; halo ole ance dec eases a high empe a u e.
TAS [1,4,6,33]
MIGS-22 Oxygen equi emen uses O2 and NO3- as elec on accep o s; does no g ow
e men a i ely TAS [4]
Ca bon sou ce a ious o ganic acids, alcohols, suga s, and a oma ic
compounds TAS [1]
Ene gy me abolism chemoo gano ophic NAS
MIGS-6
Habi a
sal e n, esh wa e
TAS [4]
MIGS-15
Bio ic ela ionship
ee li ing
TAS [1]
MIGS-14
Pa hogenici y
none
NAS
Biosa e y le el 1 TAS [34]
Isola ion sola sal acili y, concen a ion mo e han 10% NaCl TAS [1]
MIGS-4
Geog aphic loca ion
Bonai e, Ne he lands An illes
TAS [1]
MIGS-5
Sample collec ion ime
June 1974
TAS [4]
MIGS-4.1 La i ude 12.25 NAS
MIGS-4.2 Longi ude -68.26 NAS
MIGS-4.3
Dep h
su ace
NAS
MIGS-4.4
Al i ude
sea le el
NAS
E idence codes - TAS: T aceable Au ho S a emen (i.e. a di ec epo exis s in he li e a u e); NAS: Non- aceable Au ho
S a emen (i.e. no di ec ly obse ed o he li ing, isola ed sample, bu based on a gene ally accep ed p ope y o he spe-
cies, o anecdo al e idence). These e idence codes a e om o he Gene On ology p ojec [35].
Genome sequencing and anno a ion
Genome p ojec his o y
This o ganism was selec ed o sequencing on he
basis o he DOE Join Genome Ins i u e P og am
DOEM 2004. The genome p ojec is deposi ed in
he Genomes On Line Da abase [19] and he com-
ple e genome sequence is deposi ed in GenBank.
Sequencing, inishing and anno a ion we e pe -
o med by he DOE Join Genome Ins i u e (JGI). A
summa y o he p ojec in o ma ion is shown in
Table 2.
S ain his o y
The his o y o s ain 1H11T begins wi h R.H.
V eeland, who deposi ed he o ganism in he
DSMZ open collec ion, whe e cul u es o he s ain
a e main ained eeze d ied as well as in liquid
ni ogen (since 1984). The s ain used o he
p ojec was p o ided by he Ca men Va gas – Joa-
quín Nie o lab in Se ille (Spain), who acqui ed i
om he DSMZ.
Copeland e al.
h p://s anda dsingenomics.o g 383
Table 2. Genome sequencing p ojec in o ma ion
MIGS ID P ope y Te m
MIGS-31 Finishing quali y Finished
MIGS-28 Lib a ies used Th ee genomic Sange lib a ies: 4 kb pUC, 8kb pMCL200 and osmid
pcc1Fos lib a ies.
MIGS-29 Sequencing pla o ms ABI3730
MIGS-31.2 Sequencing co e age 11.5 × Sange
MIGS-30 Assemble s Ph ap
MIGS-32 Gene calling me hod C i ica complemen ed wi h he ou pu o Glimme
INSDC ID CP000285
GenBank Da e o Release Ap il 16, 2006
GOLD ID Gc00371
NCBI p ojec ID 12636
Da abase: IMG 637000075
MIGS-13 Sou ce ma e ial iden i ie DSM 3043
P ojec ele ance Bio emedia ion, Bio echnology, En i onmen al
G ow h condi ions and DNA isola ion
The cul u e o s ain 1H11T, DSM 3043, used o
p epa e genomic DNA (gDNA) o sequencing was
g own in LB medium wi h 1 M NaCl. DNA was ex-
ac ed as desc ibed by O’Conno and Zusman
[39]. The pu i y, quali y and size o he bulk gDNA
p epa a ion we e assessed by JGI acco ding o
DOE-JGI guidelines.
Genome sequencing and assembly
The genome was sequenced using a combina ion o
4 kb, 8 kb and osmid DNA lib a ies. All gene al as-
pec s o lib a y cons uc ion and sequencing can be
ound a he JGI websi e [40]. D a assemblies
we e based on 44,750 o al eads. The
Ph ed/Ph ap/Consed so wa e package was used
o sequence assembly and quali y assessmen [41].
A e he sho gun s age, eads we e assembled wi h
pa allel ph ap (High Pe o mance So wa e, LLC).
Possible mis-assemblies we e co ec ed wi h Dup-
inishe o ansposon bombing o b idging clones
(Epicen e Bio echnologies, Madison, WI) [42].
Gaps be ween con igs we e closed by edi ing in
Consed, cus om p iming, o PCR ampli ica ion
(Roche Applied Science, Indianapolis, IN). A o al o
920 addi ional eac ions, 14 sha e and 18 ans-
poson bomb lib a ies we e needed o close gaps
and o aise he quali y o he inished sequence.
The e o a e o he comple ed genome sequence
is less han 1 in 100,000. Toge he all lib a ies p o-
ided 11.5 × co e age o he genome.
Genome anno a ion
Genes we e iden i ied using wo gene modeling
p og ams, Glimme [43] and C i ica [44] as pa o
he Oak Ridge Na ional Labo a o y genome anno-
a ion pipeline. The wo se s o gene calls we e
combined using C i ica as he p e e ed s a call
o genes wi h he same s op codon. Genes speci-
ying ewe han 80 amino acids ha we e p e-
dic ed by only one o he gene calle s and had no
Blas hi in he KEGG da abase a ≤1e-05, we e
dele ed. Au oma ed anno a ion was ollowed by a
ound o manual cu a ion o elimina e ob ious
o e laps. The p edic ed CDSs we e ansla ed and
used o sea ch he Na ional Cen e o Bio echnol-
ogy In o ma ion (NCBI) non- edundan da abase,
UniP o , TIGRFam, P am, PRIAM, KEGG, COG, and
In e P o da abases. These da a sou ces we e
combined o asse a p oduc desc ip ion o each
p edic ed p o ein. Non-coding genes and miscel-
laneous ea u es we e p edic ed using RNAscan-
SE [45], TMHMM [46], and signalP [47].
Genome p ope ies
The genome consis s o a 3,696,649 bp long ch o-
mosome wi h a 63.9% G+C con en (Figu e 3 and
Table 3). O he 3,412 pu a i e genes, 3,319 a e
p o ein-coding, and 93 speci y RNAs; 21 pseudo-
genes we e also iden i ied. The majo i y o he
p o ein-coding genes (76.8%) we e assigned a
pu a i e unc ion while he emaining ones we e
anno a ed as encoding hypo he ical p o eins. The
dis ibu ion o genes in o COGs unc ional ca ego-
ies is p esen ed in Table 4.

Ch omohalobac e salexigens ype s ain (1H11T)
384 S anda ds in Genomic Sciences
Figu e 3. G aphical ci cula map o he genome. F om ou side o he cen e : Genes on o wa d s and (colo by
COG ca ego ies), Genes on e e se s and (colo by COG ca ego ies), RNA genes ( RNAs g een, RNAs ed, o he
RNAs black), GC con en , GC skew.
Insigh s in o he genome
The publica ion o genome sequence s ain 1H11T
is p eceded by some publica ions ha we e based
on d a e sions o he sequence o on publicly
a ailable genome sequence and anno a ion. O en
e al. [48] ound ha he p edic ed isoelec ic
poin s o pe iplasmic p o eins o C. salexigens
1H11T a e signi ican ly mo e acidic han hose o
o hologous p o eins in mesophilic bac e ia, and
hey sugges ed ha his ea u e may con ibu e o
he halophilic cha ac e is ics o 1H11T. Analysis o
he genomic sequence indic ed ha he o ganism
has all o he enzymes o he Embden-Meye ho
glycoly ic pa hway, hexose monophospha e shun ,
and TCA cycle bu seemed o lack he s anda d
uc ose-1,6-bisphospha e phospha ase o he glu-
coneogene ic pa hway [36]. K ejcík e al. p e-
dic ed he ise hiona e o ma ion om au ine
based on he genome sequence [49]. A es e al.
ecen ly p esen ed a genome-scale econs uc ion
o a me abolic ne wo k o s ain 1H11T ocusing
on he up ake and accumula ion o indus ially
impo an o ganic osmoly es such as ec oine and
be aine [5].
Copeland e al.
h p://s anda dsingenomics.o g 385
Table 3. Genome S a is ics
A ibu e Value % o To al
Genome size (bp) 3,696,649 100.00%
DNA coding egion (bp) 3,333,410 90.17%
DNA G+C con en (bp) 2,362,597 63.91%
Numbe o eplicons 1
Ex ach omosomal elemen s 0
To al genes 3,412 100.00%
RNA genes 93 2.73%
RNA ope ons 5
P o ein-coding genes 3,319 97.27%
Pseudogenes 21 0.62%
Genes wi h unc ion p edic ion 2,621 76.82%
Genes in pa alog clus e s 402 11.78%
Genes assigned o COGs 2,842 83.29%
Genes assigned P am domains 2,928 85.81%
Genes wi h signal pep ides 689 20.19%
Genes wi h ansmemb ane helices 828 24.27%
CRISPR epea s 5
Table 4. Numbe o genes associa ed wi h he gene al COG unc ional ca ego ies
Code alue %age Desc ip ion
J 166 5.2 T ansla ion, ibosomal s uc u e and biogenesis
A 1 0.0 RNA p ocessing and modi ica ion
K 251 7.8 T ansc ip ion
L 114 3.5 Replica ion, ecombina ion and epai
B 1 0.0 Ch oma in s uc u e and dynamics
D 34 1.1 Cell cycle con ol, cell di ision, ch omosome pa i ioning
Y 0 0.0 Nuclea s uc u e
V 33 1.0 De ense mechanisms
T 152 4.7 Signal ansduc ion mechanisms
M 184 5.7 Cell wall/memb ane biogenesis
N 81 2.5 Cell mo ili y
Z 0 0.0 Cy oskele on
W 0 0.0 Ex acellula s uc u es
U 77 2.4 In acellula a icking and sec e ion, and esicula anspo
O 122 3.8 Pos ansla ional modi ica ion, p o ein u no e , chape ones
C 207 6.4 Ene gy p oduc ion and con e sion
G 227 7.1 Ca bohyd a e anspo and me abolism
E 324 10.1 Amino acid anspo and me abolism
F 81 2.5 Nucleo ide anspo and me abolism
H 152 4.7 Coenzyme anspo and me abolism
I 110 3.4 Lipid anspo and me abolism
P 175 5.4 Ino ganic ion anspo and me abolism
Q 76 2.4 Seconda y me aboli es biosyn hesis, anspo and ca abolism
R 385 12.0 Gene al unc ion p edic ion only
S 269 8.4 Func ion unknown
- 570 16.7 No in COGs
Ch omohalobac e salexigens ype s ain (1H11T)
386 S anda ds in Genomic Sciences
Acknowledgemen s
The wo k conduc ed by he U.S. Depa men o Ene gy
Join Genome Ins i u e was suppo ed by he O ice o
Science o he U.S. Depa men o Ene gy unde Con-
ac No. DE-AC02-05CH11231.
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