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Journal of Bacteriology, December 2001, p. 7027-7036, Vol. 183, No. 24
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.24.7027-7036.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
DNA Microarray Analysis of the Hyperthermophilic
Archaeon Pyrococcus furiosus: Evidence for a New Type
of Sulfur-Reducing Enzyme Complex
Gerrit J.
Schut,1
Jizhong
Zhou,2 and
Michael W. W.
Adams1,*
Department of Biochemistry and Molecular
Biology and Center for Metalloenzyme Studies, University of Georgia,
Athens, Georgia 30602,1 and
Environmental Sciences Division, Oak Ridge National
Laboratory, Oak Ridge, Tennessee 378312
Received 4 May 2001/Accepted 21 September 2001
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ABSTRACT |
DNA microarrays were constructed by using 271 open reading frame
(ORFs) from the genome of the archaeon Pyrococcus
furiosus. They were used to investigate the effects of
elemental sulfur (S°) on the levels of gene expression in cells grown
at 95°C with maltose as the carbon source. The ORFs included those
that are proposed to encode proteins mainly involved in the pathways of sugar and peptide catabolism, in the metabolism of metals, and in the
biosynthesis of various cofactors, amino acids, and nucleotides. The
expression of 21 ORFs decreased by more than fivefold when cells were
grown with S° and, of these, 18 encode subunits associated with three
different hydrogenase systems. The remaining three ORFs encode homologs
of ornithine carbamoyltransferase and HypF, both of which appear to be
involved in hydrogenase biosynthesis, as well as a conserved
hypothetical protein. The expression of two previously uncharacterized
ORFs increased by more than 25-fold when cells were grown with S°.
Their products, termed SipA and SipB (for sulfur-induced proteins), are
proposed to be part of a novel S°-reducing, membrane-associated,
iron-sulfur cluster-containing complex. Two other previously
uncharacterized ORFs encoding a putative flavoprotein and a second FeS
protein were upregulated more than sixfold in S°-grown cells, and
these are also thought be involved in S° reduction. Four ORFs that
encode homologs of proteins involved in amino acid metabolism were
similarly upregulated in S°-grown cells, a finding consistent with
the fact that growth on peptides is a S°-dependent process. An ORF
encoding a homolog of the eukaryotic rRNA processing protein,
fibrillarin, was also upregulated sixfold in the presence of S°,
although the reason for this is as yet unknown. Of the 20 S°-independent ORFs that are the most highly expressed (at more than
20 times the detection limit), 12 of them represent enzymes purified
from P. furiosus, but none of the products of the 34 S°-independent ORFs that are not expressed above the detection limit
have been characterized. These results represent the first derived from
the application of DNA microarrays to either an archaeon or a hyperthermophile.
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INTRODUCTION |
Hyperthermophiles are microorganisms
that grow optimally at temperatures of 80°C and higher, and most are
classified as archaea (65). They are a rather
diverse group with respect to their metabolic capabilities, but many of
them utilize peptides as a carbon source and reduce elemental sulfur
(S°) to H2S (66). Of these, the
majority are obligately proteolytic and show little if any growth
unless S° is added to the growth medium. The exceptions include some
species of Pyrococcus that metabolize poly- and
oligosaccharides, as well as peptides (3, 4, 15). For
example, Pyrococcus furiosus grows on a disaccharide
(maltose) to high cell densities in the absence of S° and produces
H2 as an end product rather than
H2S. On the other hand, P. furiosus
requires S° in the growth medium when it utilizes peptides as a
carbon source (1). A comparison of the activities of some
key metabolic enzymes in cells grown either on peptides (with S°) or
on maltose (with or without S°) provided the first evidence for a
highly regulated fermentative-based metabolism in P. furiosus, with S° or its metabolites playing a key regulatory
role (1). The molecular mechanism by which S° achieves
this control is not known.
The pathways by which P. furiosus metabolizes peptides and
carbohydrates are reasonably well established. The organism contains a
modified Embden-Meyerhof pathway in which hexokinase and
phosphofructokinase are ADP rather than ATP dependent (29,
68). In addition, the expected glyceraldehyde-3-phosphate
dehydrogenase and phosphoglycerate kinase are replaced by a single
phosphate-independent enzyme, glyceraldehyde-3-phosphate ferredoxin
oxidoreductase (GAPOR [42]), which is regulated at the
transcriptional level (69). The pyruvate produced by this
pathway is converted to the end product acetate in two steps involving
pyruvate ferredoxin oxidoreductase (POR [5]) and
acetyl-coenzyme A (CoA) synthetases I and II (40, 61),
with the concomitant production of ATP from ADP and phosphate. The
oxidative steps in the pathway of acetate production from glucose
therefore involve two ferredoxin-dependent enzymes (GAPOR and POR), and
a similar mechanism is present during peptide catabolism by P. furiosus. The organism contains, in addition to POR, three other,
ferredoxin-dependent, 2-keto acid oxidoreductases, and these convert
transaminated amino acids into their corresponding CoA derivatives
(5, 19, 39), which are then transformed to their
corresponding organic acids by the two acetyl-CoA synthetases with
concomitant ATP synthesis (40).
How P. furiosus and related species couple the oxidation of
the reduced ferredoxin generated by the catabolic pathways to the
reduction of S° is not known. The organism contains three different
hydrogenases: two cytoplasmic, NAD(P)H-dependent enzymes, and one as
part of a membrane-bound complex (9, 38, 59). The
cytoplasmic hydrogenases were thought to play a role in S° reduction
since they can reduce S° in vitro (37), but this appears not to be the case as the total hydrogenase activity of S°-grown cells is dramatically lower than that of cells grown without S° (1). The mechanism by which S° affects hydrogenase
activity is not understood. Moreover, the nature of the enzyme system
that reduces S° remains unknown. In mesophilic organisms, the only S°-reducing enzyme that has been well-characterized is the trimeric, membrane-bound, molybdenum (Mo)-containing, polysulfide reductase of
the mesophilic bacterium, Wolinella succinogenes
(32), but the genome of P. furiosus
(54) does not contain ORFs that would encode a homolog of
the mesophilic enzyme. Moreover, P. furiosus is not known to
utilize Mo but contains at least three enzymes that utilize the
analogous element tungsten (W [27]), perhaps suggesting
a role for W in S° reduction.
The ability of P. furiosus to grow well both with and
without S°, together with the availability of its complete genome
sequence, provide an opportunity to investigate S° metabolism by
expression analysis by using DNA microarrays. In fact, the genus
Pyrococcus is unique in that complete genome sequences are
available from three species; P. horikoshii, P. abyssi, and P. furiosus (28, 54; D. Prieur, P. Forterre, J.-C. Thierry, and J. Dietrich
[http://www.genoscope.cns.fr/Pab/]). While DNA microarrays
(60) have revolutionized functional genomics in eukaryotic
systems (e.g., references 14, 56, and 63), there have been far fewer applications with prokaryotes. Studies initially focused on pathogens (11, 17, 58, 67, 74) and
more recently have included Escherichia coli (2, 30, 53, 73, 79), Bacillus subtilus (76, 78)
and a cyanobacterium (21). As yet there have been no
reports of using this technique with either a hyperthermophile or an
archaeon. The genome of P. furiosus contains ca. 2,200 open
reading frames (ORFs) with >50% of unknown function. As a prelude to
a complete genomic analysis, we focus here on 271 ORFs that encode
proteins that are proposed to be involved in the primary metabolic
pathways, energy conservation, and metal metabolism. The results
indicate that S° or its metabolites play a major regulatory role at
the transciptional level and that S° reduction appears to be
accomplished by a new type of enzyme system involving the products of
previously uncharacterized ORFs.
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MATERIALS AND METHODS |
Primer design and PCR.
Gene sequences were obtained from the
P. furiosus genome site (http://comb5-156.umbi.umd.edu/)
(54). Of the 275 ORFs that were examined in this study, 87 of them are listed in Tables 1 to 4. A complete list of the 275 ORFs is
available at http://adams.bmb.uga.edu/pubs/sup228.pdf. Primers were
designed for the ORFs by using the Primer 3 program (Whitehead
Institute, MIT) and were purchased from Stanford University and from
MWG Biotech (High Point, N.C.). For the initial round of PCR the primer
pairs were designed to give products corresponding to the complete
ORFs, and this yielded products for 249 of them as verified by gel
electrophoresis. The remaining 42 primer pairs were designed to yield a
product of ca. 1 kb (unless the target ORF was smaller). Of these only
four ORFs did not yield suitable PCR products, and these were not
pursued further. PCR products were purified with a Strataprep 96 PCR
purification kit (Stratagene, La Jolla, Calif.) and eluted in 33%
(vol/vol) dimethyl sulfoxide. They were spotted in duplicate onto
aminosilane coated slides (Sigma, St. Louis, Mo.) in four subarrays by
using a robotic slide printer (Omnigrid; Genemachines, San Carlos,
Calif.). The slides were processed as previously described
(8).
Growth of P. furiosus and RNA preparation.
P. furiosus was grown in batch mode in a 20-liter custom
fermentor at 95°C with maltose as the primary carbon source and was harvested in mid-log phase (1). The only variable was the
presence or absence of S°. The cells that were used to prepare RNA
for the microarray analyses were from experiments that have been
described previously (1). In that case, activity assays
were determined for more than 20 enzymes involved in the primary
metabolic pathways by using cytoplasmic and membrane fractions, and
these results are referred to below. Samples (2,000 ml) of the same
cultures were cooled on ice, and total RNA was extracted by using
acid-phenol extraction (71). No significant contamination
of the RNA with genomic DNA could be detected by Northern blot
hybridization or preliminary microarray experiments (data not shown),
and so a DNase treatment was not used.
Preparation of cDNA and hybridization conditions.
Fluorescently labeled cDNA was prepared with the ARES DNA Labeling Kit
(Molecular Probes, Eugene, Oreg.). In brief, 15 µg of total RNA was
reversed transcribed in a total volume of 20 µl by using Stratascript
RT (Stratagene, La Jolla, Calif.) in the presence of 1 mM dATP, 1 mM
CTP, 1 mM GTP, 0.3 mM dTTP, 0.5 mM aminoallyl dUTP, and 1 µg of
random 9-mers (Stratagene) according to the manufacturer's
instructions. After a 1.5 h of incubation at 42°C, RNA was
destroyed by the addition of 0.1 N NaOH, followed by a 10-min
incubation at 70°C. After neutralization with 0.1 N HCl,
amine-modified cDNA was purified by using a QIAquick PCR Purification
Kit (Qiagen, Valencia, Calif.), except that the wash buffer was
replaced with 75% (vol/vol) ethanol, and the cDNA was eluted with 45 µl of distilled water and dried under vacuum. The amine modified cDNA
was labeled with Alexa 488 or Alexa 594 dyes (Molecular Probes)
according to the manufacturer's instructions. Alexa 546 was used as
third dye for the triple-labeling experiments. The labeled cDNA was
purified with the Qiagen kit as described above and was dried under
vacuum. Differentially labeled (Alexa 488 and Alexa 594) cDNA derived
from P. furiosus cells grown in the presence or absence of
S° was pooled and hybridized to the microarrays. The labeled cDNA was
dissolved in 20 µl of hybridization buffer (Sigma), and hybridization
was performed under a coverslip at 65°C in a humidity chamber
(Arrayit, Sunnyville, Calif.) for between 10 and 15 h. The slides
were then washed twice for 5 min in each of 2× SSC (1× SSC is 0.15 M
NaCl plus 0.015 M sodium citrate), 0.1% sodium dodecyl sulfate (SDS),
and 0.2× SSC-0.1% SDS and then rinsed in distilled water and blown
dry with compressed air. The intensities of the Alexa 488 and Alexa 594 dyes were measured by using a Scan Array 5000 spectrometer (Packard,
Meriden, Conn.) with the appropriate laser and filter settings and
analyzed by using Quantarray (Packard).
Data analysis.
To compute signal intensities for each ORF,
the areas surrounding each spot were taken as the background, and 17 control spots, where only spotting buffer was printed, were included on
the 384-spot array. The control spots were corrected for local
background, and the average was used to correct the signals from each
ORF after their background correction. The relative amounts of the transcripts (with or without S°) are presented in a linear fashion by
converting all ratios to a log2 function. For the
two different growth conditions tested, a negative value of less than
2.3 represents a >5-fold downregulation of expression from a given
ORF by S°, and this corresponds to a green color in a false color
overlay. Conversely, a positive value graeater than 2.3 represents a
>5-fold upregulation and corresponds to a red color in a false color
overlay. ORFs that display log2 values between
approximately 1 and 1 are minimally affected by S° and show a
yellow color on the overlay. The detection limit of fluorescent signals
was set arbitrary to 1,000 intensity units (see Fig.
1) and such spots are not visible on the
false overlay. Only ORFs that display intensities more than twice the
detection limit are considered valid. Conversion of intensity ratios
enables the data to be presented as a log2 value
with a standard deviation (SD). This represents an average of six
hybridization experiments with cDNA derived from five different cultures of P. furiosus: two grown with S° and three grown
without it. In preliminary experiments we did not observe any
significance difference in the quality of the data between different
Alexa dyes (Molecular Probes) nor when the Alexa dyes were replaced with CY3 and CY5 (Amersham, Piscataway, N.J.), dyes that are more usually used as fluorescent labels (12). The advantage of
the Alexa dyes is that labeling with three dyes is possible with the lasers and filters available in the Scanarray 5000 (Packard). Statistical significance of the observed fluorescence signal ratios was
obtained by a paired t test analysis by using the
statistical software JMP4 (SAS Institute, Cary, N.C.).
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FIG. 1.
Fluorescence intensities of DNA microarrays. (A) cDNA
versus cDNA derived from the same cultures of cells grown on maltose
(no S°). (B) cDNA versus cDNA derived from cells grown on maltose
with or without S°. The upper and lower diagonal lines indicate
fivefold changes in the signal intensities. See the text for details.
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RESULTS AND DISCUSSION |
Selection of ORFs.
Of the ca. 2,200 ORFs in the P. furiosus genome (54), 271 were selected for
microarray analysis. The targets were putative genes involved in the
primary metabolic pathways of carbon, nitrogen, and energy metabolism,
and those that are, or might be, related to the metabolism of S° and
H2. They included ORFs that are proposed to
encode various enzymes and proteins involved in the pathways of sugar
and peptide catabolism; the utilization of metals (such as Fe, Ni, W,
and Mo); and the biosynthesis of cofactors, amino acids, lipids,
polyamines, ribosomes, and nucleotides, together with putative
chaperonins, ATPases, and transcriptional regulators. Unless
otherwise indicated, each ORF is referred to by its end nucleotide
number and by its current annotation, which is based on homology
searches (depicted in brackets [see Table 1 and reference 54]). It is specifically noted in Tables 1 to 4 where
there are experimental data derived by using P. furiosus to
support the ORF assignment (given without brackets and with a
reference, see Table 1).
Experimental protocols and data analysis.
The efficacy of the
microarray experiment with mRNA derived from P. furiosus
cells is shown in Fig. 1. Figure 1A shows data with differentially
labeled cDNA samples of RNA prepared from the same batch of cells,
grown with maltose in the absence of S°, and hybridized to the same
microarray. The intensity of the fluorescence signals varied over a
range of more than 103, a finding consistent with
what has been reported with other organisms (26, 34, 46,
78). Those ORFs with intensities of below ca. 2,000 (Fig. 1A)
are considered below the detection limit, and it cannot be concluded
that these ORFs are expressed to a significant degree. From Fig. 1A it
is clear that ORFs that give rise to low-intensity signals show a high
deviation because of background fluorescence, whereas the data derived
from ORFs that give high signal intensities lie near or on the diagonal.
As shown in Fig.
1B, S° and/or its metabolites clearly have a
dramatic effect on the expression of many of the 271 ORFs that
were
examined. It was previously shown that the presence of S°
in the
growth medium affects the activities of several metabolic
enzymes
(
1). The microarray data indicate that this regulation
is
primarily at the transcriptional level. We focus below on ORFs
whose
expression appears to be strongly regulated by S°, where
the signal
intensity changes by at least fivefold (shown by the
upper and lower
diagonal lines in Fig.
1B). The standard deviations
(log
2 intensity ratios) for almost all of these
ORFs are <2, and
they are differentially regulated with at least a
95% confidence
level as determined by using a paired
t test
analysis (
P > 0.95).
However, there are three
exceptions. These are ORFs 1419794 (formate
dehydrogenase
-chain),
156299 (3-isopropyl malate dehydrogenase),
and 438413 (phosphoribosylglycinamide formyltransferase) whose
log
2 intensities showed deviations of >2
(
P < 0.90). For these
ORFs a large variation in the
ratios was obtained by using the
same growth condition (maltose without
S°). This was verified
when cDNA samples from three independently
grown cultures were
separately labeled with the Alexa 488, Alexa 546, or Alexa 594
dyes, and all three cDNAs were hybridized to the same
slide. Only
these three (of 271) ORFs showed distinct false colors,
indicating
differential expression in one of the three samples. The
reasons
for this are not clear, but these ORFs appear not to be
regulated
by S° and are not considered
further.
ORFs strongly downregulated by S°.
The expression of 21 of
the 271 ORFs examined decreased by more than a factor of 5 when RNA was
derived from cells grown with S° (Fig. 1B), and these are listed in
Table 1. Of these, 18 ORFs encode
subunits associated with the three different hydrogenase systems that
have been characterized from P. furiosus. These include those encoding six of the eight subunits of the two cytoplasmic hydrogenases (I and II [38, 50]), whose expression
decreases between 6- and 14-fold in the presence of S° (Table 1).
Expression of the other two ORFs (encoding the - and 
-subunits of
hydrogenase I) decrease by 2.2- to 4.8-fold. These data are in accord
with the report that the presence of S° in the medium decreased the total hydrogenase activity of the cytoplasmic fraction by ca. 16-fold
(1). Since the specific activity of hydrogenase I is ca.
10-fold higher that of hydrogenase II in in vitro assays, it was not
possible to determine from the activity analyses if hydrogenase II
activity was regulated by S°. However, the microarray data clearly
show that both hydrogenases are strongly regulated. Moreover, S°
regulates the expression of the genes encoding the cytoplasmic
hydrogenases in a negative fashion, rather than S° (or its
metabolites) having some inhibitory effect on activity. Obviously,
these hydrogenases are unlikely to play a role in S° reduction, as
originally suggested (37).
It was previously shown that the activity of the membrane-bound
hydrogenase of
P. furiosus decreases by almost 30-fold when
cells are grown in the presence of S° (
1). After
solubilization
and purification from cells grown in the absence of
S°, the enzyme
contained two subunits (Mbh11 and Mbh12) and, based on
their N-terminal
sequences, the genes encoding them were proposed to be
part of
a 14-ORF operon (Mbh1 to -14 [
59]). All but one
(Mbh7) of the
14 ORFs were included on the DNA microarray. As shown in
Table
1, the expression of all but one of them decreased by more than
5-fold in the presence of S° (the remainder, Mbh9, decreased by
4.2-fold). These data confirm that the 14 ORFs do indeed constitute
an
operon and that its products are involved in the metabolism
of
H
2. The average fluorescence intensities
associated with the
13 ORFs of the membrane-bound hydrogenase operon
are shown in
Fig.
2, along with the
values for the eight subunits that encode
the two cytoplasmic
hydrogenases. The ORFs are depicted according
to their order in their
respective operons. All show a similar
pattern in that they are
downregulated to close to the detection
limit (see Fig.
1B) in the
presence of S°, a finding consistent
with barely detectable
hydrogenase activities in cell extracts
(
1). The apparent
exception is the ORF encoding the
-subunit
of hydrogenase I. Although this ORF is expressed at about five
times the detection limit,
the results show that it is regulated
by S° as determined by the
paired
t test analysis (
P > 0.98).
However,
the 3' end of this ORF (ORF 867811) overlaps by two nucleotides
with
the 3' end of an ORF (ORF 867808) on the opposing strand.
If the
untranslated 3' end of ORF 867811 mRNA extends far enough
into the
complementary strand of
hyhA, this could give rise to
a
"false signal" due to cross-hybridization. ORF 867811 encodes
a
putative ABC transporter (ATP binding protein) and was not included
on
the microarray. The interference of overlapping mRNAs in microarray
analyses has been reported (
30), although it has not been
systematically
analyzed with any genome. With the 18 hydrogenase-related ORFs
(Fig.
2), there is considerable variation in
the absolute signal
intensities, but this appears to arise to some
extent from the
differences in ORF sizes, several of which are <500
bp.
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FIG. 2.
Expression of ORFs encoding hydrogenase-related
subunits with or without S°. The ORFs encoding the subunits of the
cytoplasmic hydrogenase I (A), cytoplasmic hydrogenase II (B), and the
membrane-bound hydrogenase (C) are arranged according to their
positions in their respective operons. These are plotted against signal
intensities for cDNA obtained from cells grown with (solid bars) or
without (shaded bars) S°. The results with all subunits have a
confidence level of at least 96% (P > 0.98 for
the cytoplasmic hydrogenases and P > 0.96 for the
membrane-bound enzyme).
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Hence, of the 21 ORFs (from the total 271 ORFs) that are strongly
downregulated (>5-fold) in the presence of S°, 18 represent
structural genes for the three hydrogenases. Of the other three
S°-responsive ORFs, one (ORF 577932) showed an almost 40-fold
decrease in expression, the largest of any of the downregulated
ORFs
(Table
1). ORF 577932 is annotated as
hypF and shows 37%
sequence identity to its closest homolog, HypF, from the aerobic
bacterium
Ralstonia eutrophicus (
75). HypF is
thought to be
involved in biosynthesis of the metal site of
[NiFe]-hydrogenases.
There is only one gene of this type in
P. furiosus so it appears
that, as in
E. coli
(
25), one HypF protein is involved in the
maturation of
three different hydrogenases (two cytoplasmic and
one membrane bound).
One would expect HypFA, as a processing enzyme,
to be present at low
intracellular concentrations, and the reason
why its gene appears to be
so strongly regulated by S° is not
clear at this point.
P. furiosus contains homologs of several
other genes that are
involved in hydrogenase maturation and metal
insertion in mesophilic
bacteria. These include
hycI (ORF 637303)
and
hypACDE (ORFs 636092, 566869, 567973, and 626147; a version
of
hypB appears not to be present in the
P. furiosus genome).
These ORFs were also included on the microarray.
The expression
of the
hyp genes, which are all expressed at
significant levels
(1.5 to 9.6 times the detection limit; see Fig.
1B)
decreased
by two- to threefold when S° was added, but the expression
of
hycI was below the detection limit under both
conditions.
One of the two remaining ORFs that are strongly downregulated by S°
(Table
1) may also be related to hydrogenase biosynthesis.
This is ORF
615154, which encodes ornithine carbamoyltransferase
(OTCase). This
enzyme has been purified from
P. furiosus, its
gene has been
cloned and expressed, and its crystal structure
is available (
36,
70). OTCase catalyzes the transfer of a
carbamoyl group from
carbamoyl phosphate to ornithine, generating
citrulline in the arginine
biosynthetic pathway. The potential
link between OTCase and the
hydrogenases stems from the recent
proposal that the source of the C1
(CO and CN) ligands to the
metals at the active site of hydrogenases is
the carbamoyl group
of carbamoyl phosphate (
48). In view
of the regulation of both
OTCase and hydrogenase expression by S° in
P. furiosus, we speculate
that citrulline is the precursor
of the carbamoyl group for the
synthesis of the active sites of the
three hydrogenases in this
organism. Citrulline may well be preferred
to carbamoyl phosphate
because of the high thermal lability of the
latter compound (
35).
One would expect citrulline also to
be needed for arginine biosynthesis
even in the presence of S°, and
this appears to be the case, since
OTCase is expressed at close to
three times the detection level
in S°-grown
cells.
The remaining ORF that is strongly downregulated by S° is ORF 51760, and this is annotated as a conserved hypothetical protein
(Table
1). It
is adjacent to the gene (ORF 49183) encoding PEP
synthetase, a
gluconeogenic enzyme recently purified from
P. furiosus (
24; see also below), and an ORF (ORF 53135) encoding a
conserved
hypothetical protein, neither of which showed significant
(<2-fold)
changes in expression according to the microarray data. The
function
of the S°-responsive ORF 51760 is therefore unclear at
present.
ORFs strongly upregulated by S°.
Of the 271 ORFs examined,
the expression of 12 of them was strongly upregulated (>5-fold) when
S° was present in the growth medium (Table
2). In contrast to the ORFs that were
downregulated by S°, all of the upregulated ORFs represent putative
proteins since none of their products have been characterized from
P. furiosus. Moreover, the two most strongly regulated ORFs,
1871822 and 1872873, which are upregulated more than 25-fold, are next
to each other on the genome. The products of these two ORFs show no
sequence similarity to any known protein. Nevertheless, they will be
referred to as SipA (1871822) and SipB (1872873) for "sulfur-induced
protein" since expression of their genes clearly responds to S° or
its metabolites. Both genes are also very tightly regulated, since there appears to be little or no expression from them when cells are
grown in the absence of S° (their signal intensities are less than
the detection limit).
What is now termed
sipA was included in the list of ORFs for
microarray analysis because its product had been previously identified
(from the N-terminal sequence) after gel electrophoresis of
P. furiosus extracts. The protein was present in washed membrane
preparations of cells grown with S° but was not detected in cells
grown without S° (
23). SipA is a 19-kDa protein that
contains
a single Cys residue. Although it is strongly associated with
the membrane, sequence analyses do not reveal any transmembrane
helix
motifs (
23). What we now term
sipB is currently
annotated
as a "putative polyferredoxin," and it contains Cys
motifs that
could coordinate two [4Fe-4S] clusters. However, the
closest homolog
(25% sequence identity) to this putative 15-kDa
protein is actually
the
-subunit of the
P. furiosus
enzyme 2-ketoisovalerate ferredoxin
oxidoreductase (VOR; see below), a
protein that also contains
two [4Fe-4S] centers (see reference
41). Interestingly, although
sipA and
sipB are side by side, they are transcribed in opposite
directions. Figure
3 shows a detailed
analysis of putative transcriptional
and ribosomal binding sites for
sipA and
sipB, and these strongly
suggest that
both genes are expressed from a shared promoter domain,
as previously
shown for the genes encoding
-glucosidase and alcohol
dehydrogenase
of
P. furiosus (
72). Homologs of
sipA and
sipB are present in the genomes of the
S°-reducing
Pyrococcus species,
P. abyssi
(PAB1692 and PAB0578), and
P. horikoshii (PH1227 and
PH0982), but their genes are not arranged back to back such since
they
are in
P. furiosus and SipB is much larger (by ~15 kDa) in
these other two species. In
P. furiosus, this SipB 15-kDa
extension
is encoded by a separate ORF (1873389) that is adjacent to
sipB (see Fig.
3). The product of ORF 1873389 shows no
significant
sequence similarity to any characterized protein. This ORF
was
included in the microarray analyses, but it is not expressed above
the detection limit when cells were grown with or without S° (Table
4). This difference in genome arrangement among the
Pyrococcus species might be related to the ability of
P. furiosus, but not
P. abyssi and
P. horikoshii, to grow in the absence of S°.
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FIG. 3.
Genome organization of sipA and
sipB. The back to back ORFs coding for SipA (179 amino
acids) and SipB (115 amino acids) are shown with the indicated
directions for transcription. Putative transcription stop sites are
indicated with a "ball on a stick," and putative translational
start sites are indicated with right-angled arrows. The putative start
sites are preceded with possible ribosomal binding sites (underlined)
and TATA boxes (boxed). The start site for sipB shown
here is not the same as that given in the genome annotation (which is
indicated with an asterisk [54]). Both
sipA and sipB contain an inverted repeat
(I-1 and I-2) directly next to the proposed TATA boxes. The position of
the ORF (1873389) encoding the SipB extension (142 amino acids) is
indicated.
|
|
Considering the tight regulation of
sipA and
sipB
by S° and the likely electron transfer ability of SipB, it seems
reasonable
to suggest that SipA and SipB form part of a
membrane-associated
complex that directly reduces S° to
H
2S. Two of the other ORFs
listed in Table
2 that
are strongly upregulated by S°, ORFs 1131551
and 102519, may also be
directly involved in S° reduction since
both show sequence similarity
to the disulfide oxidoreductase
family of enzymes. ORF 1131551 is
annotated as NADH oxidase (
noxA-2)
and would encode a 49-kDa
cytoplasmic protein that shows significant
sequence similarity to NADH
peroxidase, a flavoprotein with an
active site cysteine
(
64). ORF 102519 is predicted to encode
a 26-kDa
cytoplasmic protein that contains four Cys residues (as
two -CxxC-
motifs). Both ORFs are expressed at near the detection
limit in the
absence of S° but increase to the "highly expressed"
category
(>10-fold the detection limit; see below) in S°-grown
cells, and
neither appears to be part of an operon. Homologs of
both ORFs are also
present in
P. abyssi (PAB0936 and PAB2245)
and
P. horikoshii (PH0572 and
PH0178).
Two of the other S°-induced proteins that might be related to S°
metabolism are ORF 1805557, which is annotated as a conserved
hypothetical protein, and ORF 1352727, which is annotated as an
NADH
dehydrogenase (Table
2). The expression of ORF 1805557 changes
from
below the detection limit to twice the detection limit when
cells are
grown with S°. This ORF would encode a 27-kDa cytoplasmic
protein
where the N-terminal part has high sequence similarity
to
ATP/GTP-binding proteins (
13), while the C-terminal half
contains six Cys residues, including a CxxCxxxC motif. ORF 1352727
is
part of a putative operon containing 13 ORFs, most of which
have
homology to membrane-bound NADH dehydrogenases. Hagen and
coworkers
suggested that this operon encodes a (fourth) hydrogenase
system in
P. furiosus (
62), but this seems unlikely in
view
of the upregulation of ORF 1352727 by S° and the corresponding
dramatic decrease in membrane-associated hydrogenase activity
(
1). Moreover, two other ORFs (1350388 and 1359534) in
this
putative operon were included in the microarray analysis, and
these are also upregulated by S° (three- and fourfold [data not
shown]). It is tempting to postulate that ORFs 1805557 and 1352727
(and possibly other ORFs in the putative operon) are involved
in
nucleotide binding and electron transfer during S° reduction,
although further evidence is obviously required to substantiate
this.
Four of the remaining six ORFs that are upregulated more than fivefold
by S° (Table
2) appear to encode proteins that are
involved in amino
acid metabolism. These are annotated as an oligopeptide
permease (ORF
204761), acetolactate synthase (which is involved
in the biosynthesis
of branched chain amino acids [ORF 900019]),
and as subunits of
aspartokinase (ORF 1008251) and tryptophan
synthase (ORF 1487371).
Although all are putative proteins at
present, these data support the
relationship between S° and amino
acid metabolism that was recently
established by growth studies
of
P. furiosus, in which it
was shown that peptides can only serve
as the primary carbon source if
S° is present (
1). All four
of these ORFs are expressed
at significant levels in the absence
of S° (more than twice the
detection limit), but all four fall
into the highly expressed category
(>10-fold the detection limit
[see below]) in S°-grown cells. Of
the other two upregulated ORFs
in Table
2, one is annotated as the
thermosome (ORF 1825269).
This protein is highly conserved in archaea
and is a type II chaperonin,
a class that also includes the eukaryotic
TCP-1(CCT) proteins
(
7). The thermosome is expressed at
about twice the detection
limit in the absence of S°, although why
its expression should
increase ~7-fold in the presence of S° is not
clear.
The other S°-responsive ORF in Table
2 is annotated as a
fibrillarin-like protein (ORF 62257). It shows more than 50% sequence
identity to a key eukaryotic nucleolar protein that associates
with
small nucleolar RNAs (snoRNAs) directing 2'-O-ribose methylation
of the
rRNA. Homologs of fibrillarin and some of its associated
proteins, such
as Nop56/58, as well as snoRNAs, have only recently
been identified in
archaea, although their function in these organisms
is not clear
(
16,
33,
44,
45,
47). In
P. furiosus, the
ORF
encoding the fibrillarin homolog is directly adjacent to a
homolog of
Nop56/58 (ORF 66216). The microarray data indicate
that these ORFs are
expressed at twice and six times the detection
limit, respectively, in
the absence of S°, but their expression
increases a further six- and
threefold, respectively, when S°
is present. Indeed, the microarray
experiment included two copies
of the ORF encoding the fibrillarin
homolog, and both gave remarkably
similar results with RNA from cells
grown with (an intensity of
22,237 ± 1,074, see Fig.
1B) and
without S° (3,590 ± 185).
P. furiosus also contains
five homologs of a family of RNA m
5C methyl
transferases (
52), and four were included in the
microarray
analysis (ORFs 1191037, 674979, 179810, and 1197174). All of
them
are annotated as a nucleolar NOL1-NOP2-sun family protein, but
none appear to be regulated by S°. Thus, it is not obvious why
S°
should cause the genes encoding homologs of eukaryotic nucleolar
rRNA
processing proteins to be expressed at such high levels.
Fibrillarin is
also found in archaea that do not metabolize S°,
such as methanogens
(
20), so this protein must have a more general
role,
albeit one that is associated with S° metabolism, at least
in
P. furiosus.
Sulfur-independent ORFs.
In the preceding discussion we have
considered all 33 of the 271 ORFs that show a >5-fold change in
expression when the cells were grown in the presence of S°. In
addition to these 33, there are 84 ORFs that showed between a two- to
fivefold response to S° (data not shown). At this point further
comment on the nature and function of these ORFs seems premature since
there are no obvious trends and corroborating experimental data are
required. That leaves 154 of the 271 ORFs remaining that show little if any response to S° (<2-fold), and these can be separated into three
groups: (i) 20 ORFs that appear to be the most highly expressed, where
the transcripts are at >10 times the detection level (> 20,000 intensity units [see Fig. 1B]); (ii) 34 ORFs that appear to be poorly
expressed, if at all, where the transcripts are below the detection
level (<2,000 intensity units); and (iii) 100 ORFs that appear to be
moderately expressed (2,000 to 20,000 intensity units). It should be
noted that the relative signal intensities do not necessarily correlate
with the degrees of expression of different ORFs because intensity
depends on many factors, including PCR product size and efficiencies of
labeling and hybridization. However, the results and discussion below
show that there may be some merit in this general assumption.
As discussed above, it was anticipated that S° reduction by
P. furiosus would involve Mo- or W-containing proteins, analogous
to
the situation in mesophilic S° reducers. For example, the polysulfide
(sulfur) reductase of the mesophilic bacterium,
W. succinogenes,
is a molybdoenzyme (
32). Consequently,
the microarray for
P. furiosus included numerous ORFs
involved in the metabolism of
these metals, as well as putative
molybdo- and tungstoenyzmes.
In fact, the genome contains two ORFs
(1175771 and 1419794) that
show sequence similarity to members of the
ubiquitous molybdenum-containing
family of enzymes (
22).
However, both ORFs are expressed either
below or close to the detection
limit and show no response to
the presence of S° (data not shown). In
addition,
P. furiosus appears to contain five distinct
tungstoenzymes. Three of them
have been purified and characterized
(abbreviated AOR, GAPOR,
and FOR and encoded by ORFs 356987, 478142, and 1145403, respectively),
and two are putative (WOR4 and WOR5,
encoded by ORFs 1812948 and
1385199, respectively [see reference
57]). FOR, GAPOR, and AOR
are in the highly expressed
category (>10-fold detection limit
[see Table
3]), while WOR4 and WOR5 are moderately
expressed
and are only slightly upregulated in the presence of S°
(data
not shown). Similarly,
P. furiosus contains several
ORFs that
would encode homologs of proteins involved in the
biosynthesis
of pterin (designated
moa, mob, and
moe [see reference
51]),
the cofactor that
binds the Mo and W in molybdo- and tungstoenzymes.
None of these ORFs
showed any significant changes in expression
when cells were grown with
S°, with the exception of ORF 1805557
(discussed above). In light of
these data, it is concluded that
S° reduction in
P. furiosus does not involve Mo-, W-, or pterin-containing
proteins
or at least those that can be identified by sequence
similarity to known proteins of this type.
Some comment is also necessary on the nature of the proteins encoded by
the ORFs that are designated as either poorly or highly
expressed. For
example, the ORF with the highest intensity on
the microarray (~23
times the detection limit) encodes phosphoenolpyruvate
synthase
(
ppsA). This enzyme was recently purified from
P. furiosus and was shown to be present at extremely high cellular
concentrations
(
24), a finding consistent with the highly
expressed connotation.
In fact, of the 20 ORFs that show the highest
intensities on the
microarray (>10 times the detection limit; see
Table
3), 12 of
them encode proteins that have been purified from
P. furiosus. The biochemical data therefore supports the
notion that, in general,
these ORFs encode proteins that are indeed
present at relatively
high cellular concentrations and that signal
intensity is a reasonable
measure of this. Further evidence for this
qualitative relationship
comes from the 34 ORFs that are in the poorly
expressed category,
since none of the proteins that they are proposed
to encode have
been characterized from
P. furiosus, see
Table
4 (although one,
encoding a
putative phosphoglycerate kinase, has been cloned and
expressed from
the related organism,
P. woesei
[
10]). Of the
100 (of 271) ORFs that are in the
moderately expressed category,
the products of 12 of them have been
characterized (data not shown).
Four of the highly expressed ORFs listed in Table
3 encode the two
-subunits and the two
-subunits of two closely related,
heterotetrameric enzymes, POR (
6) and VOR
(
19). Both of these
enzymes have been purified from
P. furiosus. The genes encoding
them form three adjacent
operons, in which the third is a common
gene encoding their
subunits (
31). The gene arrangement and
their transcript
intensities in cells with or without S° are shown
in Fig.
4. Although the twofold difference in
expression of
porG is statistically significant
(
P > 0.95), S° does not dramatically
affect the
expression of POR and, accordingly, the specific activity
of POR is
comparable in the two cell types (
1). Moreover, while
the
apparent amounts of the mRNA species encoding the two
-subunits
and
the two
-subunits are consistent with the high cellular
concentrations
of the enzymes (
1,
6,
19), this is not true
for the other
subunits and particular for the ORF encoding the
-subunit of
VOR, which is barely above the detection limit. How
these three
operons are regulated such that they produce two functional
enzymes
at high cellular concentrations and with a common subunit is
not
clear from the transcript levels measured by using the DNA
microarray.
Thus, while there may be some overall relationship between
transcript
intensities on the microarray slide and cellular
concentration,
that encoding the
-subunit of VOR is a notable
exception. On
the other hand, the biochemical and electrophoretic data
strongly
support the dramatic downregulation of the expression of the
three
hydrogenases by S°, and the dramatic upregulation of at least
one of the "conserved hypothetical" ORFs (
sipA) that are
now proposed
to form a new type of S°-responsive complex. Further
analyses
of this type are now required to determine the nature of this
complex, whether it is directly involved in S° reduction, and
the
nature of the effector molecule that mediates the S° response.
View larger version (30K):
[in this window]
[in a new window]
|
FIG. 4.
Expression of the ORFs encoding POR and VOR with or
without S°. The genes (A, B, and D) encoding the -, -, and
-subunits of POR and of VOR are arranged according to their
positions in their respective operons, along with the gene (G) encoding
the -subunit that is shared by the two enzymes (31).
For each ORF, the signal intensities are indicated for cDNA obtained
from cells grown with (solid bars) or without (shaded bars) S°.
|
|
| |
ACKNOWLEDGMENTS |
This research was funded by grants to M.W.W.A. from the National
Institutes of Health (GM 60329), the National Science Foundation (MCB
9904624, MCB 9809060, and BES-0004257), and the Department of Energy
(FG05-95ER20175 and contract 992732401 with Argonne National
Laboratory) and to J.Z. from the Department of Energy under the
Microbial Genome Program and the Natural and Accelerated Bioremediation
Research Program of the Office of Biological and Environmental
Research. Oak Ridge National Laboratory is managed by the University of
Tennessee-Battelle LLC for the Department of Energy under contract
DE-AC05-00OR22725.
We thank Gary Li, Scott Lee, and Marc Sudman for their help in
preparing the microarray slides; F. Robb and R. Weiss for making the
P. furiosus sequence available prior to publication; and
Frank E. Jenney, Jr., Angeli Lal Menon, James F. Holden, Eleanor Green, and Rajat Sapra for many helpful discussions.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biochemistry and Molecular Biology, Life Sciences Bldg., University of Georgia, Athens, GA 30602-7229. Phone: (706) 546-2060. Fax: (706) 542-0229. E-mail: adams{at}bmb.uga.edu.
| |
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Journal of Bacteriology, December 2001, p. 7027-7036, Vol. 183, No. 24
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.24.7027-7036.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
