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Proc Natl Acad Sci U S A. 2000 October 24; 97(22): 12176–12181. Published online 2000 October 3. | PMCID: PMC17314 |
Copyright © 2000, The National Academy of Sciences Genetics Genome sequence of Halobacterium species NRC-1 Wailap Victor Ng,ab Sean P. Kennedy,c Gregory G. Mahairas,ab Brian Berquist,c Min Pan,ab Hem Dutt Shukla,c Stephen R. Lasky,ab Nitin S. Baliga,c Vesteinn Thorsson,ab Jennifer Sbrogna,c Steven Swartzell,a Douglas Weir,c John Hall,a Timothy A. Dahl,ab Russell Welti,ab Young Ah Goo,ab Brent Leithauser,a Kim Keller,a Randy Cruz,a Michael J. Danson,d David W. Hough,d Deborah G. Maddocks,d Peter E. Jablonski,e Mark P. Krebs,f Christine M. Angevine,f Heather Dale,f Thomas A. Isenbarger,f Ronald F. Peck,f Mechthild Pohlschroder,g John L. Spudich,h Kwang-Hwan Jung,h Maqsudul Alam,i Tracey Freitas,i Shaobin Hou,i Charles J. Daniels,j Patrick P. Dennis,k Arina D. Omer,k Holger Ebhardt,k Todd M. Lowe,l Ping Liang,m Monica Riley,m Leroy Hood,abn and Shiladitya DasSarmacn aDepartment of Molecular Biotechnology, University of Washington, Seattle, WA 98195; cDepartment of Microbiology, University of Massachusetts, Amherst, MA 01003; dCentre for Extremophile Research, Department of Biology and Biochemistry, University of Bath, Bath, BA2 7AY, United Kingdom; eDepartment of Biological Sciences, Northern Illinois University, DeKalb, IL 60115; fDepartment of Biomolecular Chemistry, University of Wisconsin Medical School, Madison, WI 53706; gDepartment of Biology, University of Pennsylvania, Philadelphia, PA 19104; hDepartment of Microbiology and Molecular Genetics, University of Texas Medical School, Houston, TX 77030; iDepartment of Microbiology, University of Hawaii, Honolulu, HI 96822; jDepartment of Microbiology, Ohio State University, Columbus, OH 43210; kDepartment of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, Canada V6T 1Z3; lDepartment of Genetics, Stanford University School of Medicine, Stanford, CA 94305; mMarine Biological Laboratory, Woods Hole, MA 02543; and bInstitute for Systems Biology, Seattle, WA 98105 Accepted July 20, 2000. We report the complete sequence of an extreme halophile,
Halobacterium sp. NRC-1, harboring a dynamic
2,571,010-bp genome containing 91 insertion sequences representing 12
families and organized into a large chromosome and 2 related
minichromosomes. The Halobacterium NRC-1 genome codes
for 2,630 predicted proteins, 36% of which are unrelated to any
previously reported. Analysis of the genome sequence shows the presence
of pathways for uptake and utilization of amino acids, active
sodium-proton antiporter and potassium uptake systems, sophisticated
photosensory and signal transduction pathways, and DNA replication,
transcription, and translation systems resembling more complex
eukaryotic organisms. Whole proteome comparisons show the definite
archaeal nature of this halophile with additional similarities to the
Gram-positive Bacillus subtilis and other bacteria. The
ease of culturing Halobacterium and the availability of
methods for its genetic manipulation in the laboratory, including
construction of gene knockouts and replacements, indicate this
halophile can serve as an excellent model system among the archaea. Halobacterium
species o are obligately halophilic microorganisms
that have adapted to optimal growth under conditions of extremely high
salinity—10 times that of sea water. They contain a correspondingly
high concentration of salts internally and exhibit a variety of unusual
and unique molecular characteristics. Since their discovery, extreme
halophiles have been studied extensively by chemists, biochemists,
microbiologists, and molecular biologists to define both molecular
diversity and universal features of life. A notable list of early
research milestones on halophiles includes the discovery of a cell
envelope composed of an S-layer glycoprotein, archaeol ether lipids and
purple membrane, and metabolic and biosynthetic processes operating at
saturating salinities ( 1). These early discoveries established the
value of investigations directed at extremophiles and set the stage for
pioneering phylogenetic studies leading to the three-domain view of
life and classification of Halobacterium as a member of the
archaeal domain ( 2, 3). The Halobacterium genome was originally studied in the 1960s
and found to be composed of two components, a GC-rich (68%) major
fraction and a relatively AT-rich (58% GC) satellite ( 4, 5).
Subsequent work showed that satellite DNA corresponded to the presence
of large and variable covalently closed extrachromosomal circles and a
large number of transposable insertion sequence (IS) elements, which
explained the observed genetic plasticity of halophiles ( 6, 7). For
Halobacterium NRC-1, 3 circular replicons were mapped, a
≈2-Mbp chromosome and 2 large replicons, pNRC200 and pNRC100, about
350 and 200 Kbp in size ( 8– 11). We sequenced pNRC100 as a preliminary
step in this genome project ( 12) and found a dynamic 191,346-bp
replicon containing 176 putative genes, several of which are likely to
be essential. The complete genome sequence of Halobacterium NRC-1 is
notable because of the excellent characteristics of halophiles as
experimental organisms among the archaea ( 13). Culturing is facile,
because they are both aerobic and mesophilic. DNA-mediated
transformation may be accomplished at high efficiency, and cloning and
expression vectors with selectable markers are readily available.
Several gene replacement and knockout strategies have been used
successfully, including a recently developed selectable and
counterselectable method by using the yeast ura3 gene
homolog, which should permit systematic knockout of all nonessential
genes ( 14). Moreover, large-scale PCR amplification has been conducted
successfully and DNA arrays constructed for interrogating patterns of
gene expression. For biochemical analysis, Halobacterium
proteins can be released by lysis in hypotonic medium and stabilized by
addition of salts and other compatible solutes. Both membrane and
soluble proteins have been useful for structural studies using electron
and x-ray methods ( 15– 17). These characteristics, coupled with the
complete genome sequence, make Halobacterium NRC-1 an
excellent experimental model among the archaea. Genome Sequence, Annotation, and Organization We sequenced the Halobacterium NRC-1 genome by using a
whole genome shotgun strategy. Approximately 45,000 high-quality
sequences were obtained by using automated Applied Biosystems
sequencers, which provided ×7.5 coverage of the large chromosome. We
used 505 oligonucleotides for directed sequencing of lower-quality
regions or regions with single coverage. The remaining low-quality
regions were covered by sequencing both ends of 124 PCR amplified
genomic fragments. The shotgun Halobacterium NRC-1 sequences
were assembled by using the phredphrap programs
( 19– 21). Initially, all of the known and putative new IS elements were
masked in the assembly. This resulted in 84 high-quality contigs, which
were subsequently merged into groups of 2–10 adjacent contigs by a
second round of assembly without repeat masking. Finally, the grouped
contig consensus sequences were merged into three circular contigs by
using a third round of assembly with the
phredphrap programs. The sequences have been
deposited in GenBank and assigned the following accession numbers:
AE004437, AE004438, and AF016485. Our results confirmed the expected size and structure of the
Halobacterium NRC-1 genome. The genome was found to be
2,571,010 bp in size and composed of 3 circular replicons, a
2,014,239-bp-large chromosome and 2 smaller replicons, pNRC100 (191,346
bp) ( 12) and pNRC200 (365,425 bp). Interestingly, pNRC100 and pNRC200
contained a 145,428-bp region of identity, including 33- to 39-kb
inverted repeats that mediate inversion isomerization ( 10). These two
replicons were substantially less GC rich than the largest replicon
(57.9% and 59.2% vs. 67.9%). The genome contained 91 IS elements
representing 12 families, including 29 on pNRC100 ( 12), 40 on pNRC200,
and 22 on the large chromosome. Two new elements, ISH5 and ISH10, were
identified. The program glimmer ( 22, 23) was used for gene prediction
on the finished Halobacterium NRC-1 genome sequence.
Predicted genes were translated and the resulting sequences used to
search the nonredundant database of proteins (translation of GenBank
CDS, Protein Data Bank, SwissProt, and Protein Identification Resource
databases) available on the National Center for Biotechnology
Information web site by using the netblast
program ( 24) in the GCG software package (Genetics Computer Group,
Madison, WI). To aid in the processing of large numbers of data files,
we developed perl-based scripts to handle
recursively the input of sequences and their analysis. Additional
analysis was conducted by a consortium of 12 laboratories
( http://zdna.micro.umass.edu/haloweb). Our analysis identified 2,682 likely genes (including 52 RNA
genes) in the Halobacterium NRC-1 genome, of which 1,658
coded proteins with significant matches to the databases. Of the
matches, 591 were to conserved hypothetical proteins, and 1,067 were to
proteins with known or predicted function. The large chromosome
contained 2,111 putative genes, pNRC200 contained 374, and pNRC100
contained 197. A significantly larger fraction of the genes on the
large chromosome (45%) matched to genes of known function in the
databases than did genes on either pNRC200 (32%) or pNRC100 (26%).
The complete genetic map and table of genes and genetic elements are
available on the PNAS web site as supplementary material
( www.pnas.org). Interestingly, about 40 genes on pNRC100 and pNRC200 coded for
proteins likely to be essential or important for cell viability such as
a DNA polymerase, seven TBP and TFB transcription factors, and the
arginyl-tRNA synthetase, indicating that these replicons are
minichromosomes ( 12). A fraction of these genes have a G + C
composition that is significantly higher than the minichromosome
average, e.g., those coding for potassium and phosphate uptake,
thioredoxin reductase, cytochrome oxidase, and Orc/Cdc6 cell division
proteins. These results and the finding of many IS elements on pNRC100
and pNRC200 indicate that the minichromosomes contribute to
Halobacterium genome evolution by facilitating the
acquisition of new genes ( 12). Halobacterium NRC-1 is an aerobic chemoorganotroph,
growing on the degradation products of less halophilic organisms as the
salinity reaches near saturation. In the laboratory, cells are cultured
best in a complex medium ( 13, 25). A minimal medium described for
Halobacterium includes all but 5 of the 20 amino acids for
growth ( 26). Several amino acids may be used as a source of energy,
including arginine and aspartate, which are passed to the citric acid
cycle via 2-oxoglutarate and oxaloacetate, respectively (Fig.
). Under aerobic conditions, arginine is
presumably converted to glutamate via the arginine deiminase pathway,
and this amino acid then enters the cycle via glutamate dehydrogenase.
The arginine deiminase pathway is coded by the arcRACB genes
( 27), which are found on pNRC200.
In accordance with the ability of Halobacterium NRC-1 to
grow on amino acids, which ultimately are catabolized by the citric
acid cycle, the genes coding all of the enzymes for an aerobic cycle
are present (Fig. ). In common with all archaea, the conversion of
pyruvate to acetyl-CoA (before the citric acid cycle) and of
2-oxoglutarate to succinyl-CoA are catalyzed by the respective
2-oxoacid ferredoxin oxidoreductases ( 28, 29). Interestingly, genes
encoding malate ferredoxin oxidoreductase and fumarate reductase are
also present, so that when combined with the 2-oxoglutarate
oxidoreductase, they could form a partial reverse citric acid cycle
from oxaloacetate to 2-oxoglutarate under anaerobic conditions, as has
been found in a number of methanogenic archaea ( 30, 31). In connection
with the citric acid cycle, the key enzymes of the glyoxylate cycle,
isocitrate lyase, and malate synthase, could not be identified in the
genome sequence. This is in accord with an inability of
Halobacterium to grow on acetate ( 32, 33). Growth on amino acids requires a gluconeogenic pathway for carbohydrate
synthesis, and the genes for a reverse Embden–Meyerhof glycolytic
pathway have been identified except for fructose-1,6-bisphosphate
aldolase. The inability to find this gene was unexpected, particularly
as those for triose-phosphate isomerase and fructose-1,6-bisphosphatase
are present. However, an unusual class I aldolase found in eukaryotic
organisms has been detected in some related Haloarcula
species ( 34), and it may be that a similar enzyme is present in
Halobacterium NRC-1 but is too divergent in sequence to
permit assignment. Although Halobacterium is reported to be unable to
metabolize sugars, genes coding for glucose dehydrogenase and
2-keto-3-deoxygluconate kinase appear to be present in NRC-1. These are
enzymes of the semiphosphorylated Entner–Doudoroff pathway shown to be
present in several halophilic archaea ( 25, 35), although the gene for
2-keto-3-deoxy-6-phosphogluconate aldolase remains to be assigned in
NRC-1. With respect to glucose catabolism via an Embden–Meyerhof
glycolytic pathway, a 6-phosphofructokinase gene could not be found by
using both ATP- and ADP-dependent homologs as queries. The genes for
the catabolism of glyceraldeyde 3-phosphate (the product of glucose
catabolism via Entner–Doudoroff and/or Embden–Meyerhof pathways) to
pyruvate are all present, and it is these same enzymes that function to
effect gluconeogenesis. Halobacterium NRC-1 also possesses genes encoding enzymes of
the bacterial-like fatty acid β-oxidation pathway. Both medium-chain
and long-chain acyl-CoA ligases, 3 acyl-CoA dehydrogenases, enoyl-CoA
hydratase, 2 3-hydroxyacyl-CoA dehydrogenases, and 2 3-ketoacyl-CoA
thiolases are present. However, despite the presence of these genes,
there are no reports of the oxidation of fatty acids by NRC-1. Finally,
a gene cluster coding for proteins similar to a 2-oxoacid dehydrogenase
complex in Bacillus species was identified in NRC-1,
including pyruvate decarboxylase (a and b chains), lipoyl
acyltransferase, and dihydrolipoamide dehydrogenase, as has also been
reported in Haloferax volcanii ( 36, 37). Cell Envelope Components and Transport The cell envelope of Halobacterium NRC-1 consists of a
single lipid bilayer membrane surrounded by an S-layer assembled from
the cell-surface glycoprotein ( 38). Although the cytoplasm is in
osmotic equilibrium with the hypersaline environment, the cell
maintains a high (≈4 M) intracellular K +
concentration that is equivalent to the external
Na + concentration ( 39). The passive permeability
of the membrane to K + and
Na + ions is low ( 40), so active transport is
required to maintain the ionic distribution. Accordingly, NRC-1 has
multiple active K + transporters, including
KdpABC, an ATP-driven K + transport system, and
TrkAH, a low-affinity K + transporter driven by
the membrane potential (Fig. ). Active Na +
efflux is probably mediated by NhaC proteins, which likely correspond
to the unidirectional
Na +/H + antiporter
activity described previously ( 41). Interestingly, KdpABC, TrkA (three
of five copies), and NhaC (one of three copies) are coded by pNRC200. At least 27 members of the ABC transporter superfamily are
present in Halobacterium NRC-1. Among active transporters
for nutrient uptake identified were those for cationic amino acids
(Cat) and proline (PutP), dipeptides (DppABCDF), oligopeptides
(AppACF), and a sugar transporter (Rbs) (Fig. ). Among small-ion
transporters, most were closely related to bacterial proteins. Genes
for exporting heavy metals (arsenite and cadmium) and other toxic
compounds (multidrug-resistance homologs) are present (Fig. ). Four
ars genes ( arsRDAC) are clustered on pNRC100
( 12), whereas a fifth gene ( arsB) resides on the large
chromosome. Phosphate transport is mediated by at least two systems,
including PstABC (two copies) and phosphate permease (three copies);
all but one copy of pstABC are coded by pNRC200. For polypeptide translocation across the membrane, the general
secretory (Sec) machinery for Halobacterium NRC-1 appears to
be a hybrid of the eukaryotic and bacterial systems ( 44). The core
components, Sec61α/SecY and Sec61γ/SecE, as well as those of
the signal recognition particle, SRP54/Ffh and its 7S RNA scaffold,
are related to the corresponding eukaryotic factors (Fig. ). The SRP
complex also includes SRP19, a subunit found in eukaryotes but not in
bacteria. On the other hand, like bacteria, NRC-1 contains the
universally conserved SRP-receptor subunit SRPα/FtsY and lacks the
eukaryotic β-subunit homolog. The bacterial translocase protein
homologs SecD and SecF are also present, but the essential bacterial
ATPase SecA is absent. In addition, a gene closely related to
tatC of A. fulgidus ( 45) was found, suggesting
the presence of the twin-arginine protein export pathway. The polar lipids of Halobacterium include phospholipids and
glycolipids based on archaeol, a glycerol diether lipid containing
phytanyl chains derived from C 20 isoprenoids
( 46). All of the key enzymes of isoprenoid synthesis were identified,
including HMG-CoA reductase (MvaA), the target of the growth inhibitor
mevinolin ( 47). Interestingly, two genes for this pathway, coding for
mevalonate pyrophosphate decarboxylase and isopentenyl pyrophosphate
isomerase, have not been found in the genomes of other archaea. Enzymes
catalyzing formation of polar lipids, which have been outlined by
metabolic labeling from mevalonate and dihydroxyacetone ( 48), are coded
in NRC-1. For synthesis of phospholipids, proteins related to bacterial
and archaeal phosphatidyl transferases (PgsA and PssA) are present,
although CDP-archaeol synthase has not been identified. Because the apolar lipids of Halobacterium are
isoprenoids, their synthesis likely requires some of the same machinery
needed to synthesize the phytanyl chains of archaeol. Additional
enzymes are required to synthesize the C 30
isoprenoid squalene, the C 40 retinal-precursor
β-carotene, and C 50 bacterioruberins, which are
thought to act as photoprotectants ( 49). We identified two phytoene
synthase and three phytoene dehydrogenase homologs in NRC-1. Signal Transduction and Photobiology Halobacterium inhabits a harsh environment with extreme
solar radiation and dynamic nutritional conditions. Accordingly,
Halobacterium cells have developed sophisticated sensory
pathways for color-sensitive phototaxis, chemotaxis to a large variety
of substances, aerotaxis, osmotaxis, and thermotaxis (Fig. ). Compared
with the 5 methyl-accepting taxis transducers in Escherichia
coli, the NRC-1 genome reveals at least 17 homologous
methyl-accepting proteins, 13 of which had been previously identified
( 50– 52). One transducer reported in other Halobacterium
strains ( htrXI or car) is not present in NRC-1.
Unlike in bacteria, transducer and flagellin genes are not clustered in
one or two operons, although a single large cluster of genes in
Halobacterium NRC-1 includes nine che genes and
two fla genes. The cluster includes a complete set of
Bacillus subtilis che gene homologs, consisting of
cheA, B, C (named cheJ in
Halobacterium), D, R, Y, W, as well as a second
cheC ( cheC1 and cheC2) and a second
cheW ( cheW1 and cheW2). There is no
cheZ, which encodes a phospho-CheY phosphatase important in
Escherichia coli taxis adaptation that is also present in
several other bacteria, but not in B. subtilis. There are
six flagellin genes, but the numerous flagellar apparatus and motility
genes of bacteria are not evident in the sequence. Halobacterium has been studied heavily with regard to its
photoactive visual pigment-like seven-transmembrane-helix retinal
proteins, the archaeal rhodopsins, which have been demonstrated in
several archaeal halophiles as well as in the eukaryote
Neurospora crassa and other fungi ( 53). Only the four
members of this family previously identified in
Halobacterium are present [the light-driven ion
transporters bacteriorhodopsin and halorhodopsin,
and the phototaxis receptors, sensory rhodopsins I and II (Fig. )].
Other possible photoreceptor genes identified include those homologous
to genes encoding the flavoprotein cryptochromes, which serve as
circadian photoregulators in Arabidopsis and mammals ( 54).
There are two homologous genes in NRC-1; however, it should be noted
that they are also homologous to E. coli photolyase, and
photolyase and cryptochromes cannot be unequivocally distinguished on
the basis of primary sequence alone. A homolog of KaiC that generates
circadian oscillation in cyanobacteria is present in NRC-1 ( 55). At
least 6 response regulator genes and 14 histidine kinase-encoding genes
were found in the NRC-1 genome. DNA Replication, Repair, and Recombination The Halobacterium NRC-1 genome revealed three DNA
polymerase types ( 56), two family B polymerases (one coded by pNRC200),
a bacteriophage-like family A polymerase, as well as the heterodimeric
family D polymerase. The large subunit of the latter contains an intein
similar to the hyperthermophilic archaeon Pyrococcus
horikoshii. Additional proteins that may be active at the
replication fork include a putative DNA ligase, primase, type I
topoisomerase (TopA), and two type II topoisomerases (GyrA and B, and
Top6A and B). We also observed the presence of the following: Pcna,
sliding clamp, Rfc, clamp loader, and Rpa, replication protein A
involved in single-strand DNA binding, Mcm minichromosome maintenance
protein, and Orc/Cdc6, origin recognition complex proteins. Nine
copies of orc are present including three scattered on the
large chromosome, suggesting the possibility of multiple replication
origins. For DNA repair, Halobacterium NRC-1 possesses two of the
three genes involved in the guanine oxidization pathway,
mutT and mutY. In addition, both the nucleotide
and base excision pathways appear to be complete as copies of the
uvrABC nuclease and uvrD helicase, and
endonucleases and glycosylase genes are present. Two of the three genes
of methyl-directed mismatch repair were found, mutL and
mutS (three copies), but the nuclease gene mutH
was missing. The E. coli-type dam methylase
(recognizing GATC) is absent in NRC-1. However, a putative
CTAG-specific methylase gene is present, which has also been found in
Methanobacterium thermoformicicum ( 57). Repair genes similar to those in yeast are present in
Halobacterium NRC-1, including rad2,
rad3, rad24, and rad25. Several of these
proteins appear to be active in the excision repair pathway. Products
of rad3 and rad25 have been identified as repair
helicases and Rad2 is a single-stranded DNA endonuclease. This suggests
that Halobacterium NRC-1 has developed multiple pathways to
repair UV-induced damage as a means for survival. Cell-cycle genes in
Halobacterium NRC-1 include five copies of cdc48,
one of which is on pNRC200. The search for genes encoding proteins involved in recombination
yielded two RadA genes, with homology to both the yeast protein Rad51
and the E. coli protein RecA ( 58) and a homolog of the
putative Holliday junction resolvase from Pyrococcus
furiosus ( 59). Halobacterium NRC-1, like other archaea, drives
regulated transcription by using a single version of a eukaryotic RNA
polymerase II-like transcription system. The information for the
multisubunit RNA polymerase II is coded by 12 genes located at 6 loci.
Genes encoding Rpo subunits A, C, B′, B′′, and H are present in a gene
cluster ( 60), as are the genes for subunits E′ and E′′, and subunits K
and N. Subunit M, which has also been annotated as TFIIS ( 61), is also
present. An interesting finding is the presence of multiple copies of TBP
and TFB transcription factor genes. Five complete tbp genes
and one partial gene that has one-half of the two stirrups were
identified. Four of the six tbp genes were reported
previously on pNRC100 ( 12); additional single genes were found on both
the large chromosome and pNRC200. In contrast, five of the seven
tfb genes are present on the large chromosome, and the other
two are on pNRC200. The possibility of a novel regulatory system
involving up to 42 different TBP-TFB combinations has been discussed
recently ( 62). The finding of alternate TATA box and possibly BRE
sequences on the basis of saturation mutagenic analysis of the
bacterio-opsin gene ( bop) promoter supports this hypothesis
( 63, 64). At least 27 transcriptional regulators were also identified.
Transcription factors known to be required for polymerase II
transcription in other systems (TFIIF, TFIIH, and TFIIEβ) were not
evident. A TFIIEα homolog was identified by using the
pfam search tool ( 65). Additional factors present
include termination/antitermination factor homologs NusA and NusG
( 66). Translational components of Halobacterium NRC-1, like
other archaea, have both bacterial and eukaryotic homologs. We
identified 47 tRNA genes for all 20 amino acids and all 61 possible
codons, by using the tRNA scan-se program ( 67),
including tRNAs with 44 unique anticodons, 1 methionine initiator tRNA,
1 redundant tRNA (Ala-CGC), and 1 tRNA (anticodon CAU), which is
predicted to be converted from methionine to isoleucine specificity
posttranscriptionally as in E. coli ( 68). Three tRNA genes
contain introns, Trp-tRNA-CCA, elongator Met-tRNA-CAU, and
Ile-tRNA-CAU. Aminoacyl tRNA synthetases are present for all amino
acids except asparagine and glutamine, which likely require
amidotransferases. Homologs of the gatA, gatB,
and gatC genes, similar to other archaea that lack AsnRS and
GlnRS genes, are present ( 69). Interestingly, one aminoacyl tRNA
synthetase, ArgRS, closely related to the E. coli and other
Gram-negative bacterial and yeast mitochondrial enzymes, is coded by
pNRC200. The single-copy rRNA operon is bacterial-like in its organization and
gene content: 5′ 16S, tRNA (Ala-UGC), 23S, 5S, tRNA (Cys-GCA) ( 70). The
RNA component but not protein components of RNaseP was detected. Genes
coding homologs of the eukaryotic nucleolar proteins fibrillarin and
Nop56/58 were also identified in NRC-1. The occurrence of these
proteins in other archaea and the recent identification of C/D box
snoRNAs in thermophilic archaea ( 71) suggest that the snoRNA-mediated
2- O-methylribose modification system is generally present,
although none could be identified in NRC-1. Generally, the protein components of the translation apparatus of
archaea resemble more closely those of eukaryotes than those of
bacteria ( 72). In our annotation of ribosomal (r-) proteins, we used
the nomenclature for Haloarcula marismortui ( 71), a related
halophile where 25 30S subunit and 28 50S subunit r-proteins have been
enumerated by purification, partial or complete amino acid sequence
analysis, and gene sequence analysis ( 73), and where the crystal
structure of the 50S subunit has been determined ( 17). Despite their
generally higher sequence similarity to eukaryotes, the r-protein genes
of Halobacterium NRC-1 are organized into multigene clusters
that resemble operons of E. coli. In one of these clusters,
the L1P, L10P, and L12P genes are cotranscribed, and the 5′ leader of
the mRNA contains a bacterial-like L1 translational operator, a
structural mimic of the site in 23S rRNA that is used to autogenously
regulate translation of the mRNA ( 74). Genes coding homologs of
eukaryotic eIF1A, eIF2 α, β, and γ subunits, eIF4, eIF5, and
eIF2B α and δ are also present. The Halobacterium NRC-1-predicted proteome was compared
with 11 other complete microbial genomes by using the
darwin suite of programs ( 75, 76). The results
shown in Table 1 confirm the archaeal
nature of Halobacterium NRC-1, showing closest similarities
to Archaeoglobus fulgidus and Methanococcus
jannaschii. We also found homologs to many of the archaeal
“signature” proteins recently reported ( 77). The NRC-1-predicted
proteins were also similar to the Gram-positive bacterium, B.
subtilis, more than to any other bacteria, and displayed a large
number of unique homologs with the radiation-resistant bacterium
Deinococcus radiodurans, suggesting that NRC-1 may have
acquired a substantial number of genes from certain bacteria, possibly
by lateral gene transfer. Additional findings were that the NRC-1
proteome is highly acidic (average pI of 5.1), consistent with protein
stabilization and adaptation to a high-salt environment ( 41), and that
there is a high degree of redundancy among many protein classes. A more
detailed comparative genomics investigation should provide further
insights into evolutionary and adaptative forces operating in this
extremophile.
| Table 1Pairwise comparison of Halobacterium NRC-1
proteome and 11 other microbial
proteomes |
The sequence of Halobacterium NRC-1 has revealed
3 large replicons, a large chromosome and 2 novel minichromosomes, and
2,682 putative genes, including 972 novel genes, with no homologs in
the databases. Because this halophile is amenable to experimental
analysis by using a battery of approaches such as gene knockouts, DNA
arrays, and proteomics, future studies should yield significant
insights into the functions of conserved unknown and hypothetical genes
among the archaea. Moreover, because the halophilic proteins are highly
negatively charged with enhanced solubility, they lend themselves
readily to the determination of high-throughput three-dimensional
structure by experimental and theoretical approaches (structural
genomics). Also, this system should serve as an excellent model of
aspects of eukaryotic biology, e.g., DNA replication, transcription,
and translation. Comparison of a halophile genome to other prokaryotic
genomes should lead to a better understanding of microbial adaptation
to extreme conditions, such as hypersalinity, damaging radiation, and
an oxidizing atmosphere. Indeed, the availability of the complete
genome sequence for this easily cultured and tractable microbe should
facilitate a wide range of studies and establish this halophile as a
model organism among the archaea. This work was supported by collaborative research grants from the
National Science Foundation to S.D. (MCB-97022066 and MCB-9812330) and
L.H. (MCB-9900497). 1. Bayley S T, Morton R A. CRC Crit Rev Microbiol. 1978;6:151–205. [PubMed] 2. Woese C R. Microbiol Rev. 1987;51:221–271. [PubMed] 3. Doolittle W F. Science. 1999;25:2124–2129. [PubMed] 4. Joshi J G, Guild W R, Handler P. J Mol Biol. 1963;6:34–38. 5. Moore R L, McCarthy B J. J Bacteriol. 1969;99:248–254. [PubMed] 6. Charlebois R L, Doolittle W F. In: Mobile DNA. Berg D E, Howe M M, editors. Washington, DC: Am. Soc. Microbiol.; 1989. pp. 297–307. 7. DasSarma S. Experientia. 1993;49:482–486. [PubMed] 8. Bobovnikova Y, Ng W-L, DasSarma S, Hackett N R. Sys Appl Microbiol. 1994;16:597–604. 9. Hackett N R, Bobovnikova Y, Heyrovska N. J Bacteriol. 1994;176:7711–7718. [PubMed] 10. Ng W-L, Kothakota S, DasSarma S. J Bacteriol. 1991;173:1958–1964. [PubMed] 11. Ng W-L, Arora P, DasSarma S. Syst Appl Microbiol. 1994;16:560–568. 12. Ng W V, Ciufo S A, Smith T M, Bumgarner R E, Baskin D, Faust J, Hall B, Loretz C, Seto J, Slagel J, Hood L, DasSarma S. Genome Res. 1998;8:1131–1141. [PubMed] 13. DasSarma S, Robb F T, Place A R, Sowers K R, Schreier H J, Fleischmann E M. Archaea: A Laboratory Manual—Halophiles. Plainview, NY: Cold Spring Harbor Lab. Press; 1995. 14. Peck R F, DasSarma S, Krebs M P. Mol Microbiol. 2000;35:667–676. [PubMed] 15. Subramaniam S, Henderson R J. J Struct Biol. 1999;128:19–25. [PubMed] 16. Luecke H, Schobert B, Richter H T, Cartailler J P, Lanyi J K. Science. 1999;286:255–261. [PubMed] 17. Ban N, Sissen P, Hansen J, Capel M, Moore P B, Steitz T A. Nature (London). 1999;400:841–847. [PubMed] 18. Tindall B J. In: The Prokaryotes, A Handbook on the Biology of Bacteria. Balows A, Truper H J, Dworkin M, Harder K-H, Schleifer K-H, editors. New York: Springer; 1992. pp. 768–808. 19. Ewing B, Green P. Genome Res. 1998;8:186–194. [PubMed] 20. Ewing B, Hillier L, Wendl M C, Green P. Genome Res. 1998;8:175–185. [PubMed] 21. Gordon D, Abajian C, Green P. Genome Res. 1998;8:195–202. [PubMed] 22. Salzberg S L, Delcher A L, Kasif S, White O. Nucleic Acids Res. 1998;26:544–548. [PubMed] 23. Delcher A L, Harmon D, Kasif S, White O, Salzberg S L. Nucleic Acids Res. 1999;27:4636–4641. [PubMed] 24. Altschul S F, Madden T L, Schaffer A A, Zhang J, Zhang A, Miller W, Lipman D J. Nucleic Acids Res. 1997;25:3389–3402. [PubMed] 25. Rawal N, Kelkar S M, Altekar W. Ind J Biochem Biophys. 1988;25:674–686. [PubMed] 26. Grey V L, Fitt P S. Can J Microbiol. 1976;22:440–442. [PubMed] 27. Ruepp A, Soppa J. J Bacteriol. 1996;178:4942–4947. [PubMed] 28. Plaga W, Lottspeich F, Oesterhelt D. Eur J Biochem. 1992;205:391–397. [PubMed] 29. Adams M W W, Kletzin A. Adv Prot Chem. 1996;48:101–180. [PubMed] 30. Sprott G D, Ekiel I, Patel G B. Appl Environ Microbiol. 1993;59:1092–1098. [PubMed] 31. Blaut M. Antonie Leeuwenhoek. 1994;66:187–208. [PubMed] 32. Oren A, Gurevich P. FEMS Microbiol Lett. 1995;130:91–95. 33. Serrano J A, Camacho M, Bonete M J. FEBS Lett. 1998;434:13–16. [PubMed] 34. Krishnan G, Altekar W. Eur J Biochem. 1991;195:343–350. [PubMed] 35. Tomlinson G A, Koch T K, Hochstein L I. Can J Microbiol. 1974;20:1085–1091. [PubMed] 36. Danson M J, Jolley K A, Maddocks D G, Dyall-Smith M L, Hough D W. In: Microbiology and Biogeochemistry of Hypersaline Environments. Oren A, editor. Boca Raton, FL: CRC; 1999. pp. 239–248. 37. Jolley K A, Maddocks D G, Gyles S L, Mullan Z, Tang S L, Dyall-Smith M L, Hough D W, Danson M J. Microbiology. 2000;146:1061–1069. [PubMed] 38. Kushner D J. In: The Archaebacteria. Woese C R, Wolfe R S, editors. Vol. 8. Orlando, FL: Academic; 1985. pp. pp.171–215. 39. Christian J H B, Waltho J A. Biochim Biophys Acta. 1962;65:506–508. [PubMed] 40. Stoeckenius W, Lozier R H, Bogomolni R A. Biochim Biophys Acta. 1979;505:215–278. [PubMed] 41. Lanyi J K. Microbiol Rev. 1978;42:682–706. [PubMed] 42. Murakami N, Konishi T. Arch Biochem Biophys. 1990;281:13–20. [PubMed] 43. MacDonald R E, Greene R V, Lanyi J K. Biochemistry. 1977;16:3227–3235. [PubMed] 44. Pohlschroder M, Prinz W A, Hartmann E, Beckwith J. Cell. 1997;91:563–566. [PubMed] 45. Berks B C, Sargent F, Palmer T. Mol Microbiol. 2000;35:260–274. [PubMed] 46. Kates M. Experientia. 1993;49:1027–1036. [PubMed] 47. Cabrera J A, Bolds J, Shields P E, Havel C M, Watson J A. J Biol Chem. 1986;261:3578–3583. [PubMed] 48. Kamekura M, Kates M. In: Halophilic Bacteria. Rodriguez-Valera F, editor. II. Boca Raton, FL: CRC; 1988. pp. 25–54. 49. Shahmohammadi H R, Asgarani E, Terato H, Saito T, Ohyama Y, Gekko K, Yamamoto O, Ide H. J Radiat Res. 1998;39:251–262. [PubMed] 50. Yao V J, Spudich J L. Proc Natl Acad Sci USA. 1992;89:11915–11919. [PubMed] 51. Zhang W, Brooun A, McCandless J, Banda P, Alam M. Proc Natl Acad Sci USA. 1996;93:4649–4654. [PubMed] 52. Rudolph J, Nordmann B, Storch K F, Gruenberg H, Rodewald K, Oesterhelt D. FEMS Microbiol Lett. 1996;139:161–168. [PubMed] 53. Spudich E N, Yang C S, Jung K H, Spudich J L. Annu Rev Cell Dev Biol. 2000;16:365. [PubMed] 54. Cashmore A R, Jarillo J A, Wu Y J, Liu D. Science. 1999;284:760–765. [PubMed] 55. Johnson C H, Golden S S. Annu Rev Microbiol. 1999;53:389–409. [PubMed] 56. Cann I K O, Ishino Y. Genetics. 1999;152:1249–1267. [PubMed] 57. Nolling J, de Vos W M. Nucleic Acids Res. 1992;20:5047–5052. [PubMed] 58. Sandler S J, Satin L H, Clark A J. Nucleic Acids Res. 1996;24:2125–2132. [PubMed] 59. Komori K, Sakae S, Shinagawa H, Morikawa K, Ishino Y. Proc Natl Acad Sci USA. 1999;96:8873–8878. [PubMed] 60. Leffers H, Gropp F, Lottspeich F, Zillig W, Garrett R A. J Mol Biol. 1989;206:1–17. [PubMed] 61. Hausner W, Lange U, Musfeldt M. J Biol Chem. 2000;275:12393–12399. [PubMed] 62. Baliga N S, Goo Y A, Ng W V, Hood L, Daniels C J, DasSarma S. Mol Microbiol. 2000;36:1184–1185. [PubMed] 63. Baliga N S, DasSarma S. J Bacteriol. 1999;181:2513–2518. [PubMed] 64. Baliga N S, DasSarma S. Mol Microbiol. 2000;36:1175–1183. [PubMed] 65. Bateman A, Birney E, Durbin R, Eddy S R, Howe K L, Sonnhammer E L. Nucleic Acids Res. 2000;28:263–266. [PubMed] 66. Bermudez-Cruz R M, Chamberlin M J, Montanez C. Biochimie. 1999;81:757–764. [PubMed] 67. Lowe T M, Eddy S R. Nucleic Acids Res. 1997;25:955–964. [PubMed] 68. Muramatsu T, Nishikava K, Nemoto F, Kuchino Y, Nishimura S, Miyazawa T, Yokoyoma S. Nature (London). 1988;336:179–181. [PubMed] 69. Tumbula D, Vothknecht U C, Kim H S, Ibba M, Min B, Li T, Pelaschier J, Stathopoulos C, Becker H, Soll D. Genetics. 1999;152:1269–1276. [PubMed] 70. Hui I, Dennis P P. J Biol Chem. 1985;260:899–906. [PubMed] 71. Omer A D, Lowe T M, Russell A G, Ebhardt H, Eddy S R, Dennis P P. Science. 2000;288:517–522. [PubMed] 72. Dennis P P. Cell. 1997;89:1007–1010. [PubMed] 73. Engemann S, Noelle R, Herfurth E, Briesemeister U, Grelle G, Wittmann-Liebold B. Eur J Biochem. 1995;234:24–31. [PubMed] 74. Shimmin L C, Dennis P P. EMBO J. 1989;8:1225–1235. [PubMed] 75. Gonnet G H, Cohen M A, Benner S A. Science. 1992;256:1443–1445. [PubMed] 76. Riley M, Labedan B. J Mol Biol. 1997;268:857–868. [PubMed] 77. Graham D E, Overbeek R, Olsen G J, Woese C R. Proc Natl Acad Sci USA. 2000;97:3304–3308. [PubMed]
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